Compositions and methods for inducing immune response against EB virus

Abstract

Provided herein are compositions comprising an EB virus envelope protein (e.g., glycoprotein 350 (gp350)) and a saponin adjuvant, and methods of inducing an immune response against EB virus using the above compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application No. 63 / 325,316, filed on March 30, 2022. The contents of that application are incorporated by reference in their entirety for all purposes.

[0003] Description of this document submitted electronically

[0004] The contents of the electronic sequence listing (NOVV_098_01WO_SeqList_ST26.xml; size: 55.553 bytes; creation date: March 30, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure generally relates to a fusion protein comprising an Epstein-Barr Virus (EBV) envelope protein or a fragment thereof and (ii) ferritin or a fragment thereof, and nanoparticles and immunogenic compositions comprising the fusion protein, which can be used to stimulate an immune response against EBV. The nanoparticles provide antigens, which are usually produced by recombinant methods. The present disclosure also provides a composition containing nanoparticles, a method for producing nanoparticles, and a method for stimulating an immune response against EBV. Background Art

[0006] Infectious diseases continue to be a problem worldwide. In the United States, Epstein-Barr virus (EBV) causes 125,000 cases of infectious mononucleosis each year. Thirteen percent of patients diagnosed with infectious mononucleosis continue to experience fatigue six months after diagnosis, and 1% have other serious complications. Infectious mononucleosis is the leading cause of layoffs among military recruits.

[0007] EBV is also linked to approximately 200,000 cancer cases and 140,000 cancer deaths each year worldwide. Specifically, EBV is linked to 84,000 cases of gastric cancer, 78,000 cases of nasopharyngeal carcinoma, 29,000 cases of Hodgkin lymphoma, 7,000 cases of Burkitt lymphoma, and 2,000 cases of post-transplant lymphoproliferative disease.

[0008] Therefore, there is a need in the art for new compositions and methods for preventing EBV infection. SUMMARY OF THE INVENTION

[0010] The present disclosure provides a fusion protein comprising (i) an Epstein-Barr virus (EBV) envelope protein or a fragment thereof and (ii) ferritin or a fragment thereof. In an embodiment, provided herein is an immunogenic composition comprising the above-mentioned fusion protein for inducing an immune response against EBV. The present disclosure also provides a novel method for administering the immunogenic composition.

[0011] Provided herein are fusion proteins comprising (i) an Epstein-Barr virus (EBV) envelope protein or a fragment thereof and (ii) ferritin or a fragment thereof. In embodiments, the ferritin or a fragment thereof is a bulldog ferritin, a Helicobacter pylori ferritin, an Escherichia coli ferritin, or a combination thereof. In embodiments, the ferritin or a fragment thereof comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide having an amino acid sequence of any one of SEQ ID NOS: 16-20 and 25-38. In embodiments, the N-terminus of the ferritin or a fragment thereof comprises a peptide having about 6 to about 14 additional amino acids. In embodiments, the peptide comprises an amino acid sequence of any one of ESQVRQNF (SEQ ID NO: 40), ESQVRQQF (SEQ ID NO: 41), SGESQVRQQF (SEQ ID NO: 42), and SGESQVRQNF (SEQ ID NO: 43). In embodiments, the EBV envelope protein or fragment comprises one or more proteins selected from any one of gp350, gH, gp42, and gL. In embodiments, the EBV envelope protein or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide having an amino acid sequence of any one of SEQ ID NOS: 1-15, 21-22, 39, and 44. In embodiments, the EBV envelope protein or fragment thereof is gp350 or a fragment thereof. In embodiments, gp350 or a fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a polypeptide having an amino acid sequence of any one of SEQ ID NOS: 1-8, 21-22, 39 and 44. In embodiments, the EBV envelope protein or a fragment thereof comprises one, two, three or four mutations selected from any one of W162N, N164T, D208N and E210T, wherein the amino acids of the EBV envelope protein or a fragment thereof are numbered according to SEQ ID NO: 1.In an embodiment, the EBV envelope protein or a fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a polypeptide having the amino acid sequence of SEQ ID NO:1. In an embodiment, the fusion protein comprises: (i) an EBV envelope protein or a fragment thereof comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2; and (ii) ferritin or a fragment thereof comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:38. In an embodiment, the fusion protein comprises: (i) an EBV envelope protein or a fragment thereof comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:39; and (ii) ferritin or a fragment thereof comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:38. In an embodiment, the fusion protein comprises: (i) an EBV envelope protein or a fragment thereof comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:44; and (ii) ferritin or a fragment thereof comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:38.In embodiments, the fusion protein comprises: (i) an EBV envelope protein or a fragment thereof comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 1; and (ii) ferritin or a fragment thereof comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 38. In embodiments, the EBV envelope protein or a fragment thereof is located at the N-terminus of the ferritin or a fragment thereof. In embodiments, the EBV envelope protein or a fragment thereof is located at the C-terminus of the ferritin or a fragment thereof. In embodiments, the fusion protein comprises a signal peptide. In an embodiment, the signal peptide is a bovine prolactin signal peptide or a human CD5 signal peptide.

[0012] Provided herein are nucleic acids encoding fusion proteins described herein. Provided herein are vectors comprising nucleic acids described herein. Provided herein are host cells expressing fusion proteins described herein. Provided herein are nanoparticles comprising fusion proteins described herein. Provided herein are nanoparticles comprising EBV envelope proteins or fragments thereof and detergents. In embodiments, the detergent is a nonionic detergent. In embodiments, the nonionic detergent is polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65) or polysorbate-80 (PS80). In embodiments, the EBV envelope protein or its fragment comprises a transmembrane domain. In an embodiment, the transmembrane domain has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. In an embodiment, the EBV envelope protein or fragment thereof comprises any amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO: 1 or 44.

[0013] Provided herein are immunogenic compositions comprising fusion proteins described herein. Provided herein are immunogenic compositions comprising nanoparticles described herein. In embodiments, the immunogenic composition comprises an adjuvant. In embodiments, the adjuvant comprises at least two immunostimulatory complex (iscom) particles, wherein: the first immunostimulatory complex particle comprises fraction A of Quillaja Saponaria Molina, but not fraction C of Quillaja Saponaria; the second immunostimulatory complex particle comprises fraction C of Quillaja Saponaria, but not fraction A of Quillaja Saponaria. In embodiments, by weight, fraction A of Quillaja Saponaria and fraction C of Quillaja Saponaria account for about 85% and about 15% of the total weight of fraction A of Quillaja Saponaria and fraction C of Quillaja Saponaria in the adjuvant, respectively. In embodiments, by weight, fraction A of Quillaja Saponaria and fraction C of Quillaja Saponaria account for about 92% and about 8% of the total weight of fraction A of Quillaja Saponaria and fraction C of Quillaja Saponaria in the adjuvant, respectively. In embodiments, the immunogenic compositions described herein comprise about 30 μg to about 60 μg of adjuvant. In embodiments, the immunogenic compositions described herein comprise about 49 μg of adjuvant. In embodiments, the immunogenic compositions described herein comprise about 25 μg to about 75 μg of fusion protein. In embodiments, the immunogenic compositions described herein comprise about 50 μg of fusion protein.

[0014] In embodiments, provided herein is a method for stimulating an immune response against Epstein-Barr virus in a subject, comprising administering an immunogenic composition as described herein. In embodiments, the method comprises administering multiple doses of an immunogenic composition. In embodiments, the method comprises administering a first dose, a second dose, and a third dose of an immunogenic composition. In embodiments, the method comprises administering a second dose of an immunogenic composition 30 days after the first dose of an immunogenic composition. In embodiments, the method comprises administering a third dose of an immunogenic composition 180 days after the first dose of an immunogenic composition. In embodiments, the method comprises administering a third dose of an immunogenic composition 180 days after the second dose of an immunogenic composition. In embodiments, the method comprises administering an immunogenic composition intramuscularly. In embodiments, provided herein is a pre-filled syringe comprising an immunogenic composition as described herein. DETAILED DESCRIPTION

[0015] definition

[0016] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, "a protein" may refer to one protein or a mixture of such proteins, reference to "a method" includes equivalent steps and / or methods known to those skilled in the art, and so forth.

[0017] As used herein, the term "adjuvant" refers to a compound that, when used in conjunction with an immunogen, enhances or otherwise alters or modifies the immune response induced to the immunogen. Modification of the immune response may include enhancing or amplifying the specificity of both or either of the antibody and cellular immune responses.

[0018] As used herein, the term "about" or "approximately" when preceding a numerical value means a range of plus or minus 10% of the numerical value. For example, "about 100" includes 90 and 110.

[0019] As used herein, the terms "immunogen", "antigen" and "epitope" refer to substances capable of inducing an immune response, such as proteins (including glycoproteins) and peptides.

[0020] As used herein, an "immunogenic composition" is a composition comprising an antigen, wherein administration of the composition to a subject results in the production of a humoral and / or cellular immune response to the antigen in the subject.

[0021] As used herein, a "subunit" composition, such as a vaccine, includes one or more selected antigens, but not all antigens, of a pathogen. Such compositions are substantially free of whole viruses or lysates of such cells or particles, and are typically prepared from at least partially purified, usually substantially purified, immunogenic polypeptides of the pathogen. Antigens in the subunit compositions disclosed herein are typically prepared recombinantly, often using a baculovirus system.

