[go: up one dir, main page]

WO2024163327A1 - Immunogènes de la glycoprotéine 42 du virus epstein-barr pour la vaccination et la découverte d'anticorps - Google Patents

Immunogènes de la glycoprotéine 42 du virus epstein-barr pour la vaccination et la découverte d'anticorps Download PDF

Info

Publication number
WO2024163327A1
WO2024163327A1 PCT/US2024/013311 US2024013311W WO2024163327A1 WO 2024163327 A1 WO2024163327 A1 WO 2024163327A1 US 2024013311 W US2024013311 W US 2024013311W WO 2024163327 A1 WO2024163327 A1 WO 2024163327A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
protein
modified
ebv
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/013311
Other languages
English (en)
Inventor
Jeffrey I. Cohen
Wei BU
Kennichi C. DOWDELL
Joseph Marcotrigiano
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
Original Assignee
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Publication of WO2024163327A1 publication Critical patent/WO2024163327A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This disclosure concerns modified Epstein-Barr virus (EBV) glycoprotein 42 (gp42) polypeptides that retain their immunogenicity, but are significantly impaired for binding to human leukocyte antigen (HLA) class If molecules.
  • EBV Epstein-Barr virus
  • HLA human leukocyte antigen
  • This disclosure further concerns use of the modified gp42 polypeptides in immunogenic compositions and as probes to identify B cells expressing gp42-specific monoclonal antibodies.
  • Epstein-Barr virus is one of the most prevalent human viruses with approximately 95% of adults infected worldwide. As a lymphotropic virus, EBV infects primarily B cells, but also infects epithelial cells. Primary infection in adolescents or young adults causes infectious mononucleosis and subsequently the virus establishes a life-long latent infection in B cells. EBV is an oncogenic virus associated with a variety of B cell malignancies, including Burkitt lymphoma, Hodgkin lymphoma, nonHodgkin lymphoma, and post-transplant lymphoproliferative disease, as well as epithelial cell malignancies, such as gastric and nasopharyngeal carcinoma. EBV causes about 200,000 cases of cancers annually with approximately 140,000 deaths each year worldwide (Cohen et al., Sci. Transl. Med. 3(107):107fs7, 2011). Additionally, EBV is a risk factor for multiple sclerosis.
  • EBV glycoproteins gp350, gp42, gH, gL and gB are involved in B cell infection. B cell entry begins with attachment of gp350 to complement receptor 2 (CR2).
  • gp42 then binds to human leukocyte antigen (HLA) class II and activates the core fusion machinery composed of gH, gL, and gB, resulting in membrane fusion with release of viral contents into the cell.
  • HLA human leukocyte antigen
  • gH, gL and gB are conserved in all herpesviruses, while gp350 and gp42 are only present in lymphocryptoviruses like EBV.
  • EBV gp350 and gp42 are not required for epithelial cell infection; instead, the EBV protein BMRF2 is important for virus attachment to epithelial cells, but gH, gL and gB are sufficient for entry into epithelial cells.
  • EBV gp42 is a type II membrane protein and two forms of gp42 (i.e., a transmembrane anchored form and a soluble form) are detected in infected cells (Ressing et al., J. Virol. 79: 841-852, 2005). gp42 is required for virus entry into B cells, but not for epithelial cell infection. In contrast, the presence of excessive gp42 inhibits virus entry into epithelial cells (Wang and Hutt-Fletcher, J. Virol. 72(1): 158-163, 1998; Wang et al., J Virol 72:5552-5558, 1998).
  • EBV gL binds to gH to form a heterodimer, while soluble gp42 binds gH/gL to form a heterotrimer (Kirschner et al., J Virol 80:9444-9454, 2006).
  • EBV with high levels of gp42 produced in epithelial cells infects B cells more efficiently than epithelial cells, while EBV produced in B cells has relatively lower levels of gp42 and infects epithelial cells more efficiently than B cells (Borza and Hutt-Fletcher, Nat Med 8:594-599, 2002).
  • gp42 regulates tropism of EBV for entry into different cell types.
  • gp42 may help the virus to evade the immune system.
  • gp42 binds to HLA class II molecules and inhibits HLA class Il-restricted antigen presentation to T cells (Ressing et al., J. Virol. 79:841-852, 2005; Spriggs et al., J. Virol. 70:5557-5563, 1996).
  • the crystal structure of gp42 in complex with HLA class II DR1 shows that the globular C-type lectin domain (CTLD) of gp42 interacts with the
  • CTL globular C-type lectin domain
  • the receptor binding site on gp42 is composed of three distinct linear regions clustered on one side of the gp42 CTLD.
  • Binding of HLA class II broadens the hydrophobic pocket on the gp42 CTLD to enable gH/gL contact with gp42, which results in a conformational change in gH/gL and triggers virus-cell membrane fusion (Kirschner et al., Structure 17(2):223-233, 2009; Sathiyamoorthy et al., PLoS Pathog 10(8):el004309, 2014).
  • the hydrophobic pocket has been proposed to have a role in activation of virus-cell membrane fusion.
  • EBV gH and gL monoclonal antibodies have defined epitopes on gH/gL important for viral neutralization and fusion inhibition (Sathiyamoorthy et al., Proc Natl Acad Sci USA 114(41):E8703-E8710, 2017; Snijder et al., Immunity 48(4):799-811, 2018; Zhu et al., Nat Commun 12(1):6624, 2021; Chen et al., Immunity 55(11 ) :2135-2148, 2022). Since gp42 is essential for B cell entry, a need remains for the identification of sites of vulnerability on gp42 to elucidate the mechanism of virus entry into B cells and for rational design of EBV vaccines. SUMMARY
  • the present disclosure describes the identification of gp42 residues that make contact with HLA class II molecules, as well as gp42 residues that interface with known gp42-specific neutralizing antibodies. Also described are amino acid residues of gp42 that are positioned within 4 Angstroms of gp42 residues identified as being important for binding HLA class II molecules. Based on this information, modified gp42 polypeptides that retain their immunogenicity, but are severely impaired for binding to HLA class II proteins were designed. Use of the modified gp42 polypeptides in immunogenic compositions, such as in protein nanoparticle vaccine compositions, is described. The immunogenic compositions may also include EBV gp35O and/or EBV gH/gL immunogens. Also described is use of the modified gp42 polypeptides as probes to enable screening of B cells for expression of gp42 -specific monoclonal antibodies, such as for the purpose of isolating monoclonal antibodies directed against gp42.