[0022] As used herein, "substantially" refers to the separation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance constitutes the majority of a sample containing it. For example, in a sample, a substantially purified component constitutes 85%, preferably 85%-90%, more preferably at least 95%-99.5%, and most preferably at least 99% of the sample. If a component is substantially replaced, the remaining amount in the sample is less than or equal to about 0.5% to about 10%, preferably less than about 0.5% to about 1.0%.

[0023] As used herein, the terms "treat," "treatment," and "treating" refer to methods of obtaining beneficial or desired results, such as clinical results. For purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the occurrence or development of an infection or disease; ameliorating or reducing the development of infection or disease symptoms; or a combination thereof.

[0024] As used herein, "prevention" is used interchangeably with "prophylaxis" and can mean preventing an infection or disease completely, or preventing the development of symptoms of the infection or disease; delaying the onset of an infection or disease or its symptoms; or reducing the severity of a subsequently developing infection or disease or its symptoms.

[0025] As used herein, "effective dose" or "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of a pathogen infection. An effective dose or effective amount can be determined, for example, by measuring the amount of neutralizing secretory antibodies and / or serum antibodies, for example, by plaque neutralization assays, complement fixation assays, enzyme-linked immunosorbent assays (ELISA) or microneutralization assays to measure the amount of neutralizing secretory antibodies and / or serum antibodies.

[0026] The term "vaccine" as used herein refers to an immunogenic composition, such as an immunogen derived from a pathogen, that is used to induce an immune response against a pathogen to provide protective immunity (e.g., immunity that protects a subject from infection with a pathogen and / or reduces the severity of a disease or condition caused by infection with a pathogen). The protective immune response may include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" may also refer to a suspension or solution of an immunogen that is administered to a subject to produce protective immunity.

[0027] As used herein, the term "subject" includes humans and other animals. The subject is typically a human. For example, the subject can be an adult, teenager, child (2 to 14 years old), infant (birth to 2 years old) or newborn (up to 2 months). In specific aspects, the subject can be up to 4 months or 6 months old. In some aspects, an adult is an elderly person about 65 years old or older or about 60 years old or older. In some aspects, the subject is a pregnant woman or a woman who intends to become pregnant. In other aspects, the subject is not a human, such as a non-human primate, such as a baboon, chimpanzee, gorilla or macaque. In some aspects, the subject can be a pet, such as a dog or a cat.

[0028] The term "pharmaceutically acceptable" as used herein refers to approved by the U.S. federal or state government regulatory agencies, or listed in the U.S. Pharmacopeia (US Pharmacopeia), the European Pharmacopeia (European Pharmacopeia) or other recognized pharmacopeias for use in mammals, especially humans. These compositions can be used as vaccines and / or antigenic compositions for inducing protective immune responses in vertebrates.

[0029] In the context of two or more nucleic acid or polypeptide sequences, the term "percent identity" refers to two or more sequences or subsequences that, when compared, have a specified percentage of identical nucleotides or amino acid residues. Percent identity can be calculated using the CLUSTALW2 tool, which is available online. The following parameters can be used for CLUSTALW2 pairwise alignments: protein weight matrix = Gonnet; gap open = 10; gap extension = 0.1.

[0030] Immunogenic composition containing fusion protein comprising Epstein-Barr virus envelope protein and ferritin

[0031] Fusion Protein

[0032] Provided herein is an immunogenic composition comprising a fusion protein comprising (i) an Epstein-Barr virus (EBV) envelope protein or a fragment thereof and (ii) ferritin or a fragment thereof. In an embodiment, the EBV envelope protein or a fragment thereof is located at the N-terminus of ferritin or a fragment thereof. In an embodiment, the EBV envelope protein or a fragment thereof is located at the C-terminus of ferritin or a fragment thereof. Alternative fusion proteins that can be used for the immunogenic compositions described herein are described in the following documents, the entire contents of which are incorporated herein by reference for all purposes: U.S. Patent No. 10,744,199 and Kanekiyo et al. Cell. 2015 Aug 27; 162(5): 1090-100.

[0033] In an embodiment, a “fragment thereof” of a protein has a length of about 10 to about 1500 amino acids (e.g., a length of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, or about 1500 amino acids). In embodiments, fragments thereof comprise at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least about 98%, at least about 99%, or at least about 99.5% of the wild-type protein.

[0034] In embodiments, the fusion protein further comprises a protein tag for purification or detection. In embodiments, the protein tag is a polyglutamic acid tag, a FLAG tag, an HA tag, a poly-His tag (having about 5-10 histidines) (SEQ ID NO: 45), a hexahistidine tag (SEQ ID NO: 46), an 8X-His tag (having eight histidines) (SEQ ID NO: 47), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose binding protein tag, a thioredoxin tag, an Fc tag, or a C tag. In embodiments, the extension portion comprises a C tag. The C tag comprises an EPEA sequence (SEQ ID NO: 48).

[0035] EBV envelope protein

[0036] In an embodiment, the fusion protein comprises an EBV envelope protein.In an embodiment, the EBV envelope protein is selected from gp350, gH, gp42, gL, or a combination thereof.

[0037] In embodiments, the EBV envelope protein comprises gp350 or a fragment thereof. In embodiments, the EBV envelope protein comprises a full-length gp350 protein. An exemplary full-length gp350 protein from Epstein-Barr virus strain B95-8 (UNIPROTID: P03200) has an amino acid sequence of SEQ ID NO: 1. The full-length gp350 protein comprises domains 1, 2, 3, a receptor binding domain, a transmembrane domain, and a topological domain. In embodiments, the EBV envelope protein comprises one or more fragments of gp350 selected from any one of the following: gp350 domain 1, EBV gp350 domain 2, and EBV gp350 domain 3, an EBV receptor binding domain (RBD), an EBV extracellular domain, an EBV transmembrane domain, and an EBV topological domain. In embodiments, the EBV envelope protein lacks an N-terminal methionine. .

[0038] In an embodiment, the EBV envelope protein is an EBV extracellular domain. In an embodiment, the EBV extracellular domain has the sequence SEQ ID NO:2.

[0039] In embodiments, the EBV envelope protein comprises a transmembrane domain. In embodiments, the transmembrane domain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a transmembrane domain having an amino acid sequence of SEQ ID NO: 23. In embodiments, the EBV envelope protein comprises a topological domain. In embodiments, the topological domain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a topological domain having an amino acid sequence of SEQ ID NO: 24. In embodiments, the EBV envelope protein comprises a transmembrane domain and a topological domain.

[0040] In an embodiment, the fusion protein comprises an EBV envelope protein that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a protein having an amino acid sequence of any one of SEQ ID NOS: 1-15, 21-22, 39 and 44.

[0041] In an embodiment, the EBV envelope protein or a fragment thereof comprises one or more of the following mutations: W162N, N164T, D208N and E210T, wherein the amino acid sequence of the EBV envelope protein or a fragment thereof is numbered according to SEQ ID NO:1.

[0042] In an embodiment, the EBV envelope protein or fragment thereof lacks an N-terminal methionine.

[0043] Table A provides exemplary EBV envelope proteins.

[0044] Table A

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] Ferritin

[0052] In embodiments, the fusion protein comprises ferritin or a fragment thereof. In embodiments, the ferritin or a fragment thereof is bulldog ferritin, H. pylori ferritin, E. coli ferritin, or a combination thereof. In embodiments, the fusion protein comprises a ferritin that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a protein having an amino acid sequence of any one of SEQ ID NOS: 16-20 and 25-38. In embodiments, the ferritin comprises 6-14 additional amino acids at the N-terminus. In embodiments, the N-terminus of the ferritin comprises any one of ESQVRQNF (SEQ ID NO: 40), ESQVRQQF (SEQ ID NO: 41), SGESQVRQQF (SEQ ID NO: 42), and SGESQVRQNF (SEQ ID NO: 43).

[0053] Exemplary ferritins are provided in Table B. In embodiments, the UniProt ID of ferritin is P07797 or Q9ZLI1.

[0054] Table B

[0055]

[0056]

[0057]

[0058] Nanoparticles containing the fusion protein described herein

[0059] Ferritin-containing nanoparticles

[0060] In an embodiment, the fusion protein forms nanoparticles. In an embodiment, nanoparticles are produced according to the scheme outlined in the following publication, the entire contents of which are incorporated herein by reference for all purposes: Kanekiyo et al. Cell. 2015 Aug 27; 162 (5): 1090-100. In an embodiment, a host cell (e.g., 293F or Expi293F cell) is transfected with an expression vector encoding a fusion protein as described herein. In an embodiment, nanoparticles are purified by affinity chromatography. In an embodiment, purification is performed using a Galanthus nivalis lectin resin. In an embodiment, purification is performed using size exclusion chromatography.

[0061] Nanoparticles containing detergents

[0062] In embodiments, the fusion protein forms a nanoparticle comprising a fusion protein and a detergent. In embodiments, nanoparticles comprising an EBV envelope protein or a fragment thereof and a detergent are provided herein. In embodiments, the detergent is a nonionic detergent. In embodiments, the detergent is selected from polysorbate 20 (PS20), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65) and polysorbate 80 (PS80). In embodiments, the EBV envelope protein comprises an extracellular domain and a transmembrane domain. In embodiments, the transmembrane domain of the EBV envelope protein or its fragment interacts with the detergent core, and the extracellular domain of the EBV envelope protein or its fragment protrudes outward. In embodiments, a detergent exchange method is utilized to form nanoparticles, wherein a first detergent is used to separate proteins, and then the first detergent is exchanged with a second detergent to form nanoparticles.