  • modified Epstein-Barr virus (EBV) glycoprotein 42 (gp42) polypeptides that include amino acid substitutions at residues 154, 157, 160 and 220 of a wild-type gp42 amino acid sequence set forth as SEQ ID NO: 2.
  • the modified gp42 polypeptides exhibit a decrease in binding to a human leukocyte antigen (HLA) class II protein compared to the wild-type gp42 of SEQ ID NO: 2.
  • the modified gp42 polypeptide includes R154E, N157A, E160R and R220A substitutions, or RL54E, NL57A, E160R and R220E substitutions.
  • modified EBV gp42 polypeptides that have at least one amino acid substitution at any one of residues 102, 107, 109-111, 115, 118, 150-160, 162, 197 and 220 (z.e., one or more of residues 102, 107, 109, 110, 111, 115, 118, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 197 and 220) of a wild-type gp42 amino acid sequence (such as SEQ ID NO: 2), wherein the modified gp42 polypeptide exhibits a decrease in binding to HLA class II proteins compared to the wild-type gp42.
  • a wild-type gp42 amino acid sequence such as SEQ ID NO: 220
  • the at least one amino acid substitution is at any one of residues 150-160 and 220. In other aspects, the at least one amino acid substitution is at any one of residues 102, 107, 109-111, 115, 118, 162 and 197. In some examples, the modified gp42 polypeptide has at least one amino acid substitution at any one of residues 150-160 and 220 and at least one amino acid substitutions at any one of residues 102, 107, 109-111, 115, 118, 162 and 197.
  • fusion proteins that include a modified gp42 polypeptide disclosed herein and a heterologous protein.
  • the heterologous protein includes a self-assembling protein nanoparticle subunit (such as a ferritin subunit), or protein tag, fluorescent protein, or carrier protein.
  • the fusion protein further includes EBV gH and gL proteins.
  • self-assembling protein nanoparticles that include a modified gp42 polypeptide or fusion protein disclosed herein.
  • the self-assembling protein nanoparticles also include EBV gH and gL proteins.
  • nucleic acid molecules that encode a modified gp42 polypeptide, fusion protein or self-assembling nanoparticle disclosed herein.
  • the nucleic acid molecule is codon- optimized for expression in mammalian cells.
  • the nucleic acid molecule is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • immunogenic compositions that include a pharmaceutically acceptable carrier and a modified gp42 polypeptide, fusion protein, self-assembling protein nanoparticle, nucleic acid molecule, or vector disclosed herein.
  • the immunogenic composition further includes an adjuvant, an EBV gp350 immunogen, and/or EBV gH/gL immunogens.
  • the method includes administering to the subject an effective amount of a modified gp42 polypeptide, fusion protein, self-assembling protein nanoparticle, nucleic acid molecule, vector or immunogenic composition disclosed herein.
  • the method includes administering to the subject a prophylactically effective amount of a modified gp42 polypeptide, fusion protein, self-assembling protein nanoparticle, nucleic acid molecule, vector or immunogenic composition disclosed herein.
  • the method includes administering to the subject a therapeutically effective amount of a modified gp42 polypeptide, fusion protein, self-assembling protein nanoparticle, nucleic acid molecule, vector or immunogenic composition disclosed herein.
  • the method includes contacting a sample containing B cells (such as a PBMC sample) with a modified gp42 polypeptide disclosed herein and isolating B cells that bind to the modified gp42 polypeptide.
  • the sample containing B cells is obtained from a subject previously infected with EBV.
  • FIGS. 1A-1B EBV gp42 and mAbs specific for gp42.
  • FIG. 1A Schematic of EBV entry into B cells.
  • EBV gp42 is part of a complex with gH and gL on the EBV virion.
  • EBV gp42 binds to HLA class II, its receptor.
  • EBV gp42 is essential for EBV to infect B cells.
  • FIG. IB Humanized mice reconstituted with human lymphocytes were administered gp42-specific monoclonal antibody (mAb) A10 or mAb F-2-1 and subsequently infected with EBV. Animals pretreated with mAb A10 did not become viremic after EBV infection, while most of the animals pretreated with mAb F-2-1 did become viremic. These results show that mAb A10 protected the animals from EBV infection.
  • mAb monoclonal antibody
  • FIGS. 2A-2E Structural analysis of the HLA class II binding site on gp42.
  • FIG. 2A Binding of gp42 to HLA class II DR (PDB: 1KG0), mAb F-2-1, and mAb A10. Structures were determined by X-ray crystallography of purified gH/gL/gp42 trimer and Fab fragments of the mAbs. EBV gp42 is shown in complex with EBV gH/gL.
  • FIG. 2B Footprint of HLA class II DR binding site (PDB: 1KG0) on gp42.
  • FIGS. 2B-2D Footprint of mAb F-2- 1 heavy chain (HC) and light chain (LC) binding sites on gp42.
  • FIGS. 2B-2D Footprint of mAb A10 HC and LC binding sites on gp42.
  • FIGS. 2B-2D the binding footprint of HLA class II on gp42 is traced with a solid black line.
  • FIG. 2E EBV gp42 residues involved in the binding interface between gp42 and HLA class II DR, mAb F-2-1 or mAb A10. Residues contacting HLA class II are distributed in three segments and highlighted in the shaded boxes.
  • Residues forming hydrogen bonds or salt bridges are numbered as follows: “3” for HLA class II, mAb F-2-1 and mAb A10; “4” for HLA class II and mAb F-2-1; “6” for mAb F-2-1 and mAb A10; “1”, “2”, and “7” for unique residues contacted by mAb F-2-1, mAb A10, and HLA class II DR, respectively.
  • Other residues involved in the interface between gp42 and HLA class II or Fabs are labelled with “5”. Residues involved in salt bridge formation are underlined.
  • FIGS. 3A-3B Modified forms of gp42 bind to gp42-specific mAb A10, but are impaired for binding to HLA class II.
  • FIG. 3A Amino acids of EBV gp42 (far left) that have close contacts on HLA class II DR1 (left column), and mAb A10 (middle column) or mAb F-2-1 (right column) are shaded.
  • L light chain of the mAb;
  • H heavy chain of mAb.
  • the solid arrows indicate gp42 amino acids that when mutated retain binding of gp42 to the mAbs, but not to HLA class II DR1.
  • gp42 amino acid substitution mutants and the percentage of binding to HLA class II DR1, mAb A 10, mAb F-2-1, or mAb 4C12 was determined using biolayer interferometry (Octet). Boxes labelled with a single asterisk (*) are gp42 mutants that bind to HLA-class II DR1 ⁇ 10%, while boxes labelled with a double asterisk (**) bind to the indicated mAbs ⁇ 50%.
  • EARA and EARE (modified at amino acid positions 154, 157, 160, and 220) retain >65% binding to mAblO, but lose most or all of their binding to HLA class II DR1.
  • FIGS. 4A-4B Binding of wild-type (WT) gp42 or gp42 mutant polypeptides to HLA-class II on human peripheral blood mononuclear cells (PBMCs).
  • FIG. 4 A PBMCs from four donors were used in two separate experiments. Biotinylated gp42 polypeptides (EARA or EARE) were incubated with the human PBMCs and then streptavidin (SA) was added. SA without gp42 polypeptide was included as a negative control. WT gp42 polypeptide bound strongly to human PBMCs, while gp42 mutant EARA and EARE polypeptides did not show any binding to PBMCs.
  • FIG. 4B Mean fluorescent intensity (MFI) of gp42 polypeptide binding to B cells within the PBMCs was quantified. Only the WT gp42 polypeptide bound above the level of the control.
  • FIGS. 5A-5B Binding of WT gH/gL/gp42 trimer or mutant gH/gL/gp42 trimers to HLA class II on PBMCs.
  • FIG. 5A PBMCs from four donors were used in two separate experiments. Biotinylated WT gH/gL/gp42 trimer or mutant (EARA and EARE) gH/gL/gp42 trimers were incubated with the human donor PBMCs and then streptavidin (SA) was added. SA without WT or mutant gH/gL/gp42 trimer was included as a negative control.
  • SA streptavidin
  • FIGS. 6A-6B Mice immunized with WT gH/gL/gp42 trimer or mutant gH/gL/gp42 trimers produced similar levels of neutralizing antibodies to EBV.
  • Balb/c mice were administered 2 doses (at week 0 and 3) of WT or mutant gH/gL/gp42-ferritin nanoparticles (0.5 jxg) and blood was obtained 2 weeks after the second dose.
  • Neutralizing titers were measured using Raji B cells (FIG. 6A) or 293 epithelial cells (FIG. 6B). These results show that the gp42 mutants that do not bind HLA class II DR are equally immunogenic as the WT gp42.
  • FIGS. 7A-7B Structural model showing amino acid residues within 4 Angstroms of Vall51 (FIG.
  • SEQ ID NO: 1 is a codon-optimized nucleic acid sequence encoding WT gp42.
  • SEQ ID NO: 13 is a codon-optimized nucleic acid sequence encoding a modified gp42 with the
  • Epstein-Barr virus causes infectious mononucleosis and is associated with epithelial cell malignancies as well as B cell lymphomas. During primary infection, EBV infects B cells and establishes lifelong latency. EBV glycoprotein 42 (gp42), which binds to gH/gL to form a gH/gL/gp42 heterotrimer, is indispensable for virus entry into B cells. gp42 interacts with its B cell receptor, HLA class II, and activates membrane fusion with B cells (FIG. 1A).
  • gp42 amino acid residues that make contact with HLA class H molecules, as well as gp42 residues that interface with known gp42-specific neutralizing antibodies. Further described are amino acid residues that are positioned within four Angstroms of an identified HLA class II contact residue; substitutions in any of these residues alter the positioning of the HLA class II contact residues. Based on this structural information, modified gp42 polypeptides that retain their immunogenicity, but are severely impaired for binding to HLA class II proteins were designed. The findings disclosed herein facilitate the design of next-generation EBV vaccines and have therapeutic applications to reduce the burden of EBV-associated disease.
  • the modified gp42 polypeptides can be used in immunogenic compositions (such as self-assembling protein nanoparticle compositions) for immunization of subjects against EBV infection.
  • the modified gp42 polypeptides can also be used as probes to screen for B cells expressing gp42-specific monoclonal antibodies, thereby enabling the isolation of gp42-specific monoclonal antibodies.
  • a gp42 probe it was not possible to use a gp42 probe to select for gp42-specific antibodies in human blood samples because native gp42 binds HLA class II present on all B cells.
  • an antigen includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.”
  • the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:
  • Adjuvant A substance or vehicle used to enhance antigenicity, for example antigenicity of the modified gp42 polypeptides disclosed herein.
  • Adjuvants can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages).
  • a suspension of minerals alum, aluminum hydroxide, or phosphate
  • water-in-oil emulsion for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/
  • Immunostimulatory oligonucleotides can also be used as adjuvants (for example, see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199).