[0063] The fusion protein or EBV envelope protein or its fragment contained in the nanoparticle is usually produced by recombinant expression in a host cell. Standard recombinant techniques can be used. In an embodiment, a baculovirus system is used to express a fusion protein or an EBV envelope protein or its fragment in an insect host cell. In an embodiment, the baculovirus is a cathepsin-L knockout baculovirus, a chitinase knockout baculovirus. Optionally, the baculovirus is a cathepsin-L and chitinase double knockout. High-level expression can be obtained in an insect cell expression system. Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, such as Sf9, Sf21, Trichoplusiani cells, such as High Five cells, and Drosophila (Drosophila) S2 cells. In an embodiment, a fusion protein or an EBV envelope protein or its fragment described herein is produced in any suitable host cell. In an embodiment, the host cell is an insect cell. In an embodiment, the insect cell is an Sf9 cell.

[0064] Typical transfection and cell growth methods can be utilized to culture cells. According to methods known in the art, a vector, such as a vector comprising a polynucleotide encoding a fusion protein or an EBV envelope protein or its fragment, can be transfected into a host cell. For example, nucleic acid can be introduced into a eukaryotic cell by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, and transfection using a polyamine transfection reagent. In one embodiment, the vector is a recombinant baculovirus.

[0065] Methods for growing host cells include, but are not limited to, batch, fed-batch, continuous, and perfusion cell culture techniques. Cell culture refers to the growth and propagation of cells in a bioreactor (fermentation chamber) where the cells multiply and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is performed under sterile, controlled temperature and atmospheric conditions in a bioreactor. A bioreactor is a chamber for culturing cells in which environmental conditions, such as temperature, atmosphere, agitation, and / or pH, can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (e.g., Wave Biotech, Bridgewater, NJ). In another embodiment, the pre-sterilized plastic bag is a bag of about 50 L to 3500 L.

[0066] After the host cells have grown, the fusion protein or EBV envelope protein or fragments thereof can be harvested from the host cells using detergent and purification protocols. Once the host cells have been grown for 48-96 hours, the cells are separated from the culture medium and a solution containing a detergent is added to solubilize the cell membrane, thereby releasing the protein into the detergent extract. Triton X-100 and Nonylphenol polyoxyethylene ether (also known as NP-9) is each a preferred extraction detergent. The detergent can be added to a final addition of about 0.1% to about 1.0%. For example, the concentration can be about 0.1%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.8%, or about 1.0%. The range can be about 0.1% to about 0.3%. In many aspects, the concentration is about 0.5%.

[0067] In other aspects, different first detergents can be used to separate fusion proteins or EBV envelope proteins or fragments thereof from host cells. For example, the first detergent can be bis(polyethylene glycol bis[imidazolylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazolylcarbonyl]), Polyethylene glycol dodecyl ether 35, Polyethylene glycol (3) hexadecyl ether 56, Alcohol polyoxyethylene ether 72, Polyoxyl 2-stearyl ether 76, Polyethylene glycol monooleate 92V, Polyoxyethylene (10) oleyl ether 97, Polyethylene glycol cetyl ether 58P, Macrogolglycerol ricinoleate, Decaethyleneglycol monododecyl ether, N-decanoyl-N-methylglucamine, Decyl α-D-glucopyranoside, Decyl β-D-maltopyranoside, Dodecanoyl-N-methylglucamine, Dodecyl α-D-maltoside, Dodecyl β-D-maltoside, Dodecyl β-D-maltoside, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, Hexadecyl β-D-maltoside, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, methyl-6-0-(N-heptylcarbamoyl)-α-D-pyranoglucoside, nonanediol monododecyl ether, N-nonanoyl-N-glucamine, N-nonanoyl-N-glucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, octaethylene glycol monohexadecyl ether, octaethylene glycol monooctadecyl ether, octaethylene glycol monotetradecyl ether, octyl-β-D-pyranoglucoside, pentaethylene glycol monodecyl ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monohexadecyl ether ethyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monooctadecyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W-1, polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyoxyethylene bis (imidazole carbonyl), polyoxyethylene 25 propylene glycol stearate, saponin from Quillaja bark, Sorbitan laurate, Sorbitan monopalmitate, Sorbitan stearate, Sorbitan tristearate, Sorbitan monooleate, Sorbitan trioleate, 15-S-12 type Secondary alcohol polyoxyethylene ether, 15-S-30 type Secondary alcohol polyoxyethylene ether, 15-S-5 type Secondary alcohol polyoxyethylene ether, 15-S-7 type Secondary alcohol polyoxyethylene ether, 15-S-9 type Secondary alcohol polyoxyethylene ether, NP-10 type Nonylphenol polyoxyethylene ether, NP-4 nonylphenol polyoxyethylene ether, NP-40 Nonylphenol polyoxyethylene ether, NP-7 type Nonylphenol polyoxyethylene ether, NP-9 type Nonylphenol polyoxyethylene ether, TMN-10 type Branched secondary alcohol polyoxyethylene ether, TMN-6 type Branched secondary alcohol polyoxyethylene ether, TRITON™ X-100 polyethylene glycol tert-octylphenyl ether or a combination thereof.

[0068] The nanoparticles can then be separated from the cell debris using centrifugation. In embodiments, gradient centrifugation can be used, for example using cesium chloride, sucrose, and iodixanol. Alternatively or additionally, other techniques can also be used, such as standard purification techniques, including ion exchange chromatography, affinity chromatography, and gel filtration chromatography, etc.

[0069] For example, the first column can be an ion exchange chromatography resin, such as EMD methacrylate-based polymer beads TMAE (EMD Millipore), the second column can be a lentil ( Lens culinaris ) lectin affinity resin, and the third column can be a cation exchange column, e.g. EMD methacrylate-based polymer beads SO3 (EMD Millipore) resin. In other aspects, the cation exchange column can be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc). Preferably, the method disclosed herein does not use a detergent extraction column, such as a hydrophobic interaction column. Such chromatographic columns are typically used to remove detergents during purification, but may have a negative impact on the method disclosed herein.

[0070] In order to form nanoparticles, the first detergent for extracting fusion protein or EBV envelope protein or its fragment from host cells is substantially replaced by the second detergent, to reach the nanoparticle structure. NP-9 is a preferred extraction detergent. Usually, when measured by HPLC, the nanoparticles do not contain detectable NP-9. The second detergent is usually selected from PS20, PS40, PS60, PS65 and PS80. The second detergent is preferably PS80.

[0071] In a specific aspect, the detergent exchange is performed using affinity chromatography to bind the glycoprotein via the carbohydrate moiety. For example, the affinity chromatography can use a leguminous lectin column. Leguminous lectins are proteins that were originally identified in plants and found to interact specifically and reversibly with carbohydrate residues. For example, see Sharon and Lis, "Legume lectins--a large family of homologous proteins," FASEB J. 1990 Nov; 4 (14): 3198-208; Liener, "The Lectins: Properties, Functions, and Applications in Biology and Medicine," Elsevier, 2012. Suitable lectins include concanavalin A (con A), pea lectin, sainfoin, and lentil lectin. Lentil lectin is a preferred detergent exchange column due to its binding properties. Lectin columns are commercially available; for example, Capto Lentil Lectin (Capto lentil lectin) is available from GE Healthcare. In some aspects, lentil lectin columns can use recombinant lectins. At the molecular level, it is believed that the carbohydrate portion is combined with lentil lectin, thereby releasing the amino acids of the protein to condense around the detergent, thereby forming a detergent core providing nanoparticles with multiple copies of antigens, for example, glycoprotein oligomers, which can be dimers, trimers or tetramers anchored in the detergent. In embodiments, fusion protein nanoparticles or EBV envelope proteins or fragments thereof form trimers. In embodiments, fusion protein nanoparticles or trimers of EBV envelope proteins or fragments thereof are anchored in detergents. In embodiments, each fusion protein nanoparticle or EBV envelope protein or fragment nanoparticle contains at least one trimer bound to a non-ionic core.

[0072] When detergent is incubated with protein to form nanoparticles during detergent exchange, detergent can be present in an amount up to about 0.1% (w / v) in the early purification step, and in order to make the final nanoparticles have optimal stability, the amount is reduced. For example, a nonionic detergent (e.g., PS80) can be from about 0.005% (v / v) to about 0.1% (v / v), such as about 0.005% (v / v), about 0.006% (v / v), about 0.007% (v / v), about 0.008% (v / v), about 0.009% (v / v), about 0.01% (v / v), about 0.015% (v / v), about 0.02% (v / v), about 0.025% (v / v), about 0.03% (v / v), about 0.04% (v / v), about 0.05% (v / v), about 0.06% (v / v), about 0.08% (v / v), about 0.09% (v / v), about 0.10% (v / v), about 0.11% (v / v), about 0.12% (v / v), about 0.13% (v / v), about 0.14% (v / v), about 0.15% (v / v), about 0.16% (v / v), about 0.17% (v / v), about 0.18% (v / v), about 0.19% (v / v), about 0.20% (v / v), about 0.21% (v / v), about 0.22% (v / v), about 0.23% (v / v), about 0.24% (v / v), about 0.25% (v / v), about 0.26% (v / v), about 0.27% (v In some embodiments, the nanoparticles contain about 0.03% to about 0.05% PS80. In some embodiments, the nanoparticles contain about 0.01% (v / v) PS80.

[0073] In embodiments, the fusion protein or EBV envelope protein or its fragment of dialysis purification.In embodiments, dialysis is carried out after purification.In embodiments, the fusion protein or EBV envelope protein or its fragment of dialysis purification in a solution comprising sodium phosphate, NaCl and PS80.In embodiments, the dialysis solution comprising sodium phosphate contains about 5mM to about 100mM sodium phosphate, for example, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM or about 100mM sodium phosphate. In embodiments, the pH of the solution comprising sodium phosphate is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In embodiments, the dialysis solution comprising sodium chloride comprises about 50 mM NaCl to about 500 mM NaCl. NaCl, for example, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM 470mM, about 480mM, about 490mM, or about 500mM NaCl.In embodiments, the dialysis solution comprising PS80 comprises about 0.005% (v / v), about 0.006% (v / v), about 0.007% (v / v), about 0.008% (v / v), about 0.009% (v / v), about 0.01% (v / v), about 0.015% (v / v), about 0.02% (v / v), about 0.025% (v / v), about 0.03% (v / v), about 0.035% (v / v), about 0.040% (v / v), about 0.045% (v / v), about 0.050% (v / v), about 0.060% (v / v), about 0.070% (v / v), about 0.080% (v / v), about 0.090% (v / v), about 0.100% (v / v), about 0.110% (v / v), about 0.120% (v / v), about 0.130% (v / v), about 0.140% (v / v), about 0.150% (v / v), about 0.160% (v / v), about 0.170% (v / v), about 0.180% (v / v), about 0.190% (v / v), In some embodiments, the dialysis solution comprises about 25 mM sodium phosphate (pH 7.2), about 300 mM NaCl and about 0.01% (v / v) PS80.

[0074] Detergent exchange can be performed with the protein purified as described above, purified, frozen and stored, and then thawed for detergent exchange. The stability of the composition disclosed herein can be measured by a variety of methods. In one method, a peptide map is prepared to determine the integrity of the antigen protein after various treatments, and various treatments are designed to apply stress to the nanoparticles by simulating harsh storage conditions. Therefore, the measurement of stability is the relative abundance of antigen peptides in the stress sample compared with the control sample. For example, the stability of the nanoparticles can be assessed by exposing the nanoparticles containing purified fusion protein or EBV envelope protein or its fragments to various pH, protease, salt, oxidant (including but not limited to hydrogen peroxide), various temperatures, freezing / thawing cycles and stirring. It is believed that the position of the glycoprotein anchored in the detergent core enhances stability by reducing undesirable interactions. For example, the improved protective effect for protease-based degradation can be achieved by a shielding effect, whereby the glycoprotein anchored in the core at a molar ratio disclosed herein will produce a steric hindrance that prevents protease from entering. Stability can also be measured by monitoring the intact protein.

[0075] Adjuvant

[0076] In an embodiment, the immunogenic composition comprises an adjuvant.

[0077] In certain embodiments, the compositions disclosed herein can be combined with one or more adjuvants to enhance the immune response. In other embodiments, the compositions are prepared without the use of an adjuvant and can therefore be administered as a non-adjuvanted composition.

[0078] Aluminum-based adjuvants

[0079] In embodiments, the adjuvant can be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to alum. For example, the nanoparticles can be at least 80%, at least 85%, at least 90%, or at least 95% bound to alum. Typically, 92% to 97% of the nanoparticles are bound to alum in the composition. The amount of alum per dose is typically about 400 μg to about 1250 μg. For example, the amount of alum present per dose can be about 300 μg to about 900 μg, about 400 μg to about 800 μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about 400 μg to about 500 μg. Typically, corresponding to a dose of 120 μg of protein nanoparticles, the alum present is about 400 μg.

[0080] In an embodiment, the adjuvant is a saponin adjuvant.

[0081] Saponin adjuvant

[0082] Adjuvants containing saponins can also be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the Quillaja tree. Typically, saponins are prepared using a multi-step purification process to obtain multiple fractions. As used herein, the term "Quillaja saponin fraction" is used to generically describe a semi-purified or defined saponin fraction of the Quillaja tree or a substantially purified fraction thereof.

[0083] Saponin fraction

[0084] Several methods of producing saponin fractions are suitable. Fractions A, B and C are described in U.S. Pat. No. 6,352,697 and can be prepared as follows. The lipophilic fraction is extracted from Quil A (aqueous extract of crude soap bark tree) by chromatographic separation and eluted with 70% acetonitrile in water to recover the lipophilic fraction. The lipophilic fraction is then separated by semi-preparative HPLC with an acetonitrile acidic aqueous solution gradient of 25% to 60%. The fraction referred to herein as "fraction A" or "QH-A" is or corresponds to the fraction eluted with about 39% acetonitrile. The fraction referred to herein as "fraction B" or "QH-B" is or corresponds to the fraction eluted with about 47% acetonitrile. The fraction referred to herein as "fraction C" or "QH-C" is or corresponds to the fraction eluted with about 49% acetonitrile. For additional information on fraction purification, see U.S. Pat. No. 5,057,540. When prepared as described herein, Quillaja fractions A, B and C each represent chemically closely related groups or families of molecules with definable properties. The chromatographic conditions under which these fractions were obtained provide high batch-to-batch reproducibility in elution profiles and biological activity.

[0085] Other saponin fractions have been described. EP 0436620 describes fractions B3, B4 and B4b. Fractions QA1-QA22 are described in EP03632279 B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja Spikoside (lsconova AB, Ultunaallén 2B, 756 51 Uppsala, Switzerland). The fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21 and QA-22 of EP 0 3632 279 B2 can be used, in particular QA-7, QA-17, QA-18 and QA-21. The methods for obtaining them are described in EP 03632279 B2, in particular in Example 1 on pages 6 and 8 and 9.

[0086] The saponin fractions described herein for forming adjuvants are generally substantially pure fractions; that is, these fractions are substantially free of contamination from other materials. In particular aspects, substantially pure saponin fractions may contain up to 40%, 30%, 25%, 20%, 15%, 10%, 7%, 5%, 2%, 1%, 0.5% or 0.1% by weight of other compounds, such as other saponins or other adjuvant materials.

[0087] ISCOM Structure

[0088] The saponin fraction can be administered in the form of caged particles called ISCOM (immunostimulatory complex). ISCOM can be prepared as described in EP0109942B1, EP0242380B1 and EP0180546B1. In a specific embodiment, transport antigens and / or passenger antigens can be used, as described in EP 9600647-3 (PCT / SE97 / 00289).

[0089] Matrix adjuvant

[0090] In an embodiment, the ISCOM is an ISCOM matrix complex. The ISCOM matrix complex comprises at least one saponin fraction and a lipid. The lipid is at least a sterol, such as cholesterol. In a specific aspect, the ISCOM matrix complex also contains a phospholipid. The ISCOM matrix complex may also contain one or more other immunomodulatory (adjuvant active) substances, not necessarily glycosides, and may be produced as described in EP0436620B1, which is incorporated herein by reference in its entirety.

[0091] In other aspects, ISCOM is an ISCOM complex. The ISCOM complex comprises at least one saponin, at least one lipid and at least one antigen or epitope. The ISCOM complex contains an antigen bound by detergent treatment, thereby integrating part of the antigen into the particle. In contrast, the ISCOM matrix is ​​formulated in a blend with the antigen, and the binding between the ISCOM matrix particles and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0092] The saponin fraction may be incorporated into an ISCOM matrix complex or an ISCOM complex, or at least one additional adjuvant also incorporated into or mixed with an ISCOM or ISCOM matrix complex is selected from: Fraction A, Fraction B or Fraction C of Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or any purified sub-fraction, such as QA 1-21.

[0093] In specific aspects, each ISCOM particle can contain at least two saponin fractions. Any combination of weight percentages of different saponin fractions can be used. Any combination of weight percentages of any two fractions can be used. For example, the particle can contain any weight percentage of fraction A and any weight percentage of another saponin fraction, such as a crude saponin fraction or fraction C. Thus, in specific aspects, each ISCOM matrix particle or each ISCOM complex particle can contain 0.1-99.9%, 5-95%, 10-90%, 15-85%, 20-80%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 40-60% or 50% by weight of one saponin fraction, such as fraction A, and in each case of another saponin, such as any crude fraction or any other fraction, such as fraction C, the remainder up to 100%. The weight is calculated by the total weight of the saponin fractions. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in US Published Application No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0094] In embodiments, the ISCOM matrix or ISCOM complex comprises 5-99% by weight of one fraction, such as Fraction A, and the remainder up to 100% of another fraction, such as the crude saponin fraction or Fraction C. The weight is calculated based on the total weight of the saponin fractions.

[0095] In embodiments, the ISCOM matrix or ISCOM complex comprises 40% to 99% by weight of one fraction, such as Fraction A, and 1% to 60% by weight of another fraction, such as a crude saponin fraction or Fraction C. The weight is calculated based on the total weight of the saponin fractions.