  • Adjuvants include biological molecules, such as costimulatory molecules.
  • Exemplary adjuvants include IL-2, RANTES, GM- CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, immune stimulating complex (ISCOM) matrix, and toll-like receptor (TLR) agonists, such as TLR-9 agonists, Poly I:C, or PolylCLC.
  • Additional adjuvants include monophosphoryl lipid A (MPL) and Alhydroxiquim-II.
  • MPL monophosphoryl lipid A
  • Alhydroxiquim-II A variety of known adjuvants can be used (see, e.g., Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).
  • Adjuvants can be used in combination with the disclosed gp42 polypeptides, protein nanoparticles, nucleic acid molecules, vectors and compositions, for example to enhance their antigenicity.
  • the adjuvant is or includes AS03, which is composed of a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion (see, e.g., Garmon et al., Expert Rev Vaccines 11(3):349-366, 2012).
  • the adjuvant is an Army Liposome Formulation (ALF) adjuvant, such as ALFQ (ALF containing the QS21 saponin), ALFQA (ALFQ adsorbed to aluminum hydroxide) or ALFA (ALF adsorbed to aluminum hydroxide) (see Alving et al., Expert Rev Vaccines 19(3):279-292, 2020).
  • ALFQ ALF containing the QS21 saponin
  • ALFQA ALFQ adsorbed to aluminum hydroxide
  • ALFA ALF adsorbed to aluminum hydroxide
  • Administration The introduction of a composition, such as gp42 polypeptide, nucleic acid molecule, or protein nanoparticle provided herein, into a subject by a chosen route.
  • Administration can be local or systemic.
  • the chosen route is intravenous
  • the composition is administered by introducing the composition into a vein of the subject.
  • routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intraosseous, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.
  • Amino acid substitution The replacement of an amino acid in a polypeptide with one or more different amino acids.
  • a native amino acid in the EBV gp42 protein (such as SEQ ID NO: 2) is replaced with an arginine, an alanine, a glutamic acid or a serine.
  • Antibody An immunoglobulin, antigen-binding fragment, or derivative thereof, which specifically binds and recognizes an analyte (antigen), such as the disclosed gp42 polypeptides and protein nanoparticles.
  • analyte such as the disclosed gp42 polypeptides and protein nanoparticles.
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispeciflc antibodies), and antibody fragments, so long as they exhibit the desired antigenbinding activity.
  • Binding affinity Affinity of one protein for another protein (such affinity of gp42 to its receptor, HLA class II). Affinity can be calculated, for example, by a modification of the Scatchard method described by Frankel etal. (Mol. Immunol., 16:101-106, 1979). In some aspects, binding affinity is measured by competition radioimmunoassay, ELISA, flow cytometry, bio-layer interferometry (BLI) technology (such as the Octet system, Creative Biolabs), or surface plasmon resonance (SPR; such as by using a BIACORES- 2000 or a BIACORES-3000; BIAcore, Inc., Piscataway, N.J.).
  • BLI bio-layer interferometry
  • SPR surface plasmon resonance
  • Carrier protein An immunogenic protein to which an antigen can be linked. When linked to a carrier, the antigen may become more immunogenic. Carriers are chosen to increase the immunogenicity of the antigen and/or to elicit antibodies against the carrier which are diagnostically, analytically, and/or therapeutically beneficial.
  • Useful carrier proteins include polymeric carriers, which can be natural (for example, proteins from bacteria or viruses), semi- synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached.
  • the carrier protein is keyhole limpet hemocyanin (KLH), tetanus toxoid (TT), diphtheria toxoid (DT), modified cross-reacting material of diphtheria toxin (CRM197), meningococcal outer membrane protein complex (OMPC), Hemophilus influenzae protein D (HiD), or recombinant tetanus toxin heavy chain C fragment (rTTHC) (see, e.g., Ou et al., Scientific Reports 10:3032, 2020). Additional description of exemplary protein carriers for vaccines can be found in, e.g., WO 2020/061564 and Pichichero (Hum Vaccin Immunother 9: 2505- 2523, 2013).
  • Codon-optimized nucleic acid A nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species or group of species).
  • a nucleic acid sequence can be optimized for expression in mammalian cells or in a particular mammalian species (such as human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.
  • Contacting Placement in direct physical association; includes both in solid and liquid form. “Contacting” is often used interchangeably with “exposed.” For example, contacting can occur in vitro with a modified gp42 polypeptide and a biological sample (such as a PBMC sample) in solution.
  • a modified gp42 polypeptide that exhibits a decrease in binding to an HLA class H protein is a modified gp42 polypeptide that binds to the HLA class II protein with an affinity that is lower than the affinity of wild-type gp42 (such as wild-type gp42 of SEQ ID NO: 2) for the HLA class II protein.
  • the affinity is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.
  • the affinity of the modified gp42 polypeptide for HLA class II is reduced to the extent that binding is below the limit of detection.
  • Affinity can be measured using known techniques, such as by ELISA, How cytometry, surface plasmon resonance (SPR) or bio-layer interferometry (BLI).
  • Degenerate variant A nucleic acid encoding a protein (for example, a modified gp42 polypeptide) that includes a sequence that is degenerate as a result of the genetic code. There are twenty natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the protein encoded by the nucleotide sequence is unchanged.
  • Detectable label A detectable molecule (also known as a detectable marker) that is conjugated directly or indirectly to a second molecule, such as an antibody or polypeptide, to facilitate detection of the second molecule.
  • the detectable label can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination).
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (such as 35 S or 13 ’I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents (such as gadolinium chelates), nucleic acids (such as DNA barcodes), and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI).
  • radioisotopes or radionuclides such as 35 S or 13 ’I
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • Methods for using detectable labels and guidance in the choice of detectable labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).