[0096] The ISCOM matrix or ISCOM complex may comprise 70% to 95% by weight of one fraction, such as fraction A, and 30% to 5% by weight of another fraction, such as a crude saponin fraction or fraction C. The weight is calculated based on the total weight of the saponin fractions. In other embodiments, the saponin fraction of Quillaja saponaria is selected from any one of QA 1-21.

[0097] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles can each be formed using only one saponin fraction. The compositions disclosed herein can contain a plurality of particles, each of which contains only one saponin fraction. That is, certain compositions can contain one or more different types of ISCOM matrix complex particles and / or one or more different types of ISCOM complex particles, each of which contains a saponin fraction of Quillaja saponin, wherein the saponin fraction in one complex is different from the saponin fraction in the other complex particles.

[0098] In particular aspects, one type of saponin fraction or crude saponin fraction can be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction can be incorporated into another ISCOM matrix complex or particle. The composition or vaccine can comprise at least two types of complexes or particles, each type of complex or particle having one type of saponin incorporated into physically distinct particles.

[0099] In the composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles may be used, wherein one saponin fraction Quillaja saponaria and another saponin fraction Quillaja saponaria are incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles, respectively.

[0100] ISCOM matrix or ISCOM complex particles each having a saponin fraction may be present in the composition in any weight percentage combination. In a specific aspect, the composition may contain 0.1% to 99.9%, 5% to 95%, 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 30% to 70%, 35% to 65%, 40% to 60%, 45% to 55%, 40-60% or 50% by weight of an ISCOM matrix or complex containing a first saponin fraction, with the remainder being composed of an ISCOM matrix or complex containing a different saponin fraction. In some aspects, the remainder is one or more ISCOM matrices or complexes, each of which contains only one saponin fraction. In other aspects, the ISCOM matrix or complex particles may contain more than one saponin fraction.

[0101] In a specific composition, the only saponin fraction in the first ISCOM matrix or ISCOM complex particle is Fraction A and the only saponin fraction in the second ISCOM matrix or ISCOM complex particle is Fraction C.

[0102] A preferred composition comprises a first ISCOM matrix comprising fraction A and a second ISCOM matrix comprising fraction C, wherein the fraction A ISCOM matrix comprises about 70% by weight of the total saponin adjuvant and the fraction C ISCOM matrix comprises about 30% by weight of the total saponin adjuvant. In another preferred composition, the fraction A ISCOM matrix comprises about 85% by weight of the total saponin adjuvant and the fraction C ISCOM matrix comprises about 15% by weight of the total saponin adjuvant. In another preferred composition, the fraction A ISCOM matrix comprises about 92% by weight of the total saponin adjuvant and the fraction C ISCOM matrix comprises about 8% by weight of the total saponin adjuvant. Thus, in certain compositions, the fraction A ISCOM matrix is ​​present at about 70% to about 85% of the total weight of the saponin adjuvant in the composition and the fraction C ISCOM matrix is ​​present at about 15% to about 30% of the total weight of the saponin adjuvant in the composition. In an embodiment, the Fraction A ISCOM matrix comprises 50-96% by weight of the combined weight of the Fraction A ISCOM matrix and the Fraction C ISCOM matrix in the adjuvant, with the Fraction C ISCOM matrix comprising the remainder. TM ), the Fraction A ISCOM matrix is ​​present in an amount of about 85% of the total weight of the saponin adjuvant in the composition, and the Fraction C ISCOM matrix is ​​present in an amount of about 15% of the total weight of the saponin adjuvant in the composition. MATRIX-M TM Interchangeably referred to as Matrix-M1.

[0103] Exemplary QS-7 and QS-21 fractions, their production and use are described in U.S. Pat. Nos. 5,057,540, 6,231,859, 6,352,697, 6,524,584, 6,846,489, 7,776,343 and 8,173,141, which are incorporated herein by reference.

[0104] In addition or as an alternative, other adjuvants may be used. It is contemplated that the scope of the present disclosure includes any adjuvant described in "A Compendium of Vaccine Adjuvants and Excipients" (2nd Edition) by Vogel et al., which is incorporated herein by reference in its entirety for all purposes. Other adjuvants include complete Freund's adjuvant (a nonspecific immune response stimulator containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP complexes (e.g., thur-MDP and nor-MDP), CGP (MTP-PE), lipid A and monophosphoryl lipid A (MPL), MF-59, RIBI, RIBI containing 2% squalene The three components extracted from bacteria in the polysorbate 80 emulsion are MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS). In an embodiment, the adjuvant can be a paucilamellar lipid vesicle; for example They are oligolamellar, non-phospholipid vesicles with a size of about 100 nm to about 500 nm. They contain Ethoxylate 72, cholesterol, oleic acid and squalene. It is an effective adjuvant (see US Pat. Nos. 5,629,021, 6,387,373 and 4,911,928).

[0105] excipient

[0106] The immunogenic compositions described herein may include various excipients, buffers, and the like. For example, the immunogenic compositions may contain sodium phosphate, sodium chloride, and / or histidine. Sodium phosphate may be present at about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 25 mM; in specific cases, there is about 22 mM sodium phosphate. Histidine may be present at about 0.1% (w / v), about 0.5% (w / v), about 0.7% (w / v), about 1% (w / v), about 1.5% (w / v), about 2% (w / v), or about 2.5% (w / v). Sodium chloride, when present, may be about 150 mM. In certain compositions, sodium chloride may be present at a higher concentration, such as about 200 mM to about 500 mM. In embodiments, sodium chloride is present at a high concentration, including but not limited to about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM.

[0107] Administration and Dosage

[0108] Provided herein is a method for inducing an immune response against EBV in a subject in need, including administering an immunogenic composition comprising a fusion protein described herein. In embodiments, the subject is human. In embodiments, the subject's body mass index (BMI) is less than or equal to 36. In embodiments, BMI is less than about 35, less than about 34, less than about 33, less than about 32, less than about 31, less than about 30, less than about 29, less than about 28, less than about 27, less than about 26, less than about 25, less than about 24, less than about 23, less than about 22, less than about 21, less than about 20, less than about 19, less than about 18, less than about 17, less than about 16, less than about 15, less than about 14, less than about 13, less than about 12, less than about 11 or less than about 10. In embodiments, BMI is 10-36, 15-36, 18.5-36, 25.0-29.9, 18.5-24.9 or 30 or higher. In embodiments, the subject is greater than about 60 years old, greater than about 61 years old, greater than about 62 years old, greater than about 63 years old, greater than about 64 years old, greater than about 65 years old, greater than about 66 years old, greater than about 67 years old, greater than about 68 years old, greater than about 69 years old, or greater than about 70 years old. In embodiments, the subject is about 18 years old to about 29 years old. In embodiments, the subject is about 30 years old to about 65 years old. In embodiments, the subject is about 10 years old to about 17 years old. In embodiments, the subject is about 1 year old, about 2 years old, about 3 years old, about 4 years old, about 5 years old, about 6 years old, about 7 years old, about 8 years old, about 9 years old, about 10 years old, about 11 years old, about 12 years old, about 13 years old, about 14 years old, about 15 years old, about 16 years old, about 17 years old, about 18 years old, about 19 years old, about 20 years old, about 21 years old, about 22 years old, about 23 years old, about 24 years old, about 25 years old, about 26 years old, about 27 years old, about 28 years old, about 29 years old, about 30 years old, about 31 years old, about 32 years old, about 33 years old, about 34 years old, about 35 years old, about 36 years old, about 37 years old, about 38 years old, about 39 years old, about 40 years old, about 41 years old, about 42 years old, about 43 years old, about 44 years old, about 45 years old, about 46 years old, about 47 years old, about 48 years old, about 49 years old, about 50 years 6 years old, about 27 years old, about 28 years old, about 29 years old, about 30 years old, about 31 years old, about 32 years old, about 33 years old, about 34 years old, about 35 years old, about 36 years old, about 37 years old, about 38 years old, about 39 years old, about 40 years old, about 41 years old, about 42 years old, about 43 years old, about 44 years old, about 45 years old, about 46 years old, about 47 years old, about 48 years old, about 49 years old, about 50 years old, about 51 years old, about 52 years old, about 53 years old, about 54 years old, about 55 years old, about 56 years old, about 57 years old, about 58 years old, about 59 years old, about 60 years old, about 61 years old, about 62 years old, about 63 years old, about 64 years old, about 65 years old, about 66 years old, about 67 years old, about 68 years old, about 69 years old, about 70 years old, about 71 years old, about 72 years old, about 73 years old, about 74 years old, about 75 years old, about 76 years old, about 77 years old, About 78 years old, about 79 years old, about 80 years old, about 81 years old, about 82 years old, about 83 years old, about 84 years old, about 85 years old, about 86 years old, about 87 years old, about 88 years old, about 89 years old, about 90 years old, about 91 years old, about 92 years old, about 93 years old, about 94 years old, about 95 years old, about 96 years old, about 97 years old, about 98 years old, about 99 years old, about 100 years old, including any ages and ranges in between.