  • the detectable label is an affinity tag (e.g., glutathione S-transferase (GST), His tag), an epitope tag (hemagglutinin (HA), V5, FLAG, Myc), or a fluorophore (e.g., FITC, rhodamine, Cy dyes).
  • GST glutathione S-transferase
  • HA epitope tag
  • V5 FLAG
  • Myc hemagglutinin
  • a fluorophore e.g., FITC, rhodamine, Cy dyes.
  • Effective amount/ therapeutically effective amount A quantity of a specific substance (such as an immunogen, e.g., a gp42 polypeptide or nanoparticle) sufficient to achieve a desired effect, such as an immune response, in a subject being treated or immunized.
  • this can be the amount necessary to inhibit or suppress virus infection, replication and/or spread of EBV in a subject, such as by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, relative to the absence of treatment or immunization.
  • the effective amount can also be the amount necessary to reduce and/or ameliorate one or more symptoms of EBV infection.
  • the effective amount is the amount necessary to decrease the amount of EBV present in an infected subject, or it is the amount necessary to prevent or inhibit EBV infection in a subject that is not currently infected.
  • an effective amount of a disclosed immunogen can be the amount of the immunogen sufficient to elicit a priming immune response in a subject that can be subsequently boosted with the same or a different immunogen to generate a protective immune response.
  • an effective amount is the amount necessary to treat a subject with an EBV infection, such as to eliminate or reduce the titer of EBV, such as a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, as compared to a EBV titer without the treatment.
  • a prophylactically effective amount is the amount necessary to decrease the risk of contracting an EBV infection in a healthy subject by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, such as compared to a non- vaccinated subject.
  • Epitope An antigenic determinant. Epitopes are particular chemical groups or peptide sequences on a molecule that are antigenic (that elicit a specific immune response). An antibody specifically binds a particular antigenic epitope on a polypeptide, such as EBV gp42.
  • Epstein-Barr virus A member of the herpes virus family.
  • EBV is also known as human herpesvirus 4 (HHV4).
  • the genome of EBV is comprised of double-stranded DNA of approximately 172 kb, which is surrounded by a nucleocapsid, a protein tegument and a lipid envelope.
  • type 1 and type 2 which differ in their transforming and reactivation ability.
  • EBV primarily infects B lymphocytes and epithelial cells.
  • EBV infectious mononucleosis
  • Infection with EBV can cause infectious mononucleosis, which is characterized by, for example, fatigue, fever, swollen lymph nodes, inflamed throat, enlarged spleen, swollen liver and rash.
  • EBV is also associated with several non-malignant and malignant diseases (such as B cell lymphomas, e.g., Burkitt lymphoma, Hodgkin lymphoma and non- Hodgkin lymphoma) and multiple sclerosis.
  • B cell lymphomas e.g., Burkitt lymphoma, Hodgkin lymphoma and non- Hodgkin lymphoma
  • multiple sclerosis EBV is most often spread via bodily fluids, including saliva, blood and semen.
  • Ferritin nanoparticle A multi-subunit, globular shaped protein complex. In nature, ferritin proteins self-assemble into a globular structure that stores iron and releases it in a controlled fashion. Production and expression of ferritin nanoparticles based on monomeric ferritin subunits that are linked to particular antigens (such as influenza HA ectodomains) have been previously described (see, e.g., Kanekiyo et al., Nature 499, 102-106, 2013 and Zhang, Y. Int. J. Mol. Sci., 12:5406-5421, 2011; WO 2020/061564; WO 2018/005558; and WO 2013/044203).
  • An exemplary ferritin subunit amino acid sequence that can be used with the modified gp42 peptides provided herein is set forth herein as SEQ ID NO: 18.
  • Fluorescent protein A protein that emits light of a certain wavelength when exposed to a particular wavelength of light.
  • Fluorescent proteins include, but are not limited to, green fluorescent proteins (such as GFP, EGFP, AcGFPl, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP and ZsGreen), blue fluorescent proteins (such as EBFP, EBFP2, Sapphire, T-Sapphire, Azurite and mTagBFP), cyan fluorescent proteins (such as ECFP, mECFP, Cerulean, CyPet, AmCyanl, Midori-Ishi Cyan, mTurquoise and mTFPl), yellow fluorescent proteins (EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellowl and mBanana), orange fluorescent proteins (Kusabira Orange, Kusabira Orange2, mOrange, mOrange2 and m
  • Fusion protein A protein containing amino acid sequence from at least two different (heterologous) proteins or peptides. Fusion proteins can be generated, for example, by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, the nucleic acid sequences are in the same reading frame and contain no internal stop codons. Fusion proteins, particularly short fusion proteins, can also be generated by chemical synthesis.
  • Glycoprotein 42 An EBV protein that is required for infection of B cells and for mediating B cell fusion.
  • EBV gp42 is a type II membrane protein; two forms of gp42 are detectable in EBV-infected cells: a transmembrane anchored form and a soluble form. Soluble gp42 forms a trimer with the gH and gL proteins.
  • Amino acid sequences for EBV gp42 are publicly available (e.g., see GENBANKTM Accession No. P03205.1).
  • An exemplary wild-type gp42 protein is set forth herein as SEQ ID NO: 2.
  • Glycoprotein 350 The major membrane glycoprotein of EBV. This protein is one of the primary targets of neutralizing antibodies against EBV.
  • the gp350 protein initiates attachment of the virus to host cells by binding to complement receptor type 2 (CR2) on B cells.
  • Amino acid sequences for EBV gp350 are publicly available (e.g., see GENBANKTM Accession No. YP_401667.1).
  • An exemplary wildtype gp350 protein is set forth herein as SEQ ID NO: 21.
  • Glycoprotein H An EBV protein that plays a role in membrane fusion and virus entry.
  • gH is a membrane-bound glycoprotein that forms a heterodimer with gL.
  • gH/gL heterodimers can also associate with gp42 to form a heterotrimer.
  • Amino acid sequences of EBV gH are publicly available (e.g., see GENBANKTM Accession No. P03231.1).
  • An exemplary gH amino acid sequence is set forth herein as SEQ ID NO: 19.
  • Glycoprotein L An EBV protein that plays a role in membrane fusion and virus entry. gL is a soluble glycoprotein that forms a heterodimer with gH.
  • gH/gL heterodimers can also associate with gp42 to form a heterotrimer.
  • Amino acid sequences of EBV gL are publicly available (e.g., see GENBANKTM Accession No. P03212.1).
  • An exemplary gL amino acid sequence is set forth herein as SEQ ID NO: 20.
  • Heterologous Originating from a separate genetic source or species.
  • Host cells Cells in which a vector can be propagated and its nucleic acid expressed.
  • the cell may be prokaryotic or eukaryotic, such as a mammalian cell, yeast cell, insect cell, or bacterial cell.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.
  • HLA Class II molecules Proteins encoded by the major histocompatibility complex (MHC) gene complex. HLAs from MHC Class II include HLA-DM, HLA- DOA, HLA-DOB, HLA-DP, HLA-DQ, and HLA-DR genes. HLA class II molecules are heterodimeric proteins comprised of an a chain, which includes an al region and an a2 region, and a
  • MHC major histocompatibility complex
  • Immune response A response of a cell of the immune system, such as a B cell, T cell, NK cell, or monocyte, to a stimulus.
  • the response is specific for a particular antigen (an “antigenspecific response”), such as EBV gp42.
  • an immune response is a T cell response, such as a CD4 + response or a CD8 + response.
  • the response is a B cell response, and results in the production of specific antibodies, such as antibodies specific for EBV gp42.
  • Immunize To render a subject (such as a mammal) partially or fully protected from an infectious disease (for example, EBV), such as by vaccination.
  • infectious disease for example, EBV
  • the term “immunize” does not require complete (100%) protection against an infectious disease.
  • immunization of a subject decreases the subject’s risk of infection or disease by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%, compared to in the absence of immunization.
  • Immunogen A compound, composition, or substance (for example, a modified gp42 polypeptide or protein nanoparticle as disclosed herein) that can elicit an immune response in an animal, including compositions that are injected or absorbed into an animal.
  • Administration of an immunogen to a subject can lead to protective immunity against a pathogen of interest (such as EBV).
  • Immunogenic composition A composition comprising an immunogen that elicits an immune response, such as a measurable T cell or B cell response (such as production of antibodies) against an antigen included on the immunogen or encoded by a nucleic acid molecule included in the immunogen.
  • an immunogenic composition is a composition that includes a disclosed modified gp42 polypeptide, or a self-assembling protein nanoparticle comprising a modified gp42 polypeptide, that induces a measurable immune response against EBV gp42, such as a measurable B cell response (such as production of antibodies, e.g., neutralizing antibodies) against gp42, when administered to a subject.
  • the immunogenic composition typically will include the modified gp42 polypeptide or protein nanoparticles in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant (for example, AS03), gH/gL immunogen and/or gp350 immunogen.
  • an adjuvant for example, AS03
  • gH/gL immunogen for example, gH/gL immunogen
  • gp350 immunogen for example, gp350 immunogen.
  • Isolated An “isolated” biological component, such as a nucleic acid, protein (including antibodies), organelle or cell, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component occurs, for example other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.
  • B cells are isolated from other cell types in a PBMC sample.
  • Lymphoproliferative disease or disorder A disease or disorder characterized by the uncontrolled or abnormal proliferation of lymphocytes. Lymphoproliferative diseases typically occur in immunocompromised or immunosuppressed subjects. Examples of lymphoproliferative diseases/disorders include, but are not limited to, lymphomas (e.g., B cell lymphoma, T cell lymphoma, follicular lymphoma), leukemias (e.g., chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia), multiple myeloma, post-transplant lymphoproliferative disorder, autoimmune lymphoproliferative syndrome, EBV- associated lymphoproliferative disease and Castleman disease.
  • lymphomas e.g., B cell lymphoma, T cell lymphoma, follicular lymphoma
  • leukemias e.g., chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell le
  • amino acid sequence modifications include, for example, substitutions, insertions and deletions, or combinations thereof.
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues.
  • Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Substitutional modifications are those in which at least one residue has been removed and a different residue (or residues) inserted in its place. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once.
  • a gp42 polypeptide is modified by substitution of an amino acid at one or more positions of the native protein to reduce or eliminate its binding to HLA class II molecules.
  • a “modified” protein or nucleic acid is one that has one or more modifications as outlined above.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • the nature of the carrier can depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polypeptide Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymers as well as in which one or more amino acid residue is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid.
  • a “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic.
  • a polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues. Amino acids in a polypeptide generally are chemically bound together via amide linkages (CONH).
  • Preventing, treating or ameliorating a disease Inhibiting the development or progression of a disease or condition, for example, in a subject who is at risk of or has an EBV infection.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • the term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters that arc specific to the particular disease.
  • a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease for the purpose of reducing the risk of developing pathology.
  • Protein tag Small peptides fused to a protein of interest.
  • the protein tag is located at the N-terminus or C-terminus of a protein of interest (such as a modified gp42 polypeptide provided herein).
  • Types of protein tags include, but are not limited to, affinity tags (e.g., HiBiT, glutathione S-transferase (GST), or His tag), epitope tags (e.g., HA, V5, FLAG, or Myc), and fluorescent tags (e.g., GFP, or a variant thereof).
  • Recombinant A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
  • Sample (or biological sample) A biological specimen containing genomic DNA, RNA (including mRNA), protein, cells or combinations thereof, obtained from a subject.