[0109] In embodiments, each dose of the fusion protein independently comprises from about 0.1 μg to about 100 μg of the fusion protein, including all ranges and subranges therebetween. In embodiments, the method comprises administering a fusion protein described herein at a dose of 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about 1.8 μg, about 1.9 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 8μg, about 9μg, about 10μg, about 11μg, about 12μg, about 13μg, about 14μg, about 15μg, about 16μg, about 17μg, about 18μg, about 19μg, about 20μg, about 21μg, about 22μg, about 23μg, about 24μg, about 25μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, about 51μg, about 52μg, about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, about 58μg, about 59μg, about 60μg, about 61μg, about 62μg, about 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, About 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76μg, about 77μg, about 78μg, about 79μg, about 80μg, about 81μg, about 82μg, about 83μg, about 84μg, about 85μg, about 86μg, about 87μg, about 88μg, about 89μg, about 90μg, about 91μg, about 92μg, about 93μg, about 94μg, about 95μg, about 96μg, about 97μg, about 98μg, about 99μg or about 100μg. In an embodiment, the fusion protein comprises an EBV envelope protein having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOS: 1-15, 21-22, 39 and 44.In embodiments, the fusion protein comprises a ferritin having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOS: 16-20 and 25-38.

[0110] In embodiments, the immunogenic composition comprises an adjuvant. In embodiments, the dose of the adjuvant ranges from about 1 μg to about 100 μg. For example, in embodiments, the dose of the adjuvant is about 1 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about 1.8 μg, about 1.9 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 17μg, about 18μg, about 19μg, about 20μg, about 21μg, about 22μg, about 23μg, about 24μg, about 25μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μg, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, about 50μg, about 51μg, about 52μg, about 53μg, about 54μg, about 55μg, about 56μg, about 57μg, about 58μg, about 59μg, about 60μg, about 61μg, about 62μg, about 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In some embodiments, the adjuvant is a saponin adjuvant. In an embodiment, the saponin adjuvant comprises 92% w / w of Fraction A ISCOM base and 8% w / w of Fraction C ISCOM base.

[0111] In embodiments, the immunogenic compositions described herein are administered in a single dose. In embodiments, the immunogenic compositions described herein are administered in multiple doses. For example, in embodiments, one, two, three, four, five, six, seven, eight, nine, or ten doses of the immunogenic composition are administered. These doses are referred to as the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose.

[0112] In embodiments, the dosage is increased after about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about The second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth dose of the immunogenic composition is administered at about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks or about 52 weeks. In embodiments, the first dose is administered after about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 37 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 58 days, about 59 days, about 60 days, about 61 days, about 62 days, about 63 days, about 64 days, about 65 days, about 66 days, about 67 days, about 68 days, about 69 days, about 70 days, about 71 days, about 72 days, about 73 days , about 74 days, about 75 days, about 76 days, about 77 days, about 78 days, about 79 days, about 80 days, about 81 days, about 82 days, about 83 days, about 84 days, about 85 days, about 86 days, about 87 days, about 88 days, about 89 days, about 90 days, about 91 days, about 92 days, about 93 days, about 94 days, about 95 days, about 96 days, about 97 days, about 98 days, about 99 days, about 100 days, about 101 days, about 102 days, about 103 days, about 104 days, about 105 days, about 106 days, about 107 days, about 108 days, about 109 days, about 110 days, about 111 days, about 112 days, about 113 days, about 114 days, about 115 days, about 116 days, about 117 days, about 118 days, about 119 days, about 120 days, about 121 days, about 122 days, about 123 days, about 114 days, about 115 days, about 116 days, about 117 days, about 118 days, about 119 days, about 120 days, about 121 days, about 122 days, about 123 days, about 124 days, about 125 days,About 126 days, about 127 days, about 128 days, about 129 days, about 130 days, about 131 days, about 132 days, about 133 days, about 134 days, about 135 days, about 136 days, about 137 days, about 138 days, about 139 days, about 140 days, about 141 days, about 142 days, about 143 days, about 144 days, about 145 days, about 146 days days, about 147 days, about 148 days, about 149 days, about 150 days, about 151 days, about 152 days, about 153 days, about 154 days, about 155 days, about 156 days, about 157 days, about 158 ​​days, about 159 days, about 160 days, about 161 days, about 162 days, about 163 days, about 164 days, about 165 days, about 166 days, about 167 days 7 days, about 168 days, about 169 days, about 170 days, about 171 days, about 172 days, about 173 days, about 174 days, about 175 days, about 176 days, about 177 days, about 178 days, about 179 days, about 180 days, about 181 days, about 182 days, about 183 days, about 184 days, about 185 days, about 186 days, about 187 days, about 188 days, about 189 days, about 190 days, about 191 days, about 192 days, about 193 days, about 194 days, about 195 days, about 196 days, about 197 days, about 198 days, about 199 days, about 200 days, about 201 days, about 202 days, about 203 days, about 204 days, about 205 days, about 206 days, about 207 days, about 208 days, about 209 days, about 210 days, about 211 days, about 212 days, about 213 days, about 214 days, about 215 days, about 216 days, about 217 days, about 218 days, about 219 days, about 220 days, about 221 days, about 222 days, about 223 days, about 224 days, about 225 days, about 226 days, about 227 days, about 228 days, about 229 days days, about 230 days, about 231 days, about 232 days, about 233 days, about 234 days, about 235 days, about 236 days, about 237 days, about 238 days, about 239 days, about 240 days, about 241 days, about 242 days, about 243 days, about 244 days, about 245 days, about 246 days, about 247 days, about 248 days, about 249 days, about 250 days 0 days, about 251 days, about 252 days, about 253 days, about 254 days, about 255 days, about 256 days, about 257 days, about 258 days, about 259 days, about 260 days, about 261 days, about 262 days, about 263 days, about 264 days, about 265 days, about 266 days, about 267 days, about 268 days, about 269 days, about 270 days, about 271 days, about 272 days, about 273 days, about 274 days, about 275 days, about 276 days, about 277 days, about 278 days, about 279 days, about 280 days, about 281 days, about 282 days, about 283 days, about 284 days, about 285 days, about 286 days, about 287 days, about 288 days, about 289 days, about 290 days, about 291 days,about 292 days, about 293 days, about 294 days, about 295 days, about 296 days, about 297 days, about 298 days, about 299 days, about 300 days, about 301 days, about 302 days, about 303 days, about 304 days, about 305 days, about 306 days, about 307 days, about 308 days, about 309 days, about 310 days, about 311 days, about 312 days, about 313 days, about 314 days, about 315 days, about 316 days, about 317 days, about 318 days, about 319 days, about 320 days, about 321 days, about 322 days, about 323 days, about 324 days, about 325 days, about 326 days, about 327 days, about 328 days, about 329 days, about 330 days, about 331 days, about about 332, about 333, about 334, about 335, about 336, about 337, about 338, about 339, about 340, about 341, about 342, about 343, about 344, about 345, about 346, about 347, about 348, about 349, about 350, about 351, about 352, about 353, about 354, about 355, about 356, about 357, about 358, about 359, about 360, about 361, about 362, about 363, about 364, or about 365, administering a second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth dose of the immunogenic composition.

[0113] In embodiments, three doses of an immunogenic composition described herein are administered, i.e., a first dose, a second dose, and a third dose. In embodiments, the second dose is administered 30 days after the first dose, and the third dose is administered 180 days after the first dose. In embodiments, the immunogenic composition comprises about 50 μg of a fusion protein described herein and about 49 μg of an adjuvant. In embodiments, the adjuvant is a saponin adjuvant.

[0114] The administration of the compositions disclosed herein can be via systemic routes, mucosal routes, transdermal routes or directly into a specific tissue. The term "systemic administration" as used herein includes parenteral routes of administration. In particular, parenteral administration includes subcutaneous injection, intraperitoneal injection, intravenous injection, arterial injection, intramuscular injection or intrasternal injection, intravenous or renal dialysis infusion techniques. Typically, systemic, parenteral administration is intramuscular injection. The term "mucosal administration" as used herein includes oral, intranasal, intravaginal, rectal, intratracheal, intestinal and ocular administration. Preferably, administration is intramuscular.

[0115] In an embodiment, the dose is administered in a volume of about 0.1 mL to about 1.5 mL, e.g., about 0.1 mL, about 0.2 mL, about 0.25 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1.0 mL, about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, or about 1.5 mL.

[0116] In embodiments, the immunogenic composition may comprise a fusion protein at a concentration of about 1 μg / mL to about 50 μg / mL, 10 μg / mL to about 100 μg / mL, about 10 μg / mL to about 50 μg / mL, about 175 μg / mL to about 325 μg / mL, about 200 μg / mL to about 300 μg / mL, about 220 μg / mL to about 280 μg / mL, or about 240 μg / mL to about 260 μg / mL.

[0117] In some embodiments, the present disclosure provides a co-formulation (i.e., pre-filled syringe or pre-mixing) strategy for an immunogenic composition comprising a fusion protein described herein and an adjuvant (e.g., a saponin adjuvant). The typical vaccine administration strategy currently used is a bedside mixed formulation. That is, the vaccine composition and the adjuvant are stored separately and mixed before administration. Due to concerns about the stability of the antigen (e.g., the fusion protein described herein) and its subsequent immunogenicity, pre-mixing, co-formulation, or pre-filled syringe strategies for vaccines are uncommon. The present disclosure provides an immunogenic composition that can be pre-mixed and stored in advance. The disclosed vaccination strategies and formulations can improve the efficiency of vaccination and can reduce the risk of bedside mixing errors while maintaining overall safety and immunogenicity.