  • peripheral blood for example, PBMC
  • serum for example, plasma
  • tissue such as lymphoid tissue
  • cells such as lymphocytes, particularly B lymphocytes
  • urine saliva, sputum, cerebral spinal fluid, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material.
  • Self-assembling protein nanoparticle A multi-subunit protein-based nanoparticle formed from subunit monomers that self-assemble under suitable conditions to form the nanoparticle (typically globular in shape).
  • self-assembling protein nanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. hit. J. Mol. Sci., 12:5406-5421, 2011; WO 2018/005558; and WO 2013/044203), hepatitis virus antigens (including HBcAg, HBeAg and ACNcAg; see, e.g., WO 2019/028266), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct.
  • Sequence identity The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • Homologs and variants of a polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of the antibody using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence.
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.
  • Synthetic Produced by artificial means in a laboratory, for example a synthetic nucleic acid or protein (for example, a gp42 polypeptide) can be chemically synthesized in a laboratory.
  • a synthetic nucleic acid or protein for example, a gp42 polypeptide
  • Vaccine A preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of infectious or other types of disease. Vaccines may elicit both prophylactic (preventative or protective) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response.
  • a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • the vector is a virus vector, such as a lentivirus vector or an adeno-associated viral vector (AAV).
  • a vector is a plasmid vector.
  • EBV gp42 residues that make contact with its cellular receptor, HLA class II, as well as gp42 residues that interface with known gp42-specific neutralizing antibodies. Also described are amino acid residues of gp42 that are positioned within four Angstroms of an identified HLA class II contact residue and can thus alter binding of gp42 to HLA class II molecules. Based on this information, modified gp42 polypeptides that retain their immunogenicity, but are severely impaired for binding to HLA class II proteins were designed. Use of the modified gp42 polypeptides in immunogenic compositions, such as in protein nanoparticle vaccine compositions, is described.
  • the immunogenic compositions may also include EBV gp350 and/or EBV gH/gL immunogens.
  • modified EBV gp42 polypeptides that include at least one amino acid substitution at any one of residues 102, 107, 109-111, 115, 118, 150-160, 162, 197 and 220 of a wild-type gp42 amino acid sequence (such as gp42 of SEQ ID NO: 2).
  • the modified gp42 polypeptides exhibit a decrease in binding to HLA class II protein(s) compared to wild-type gp42.
  • the HLA class II proteins include one or more of HLA-DM, HLA-DOA, HLA-DOB, HLA-DP, HLA-DQ, and HLA- DR.
  • the at least one amino acid substitution is at any one of residues 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 and 220. In other aspects, the at least one amino acid substitution is at any one of residues 102, 107, 109, 110, 111, 115, 118, 162 and 197.
  • the modified gp42 polypeptide has at least one amino acid substitution at any one of residues 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 and 220 and at least one amino acid substitutions at any one of residues 102, 107, 109, 110, 111, 115, 118, 162 and 197.
  • the modified gp42 polypeptides include at least two, at least three or at least four amino acid substitutions at residues selected from 102, 107, 109, 110, 111, 115, 118, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 197 and 220 with respect to a wild-type gp42 (such as SEQ ID NO: 2).
  • the gp42 polypeptide has amino acid substitutions at least at residues 154, 157, 160 and 220 with respect to a wild-type gp42 (such as SEQ ID NO: 2).
  • the gp42 polypeptides has exactly four amino acid substitutions, which are at residues 154, 157, 160 and 220 with respect to a wild-type gp42 (such as SEQ ID NO: 2).
  • the amino acid substitution replaces a native amino acid with an alanine, arginine, glutamic acid, serine, methionine, leucine, isoleucine, glycine, valine, phenylalanine, tyrosine, tryptophan, lysine, histidine or aspartic acid.
  • the amino acid substitution replaces a native amino acid with an alanine, arginine or glutamic acid.
  • the modified gp42 polypeptide includes at least one amino acid substitution selected from the group consisting of R154A, R154E, R154S, N157A, N157E, N157R, N157S, E160A, E160R, E160S, R220A, R220E and R220S.
  • the modified gp42 polypeptides includes R154E, N157A, E160R and R220A (“EARA”) substitutions; R154E, N157A, E160R and R220E (“EARE”) substitutions; an R220A substitution; an R220E substitution; R154E, N157A and E160R substitutions; R154A, N157A and E160A substitutions; or R154A, N157A, E160A and R220A substitutions
  • the amino acid sequence of the modified gp42 polypeptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.
  • the amino acid sequence of the modified gp42 polypeptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16, and includes one or more of the following substitutions (such as 1, 2, 3, or 4 of such substitutions) R154A, R154E, R154S, N157A, N157E, N157R, N157S, E160A, E160R, E160S, R220A, R220E and R220S.
  • substitutions such as 1, 2, 3, or 4 of such substitutions
  • the amino acid sequence of the modified gp42 polypeptide includes or consists of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.
  • the heterologous protein includes a self-assembling protein nanoparticle subunit.
  • the self-assembling protein nanoparticle subunit is a ferritin subunit, a hepatitis B virus core antigen (HBcAg) subunit, a hepatitis B virus e-antigen (HBeAg) subunit, an African cichlid nackednavirus core antigen (ACNcAg) subunit, a lumazine synthase subunit, an encapsulin subunit, a DNA starvation/stationary phase protection protein subunit, a T4 fibritin subunit, a sulfur oxygenase reductase subunit, a bacteriophage Q beta capsid protein (qbeta) subunit, a dihydrolipoyl transacetylase protein (e2p) subunit, a
  • the self-assembling protein nanoparticle subunit is a ferritin subunit.
  • the amino acid sequence of the ferritin subunit is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18, or includes or consist of SEQ ID NO: 18.
  • Self-assembling nanoparticles are further described in section V.
  • the heterologous protein includes, or further includes, a protein tag, a fluorescent protein, or a carrier protein.