[0118] Various containers can be used to store and transport premixed preparations, including syringes and plastic ampoules for single administration. In some cases, plastic ampoules can be manufactured using blow-fill-seal manufacturing technology or methods. Typically, the blow-fill-seal (BFS) manufacturing method includes extruding a plastic material (e.g., a resin) to form a parison, which is then placed into a mold and cut to size. The plastic is then inflated using a filling needle or mandrel, and the plastic is then formed into a hollow ampoule that substantially conforms to the shape of the mold. After expansion, the desired volume of liquid can be injected into the ampoule, and then the filling needle or mandrel can be removed, and the ampoule can be sealed. Therefore, BFS can be an automated process that can be performed in a sterile environment without direct human intervention.

[0119] In some cases, the ability to aseptically manufacture sterile ampoules containing a desired liquid can make BFS-manufactured ampoules particularly suitable for the pharmaceutical industry. However, BFS technology is not compatible with all pharmaceutical liquids, drugs, etc. For example, some known BFS manufacturing methods involve delivering the liquid or product into the ampoule while the plastic is still relatively hot, which may adversely affect temperature-sensitive liquids and / or products such as vaccines, biologics, etc. However, advances in cooling BFS technology have increased the variety of applicable products, liquids, etc., allowing some vaccines, biologics, and / or other temperature-sensitive drugs to be contained in BFS ampoules.

[0120] In some cases, the size, shape, and / or configuration of a BFS ampoule is based at least in part on the desired use and / or the desired medicinal liquid or dose that the ampoule is configured to contain. For example, some known BFS ampoules may include a pierceable top, a twist-off top, a top including an externally or internally threaded connector, and / or the like. The size and / or shape of some known BFS ampoules may be based on the volume of the liquid or dose that is configured to be placed therein. In addition, some known BFS ampoules may be manufactured in the form of a plurality of temporarily connected ampoules, which may improve manufacturing, packaging, and / or storage efficiency, and / or the like.

[0121] In an embodiment, the immunogenic composition described herein can be provided in the form of a prefilled syringe. When the immunogenic composition is prepared in the form of a prefilled syringe, the fusion protein and the adjuvant described herein are combined before administration. In an embodiment, the prefilled syringe contains the fusion protein and the saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, wherein the first iscom particle comprises a fraction A of Quillaja spp., but not a fraction C of Quillaja spp.; the second iscom particle comprises a fraction C of Quillaja spp., but not a fraction A of Quillaja spp.; wherein, by weight, the fraction A of Quillaja spp. and the fraction C of Quillaja spp. account for about 85% and about 15% of the total weight of the fraction A of Quillaja spp. and the fraction C of Quillaja spp. in the adjuvant, respectively. In an embodiment, the prefilled syringe contains a fusion protein and a saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, wherein a first iscom particle comprises Quillaja Fraction A but not Quillaja Fraction C; and a second iscom particle comprises Quillaja Fraction C but not Quillaja Fraction A; wherein, by weight, Quillaja Fraction A and Quillaja Fraction C comprise about 92% and about 8%, respectively, of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant. In an embodiment, a prefilled syringe contains a fusion protein and a saponin adjuvant, wherein the adjuvant comprises at least two iscom particles, wherein a first iscom particle comprises Quillaja Fraction A, but not Quillaja Fraction C; and a second iscom complex particle comprises Quillaja Fraction C, but not Quillaja Fraction A; wherein, by weight, Quillaja Fraction A accounts for at least about 75% of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant, and Quillaja Fraction C accounts for the remainder.

[0122] Example

[0123] Example 1: Phase I open-label study to evaluate the efficacy of an immunogenic composition comprising a fusion protein comprising gp350 and ferritin, with the addition of a saponin adjuvant

[0124] Objective: The efficacy of an immunogenic composition comprising a fusion protein comprising gp350 protein or a fragment thereof and ferritin or a fragment thereof and a saponin adjuvant is being evaluated in adults aged 18-29 years.

[0125] Methods: The immunogenic composition was administered intramuscularly on days 0, 30 and 180. Each dose of the immunogenic composition contained 50 μg of fusion protein and 49 μg of saponin adjuvant. The saponin adjuvant contained 85% w / w of Fraction A ISCOM matrix and 15% w / w of Fraction C ISCOM matrix. The composition was evaluated for its ability to induce an immune response against EBV.

[0126] Inclusion criteria: Patients were included in the study if they met the following criteria: (1) aged 18-29 years; (2) patients agreed not to receive live vaccines within 30 days before and after each dose of the immunogenic composition; (3) patients agreed not to receive inactivated vaccines, subunit vaccines, replication-deficient vaccines, or COVID-19 vaccines within 14 days before and after administration of the immunogenic composition; (4) hemoglobin and white blood cell counts were within institutional limits; (5) total lymphocyte counts were greater than or equal to 800 cells / mm 3 ; (6) Platelet count is 125,000 to 500,000 / mm 3 ; (7) Alanine aminotransferase less than or equal to 1.25 times the upper limit of normal value; (8) Serum IgG greater than 600 mg / dL; Human immunodeficiency virus (HIV) test negative.

[0127] Exclusion criteria: (1) women who are breastfeeding or planning to become pregnant within 60 days after the last dose of the vaccine; (2) participants who have received any of the following: (a) more than 10 days of systemic immunosuppressive drugs (>=10 mg prednisone or equivalent) or cytotoxic drugs within 30 days before the first dose of the vaccine, or immunomodulatory therapy within 180 days before the first dose of the vaccine; (b) blood products, including immunoglobulin products, within 120 days before the first dose of the immunogenic composition; (c) any live attenuated vaccine within 30 days before the first dose of the immunogenic composition; (d) medically indicated subacute respiratory syndrome within 14 days before the first dose of the immunogenic composition. (e) investigational product within 30 days before the first dose, or planned to receive an investigational product during the study; (f) allergy treatment for antigen injections, unless the injection schedule is maintained no more than monthly; (3) participants with any of the following: (a) febrile illness within 14 days of the first dose of vaccine; (b) body mass index (BMI) greater than 36; (c) severe reaction to vaccine; (d) hereditary, acquired, or idiopathic forms of angioedema; (e) idiopathic urticaria in the past year; (f) asthma that was not well controlled or required emergency treatment in the past two years. (g) type 1 or type 2 diabetes, excluding history of gestational diabetes; (h) clinically significant autoimmune disease or immunodeficiency; (i) poorly controlled hypertension; (j) poorly controlled thyroid disease; (k) physician-diagnosed bleeding disorders (e.g., factor deficiency, coagulopathy, or platelet disorder requiring specific prophylaxis); (l) significant bruising or difficulty with IM injection or blood draw; (m) active malignancy, or malignancy that has been treated without reasonable assurance of continued cure, or that may recur during the study; (n) seizure disorders, except for a history of: 1) febrile seizures; 2) seizures secondary to alcohol withdrawal more than 3 years ago; or 3) seizures that did not require treatment within the past 3 years; (o) asplenia, functional asplenia, or any medical condition that resulted in the loss or removal of the spleen; (p) history of Guillain-Barré syndrome; and (q) alcohol or drug abuse or addiction.

[0128] Numbering Implementation Plan

[0129] 1. A fusion protein comprising (i) an Epstein-Barr virus (EBV) envelope protein or a fragment thereof and (ii) ferritin or a fragment thereof.

[0130] 2. The fusion protein according to embodiment 1, wherein the ferritin or fragment thereof is bulldog ferritin, Helicobacter pylori ferritin, Escherichia coli ferritin or a combination thereof.

[0131] 3. The fusion protein according to embodiment 1 or 2, wherein the EBV envelope protein is selected from gp350, gH, gp42 and gL.

[0132] 4. A fusion protein according to any one of embodiments 1-3, wherein the EBV envelope protein comprises gp350 or a fragment thereof, and the gp350 or a fragment thereof comprises one or more domains selected from EBV gp350 domain 1, EBV gp350 domain 2 and EBV gp350 domain 3, an EBV receptor binding domain (RBD), an EBV extracellular domain and a combination thereof.

[0133] 5. A fusion protein according to any one of embodiments 1-4, wherein the EBV envelope protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOS:1-15.

[0134] 6. The fusion protein of any one of embodiments 1-5, wherein the ferritin or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOS: 16-20.

[0135] 7. A nucleic acid encoding the fusion protein of any one of embodiments 1-6.

[0136] 8. A host cell expressing the fusion protein described in any one of embodiments 1-6.

[0137] 9. An immunogenic composition comprising the fusion protein of any one of embodiments 1-6.

[0138] 10. The immunogenic composition of embodiment 9, comprising an adjuvant.

[0139] 11. The immunogenic composition of embodiment 10, wherein the adjuvant comprises at least two iscom particles, wherein:

[0140] The first iscom particle comprises Quillaja fraction A but not Quillaja fraction C; and

[0141] The second iscom granule contained Quillaja fraction C instead of Quillaja fraction A.

[0142] 12. The immunogenic composition of embodiment 11, wherein Quillaja Fraction A and Quillaja Fraction C account for about 85% and about 15% by weight, respectively, of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

[0143] 13. The immunogenic composition of embodiment 11, wherein Quillaja Fraction A and Quillaja Fraction C account for about 92% and about 8% by weight, respectively, of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

[0144] 14. The immunogenic composition of any one of embodiments 9-12, wherein the adjuvant is administered at a dose of about 30 μg to about 60 μg.

[0145] 15. The immunogenic composition of any one of embodiments 9-12, wherein the adjuvant is administered at a dose of about 49 μg.

[0146] 16. The immunogenic composition of any one of embodiments 9-12, wherein the EBV envelope protein is administered at a dose of about 25 μg to about 75 μg.

[0147] 17. The immunogenic composition of any one of embodiments 9-12, wherein the EBV envelope protein is administered at a dose of about 50 μg.

[0148] 18. A prefilled syringe comprising the immunogenic composition of any one of embodiments 9-17.

[0149] 19. A method of stimulating an immune response against Epstein-Barr virus in a subject, comprising administering the immunogenic composition of any one of embodiments 9-17.

[0150] 20. The method of embodiment 19, comprising administering an adjuvant.

[0151] 21. The method according to embodiment 20, wherein the adjuvant comprises at least two iscom particles, wherein:

[0152] The first iscom particle comprises Quillaja fraction A but not Quillaja fraction C; and

[0153] The second iscom granule contained Quillaja fraction C instead of Quillaja fraction A.

[0154] 22. The method of embodiment 21, wherein Quillaja Fraction A and Quillaja Fraction C account for about 85% and about 15% by weight, respectively, of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

[0155] 23. The method of embodiment 21, wherein Quillaja Fraction A and Quillaja Fraction C account for about 92% and about 8% by weight, respectively, of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

[0156] 24. The method of any one of embodiments 20-23, wherein the adjuvant is administered at a dose of about 30 μg to about 60 μg.

[0157] 25. The method of any one of embodiments 20-24, wherein the adjuvant is administered at a dose of about 49 μg.

[0158] 26. The method of any one of embodiments 19-25, wherein the EBV envelope protein is administered at a dose of about 25 μg to about 75 μg.

[0159] 27. The method according to any one of embodiments 19-25, wherein the EBV envelope protein is administered at a dose of about 50 μg.

[0160] 28. The method of any one of embodiments 19-27, wherein multiple doses of the immunogenic composition are administered.

[0161] 29. The method of any one of embodiments 19-27, wherein a single dose of the immunogenic composition is administered.

[0162] 30. The method of embodiment 28, wherein three doses of the immunogenic composition are administered.

[0163] 31. The method of embodiment 30, wherein the first dose is administered on day 0, the second dose is administered on day 30, and the third dose is administered on day 180.

[0164] 32. The method of any one of embodiments 19-31, wherein the immunogenic composition is administered intramuscularly

[0165] Join by reference

[0166] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. However, reference to any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be, construed as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. This application hereby incorporates by reference the entire disclosure of U.S. Patent No. 10,744,199 for all purposes.

Claims

1. A fusion protein comprising (i) Epstein-Barr virus (EBV) envelope protein or a fragment thereof and (ii) ferritin or a fragment thereof.

2. The fusion protein of claim 1, wherein the ferritin or fragment thereof is bulldog ferritin, Helicobacter pylori ferritin, Escherichia coli ferritin, or a combination thereof.

3. The fusion protein of any one of claims 1-2, wherein the ferritin or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 16-20 and 25-38.

4. The fusion protein of any one of claims 1-3, wherein the N-terminus of the ferritin or fragment thereof comprises a peptide having about 6 to about 14 additional amino acids.

5. The fusion protein of claim 4, wherein the peptide comprises the amino acid sequence of any one of ESQVRQNF (SEQ ID NO: 40), ESQVRQQF (SEQ ID NO: 41), SGESQVRQQF (SEQ ID NO: 42), and SGESQVRQNF (SEQ ID NO: 43).

6. The fusion protein according to any one of claims 1 to 4, wherein the EBV envelope protein or fragment comprises one or more proteins selected from any one of gp350, gH, gp42 and gL.

7. A fusion protein according to any one of claims 1-6, wherein the EBV envelope protein or a fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a polypeptide having an amino acid sequence of any one of SEQ ID NOS: 1-15, 21-22, 39 and 44. The fusion protein according to claim 6 , wherein the EBV envelope protein or a fragment thereof is gp350 or a fragment thereof.

9. The fusion protein of claim 8, wherein the gp350 or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a polypeptide having an amino acid sequence of any one of SEQ ID NOS: 1-8, 21-22, 39 and 44.

10. The fusion protein according to any one of claims 1-10, wherein the EBV envelope protein or a fragment thereof comprises one, two, three or four mutations selected from any one of W162N, N164T, D208N and E210T, wherein the amino acids of the EBV envelope protein or a fragment thereof are numbered according to SEQ ID NO:

1.

11. A fusion protein according to any one of claims 1-9, wherein the EBV envelope protein or a fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a polypeptide having the amino acid sequence of SEQ ID NO:

1.

12. The fusion protein according to any one of claims 1 to 10, wherein: (i) the EBV envelope protein or a fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2; and (ii) the ferritin or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

38.

13. The fusion protein according to any one of claims 1 to 10, wherein: (i) the EBV envelope protein or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 39; and (ii) the ferritin or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

38.

14. The fusion protein according to any one of claims 1 to 10, wherein: (i) the EBV envelope protein or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:44; and (ii) the ferritin or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

38.

15. The fusion protein according to any one of claims 1 to 10, wherein: (i) the EBV envelope protein or a fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 1; and (ii) the ferritin or fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:

38.

16. The fusion protein according to any one of claims 1-10, wherein the EBV envelope protein or a fragment thereof is located at the N-terminus of the ferritin or a fragment thereof.

17. The fusion protein according to any one of claims 1-10, wherein the EBV envelope protein or a fragment thereof is located at the C-terminus of the ferritin or a fragment thereof.

18. The fusion protein according to any one of claims 1 to 10, comprising a signal peptide. The fusion protein according to claim 18 , wherein the signal peptide is a bovine prolactin signal peptide or a human CD5 signal peptide.

20. A nucleic acid encoding the fusion protein of any one of claims 1-19.

21. A vector comprising the nucleic acid of claim 20.

22. A host cell expressing the fusion protein of any one of claims 1-19.

23. A nanoparticle comprising the fusion protein of any one of claims 1-19.

24. A nanoparticle comprising an EBV envelope protein or a fragment thereof and a detergent.

25. The nanoparticle of claim 24, wherein the detergent is a non-ionic detergent.

26. The nanoparticle of claim 25, wherein the non-ionic detergent is polysorbate 20 (PS20), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65) or polysorbate 80 (PS80).

27. The nanoparticle of any one of claims 24-26, wherein the EBV envelope protein or fragment thereof comprises a transmembrane domain.

28. The nanoparticle of claim 27, wherein the transmembrane domain has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

23.

29. A nanoparticle according to claim 27 or 28, wherein the EBV envelope protein or a fragment thereof comprises any amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOS: 1 or 44.

30. An immunogenic composition comprising the fusion protein of any one of claims 1-19 and a pharmaceutically acceptable buffer.

31. An immunogenic composition comprising the nanoparticle of any one of claims 23-29 and a pharmaceutically acceptable buffer.

32. The immunogenic composition of claim 30 or 31, comprising an adjuvant.

33. The immunogenic composition of claim 32, wherein the adjuvant comprises at least two iscom particles, wherein: The first iscom granule comprises fraction A of Quillaja Saponaria Molina instead of fraction C of Quillaja Saponaria Molina; and The second iscom granule contained Quillaja fraction C instead of Quillaja fraction A.

34. The immunogenic composition of claim 33, wherein Quillaja Fraction A and Quillaja Fraction C comprise about 85% and about 15% by weight, respectively, of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

35. The immunogenic composition of claim 33, wherein Quillaja Fraction A and Quillaja Fraction C comprise about 92% and about 8% by weight, respectively, of the total weight of Quillaja Fraction A and Quillaja Fraction C in the adjuvant.

36. The immunogenic composition of any one of claims 32-35, comprising about 30 μg to about 60 μg of adjuvant.

37. The immunogenic composition of any one of claims 32-35, comprising about 49 μg of adjuvant.

38. The immunogenic composition of any one of claims 32-37, comprising about 25 μg to about 75 μg of the fusion protein.

39. The immunogenic composition of any one of claims 32-37, comprising about 50 μg of the fusion protein.

40. A method of stimulating an immune response against Epstein-Barr virus in a subject, the method comprising administering the immunogenic composition of any one of claims 30-39.

41. The method of claim 40, comprising administering multiple doses of the immunogenic composition.

42. The method of claim 41, comprising administering a first dose, a second dose, and a third dose of the immunogenic composition.

43. The method of claim 42, comprising administering the second dose of the immunogenic composition 30 days after the first dose of the immunogenic composition.

44. The method of claim 42 or 43, comprising administering the third dose of the immunogenic composition 180 days after the first dose of the immunogenic composition.

45. The method of claim 42 or 43, comprising administering the third dose of the immunogenic composition 180 days after the second dose of the immunogenic composition.

46. ​​The method of any one of claims 40-45, wherein the immunogenic composition is administered intramuscularly.

47. A prefilled syringe comprising the immunogenic composition of any one of claims 30-39.

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