  • the protein tag is an affinity tag (such as, but not limited to, GST, His tag or HiBiT), an epitope tag (such as, but not limited to, HA, V5, FLAG, or Myc tag).
  • the fluorescent protein is a green fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, or a red fluorescent protein.
  • the carrier protein is or includes keyhole limpet hemocyanin (KLH), tetanus toxoid (TT), diphtheria toxoid (DT), modified cross-reacting material of diphtheria toxin (CRM 197), meningococcal outer membrane protein complex (OMPC), Hemophilus influenzae protein D (HiD), or recombinant tetanus toxin heavy chain C fragment (rTTHC).
  • KLH keyhole limpet hemocyanin
  • TT tetanus toxoid
  • DT diphtheria toxoid
  • CCM 197 meningococcal outer membrane protein complex
  • HiD Hemophilus influenzae protein D
  • rTTHC recombinant tetanus toxin heavy chain C fragment
  • the fusion protein includes, or further includes, EBV gH and gL proteins.
  • the amino acid sequence of the gH protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19.
  • the amino acid sequence of the gH protein includes or consists of SEQ ID NO: 19.
  • the amino acid sequence of the gL protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20.
  • the amino acid sequence of the gH protein includes or consists of SEQ ID NO: 20.
  • self-assembling protein nanoparticles that include a modified gp42 polypeptide or fusion protein disclosed herein.
  • the self-assembling protein nanoparticles are made from self-assembling protein nanoparticle subunits, such as ferritin subunits, HBcAg subunits, HBeAg subunits, ACNcAg subunits, lumazine synthase subunits, encapsulin subunits, DNA starvation/stationary phase protection protein subunits, T4 fibritin subunits, sulfur oxygenase reductase subunits, qbeta subunits, e2p subunits, 6ccq subunits, lf52 subunits, 5U6Y subunits, HIV capsid oligomerization domain subunits, hexamer subunits, or acinetobacter phage AP205 subunits.
  • self-assembling protein nanoparticle subunits such as ferritin subunits, HBcAg subunit
  • the self- assembling protein nanoparticle subunits are ferritin subunits.
  • the amino acid sequence of the ferritin subunit is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18, or includes or consists of SEQ ID NO: 18.
  • Self-assembling nanoparticle subunits are further described in section V.
  • nucleic acid molecules encoding a modified gp42 polypeptide, fusion protein, or self-assembling nanoparticle disclosed herein.
  • the nucleic acid molecule is codon- optimized for expression in mammalian cells.
  • the nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.
  • the nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, and encodes one or more of the following substitutions (such as 1, 2, 3, or 4 of such substitutions) R154A, R154E, R154S, N157A, N157E, N157R, N157S, E160A, E160R, E160S, R220A, R220E and R220S.
  • substitutions such as 1, 2, 3, or 4 of such substitutions
  • the nucleic acid molecule includes SEQ ID NO: 3, or a degenerate variant thereof; SEQ ID NO: 5, or a degenerate variant thereof; SEQ ID NO: 7, or a degenerate variant thereof; SEQ ID NO: 9, or a degenerate variant thereof; SEQ ID NO: 11, or a degenerate variant thereof; SEQ ID NO: 13, or a degenerate variant thereof; or SEQ ID NO: 15, or a degenerate variant thereof.
  • the nucleic acid molecule is DNA or cDNA. In other aspects, the nucleic acid molecule is RNA, such as messenger RNA (mRNA).
  • mRNA messenger RNA
  • the nucleic acid molecule is operably linked to a promoter.
  • the promoter is a eukaryotic promoter, such as a mammalian promoter. In other examples, the promoter is a prokaryotic promoter.
  • the promoter can be a constitutive promoter or a non-constitutive promoter.
  • the vector is a viral vector, such as but not limited to, a lentivirus vector, an adeno-associated virus (AAV) vector or an adenovirus vector.
  • the vector is a plasmid vector.
  • isolated cells that include a nucleic acid molecule or vector disclosed herein.
  • the cells are eukaryotic cells, such as mammalian cells.
  • the cells are prokaryotic such cells, such as bacterial cells.
  • compositions that include a pharmaceutically acceptable carrier and a modified gp42 polypeptide, fusion protein, self-assembling protein nanoparticle, nucleic acid molecule or vector disclosed herein.
  • the composition includes or consists of a pharmaceutically acceptable carrier and (i) a modified gp42 polypeptide provided herein (such as one at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16) and an EBV gp350 polypeptide (such as one having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21, or includes or consists of SEQ ID NO: 21); (ii)
  • the immunogenic composition further includes an adjuvant.
  • the adjuvant includes a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion (such as the AS03 adjuvant).
  • the adjuvant includes immunostimulatory oligonucleotides (e.g., CpG), aluminum salts (e.g., aluminum hydroxide, aluminum phosphate or alum (potassium aluminum sulfate)), monophosphoryl lipid A (MPL), TLR agonist(s), or Alhydroxiquim-II. Immunogenic compositions are further described in section VII.
  • eliciting an immune response in a subject by administering to the subject an effective amount of a modified gp42 polypeptide, fusion protein, self-assembling protein nanoparticle, nucleic acid molecule, vector, or immunogenic composition disclosed herein.
  • methods of immunizing a subject against EBV infection by administering to the subject a prophylactically effective amount of a modified gp42 polypeptide, fusion protein, self-assembling protein nanoparticle, nucleic acid molecule, vector, or immunogenic composition disclosed herein.
  • Methods of treating an EBV infection in a subject by administering to the subject a therapeutically effective amount of a modified gp42 polypeptide, fusion protein, self-assembling protein nanoparticle, nucleic acid molecule, vector, or immunogenic composition disclosed herein are also provided. Methods of eliciting an immune response, immunization and treatment are further described in section VIII.
  • the method includes contacting a sample containing B cells with a modified gp42 disclosed herein and isolating B cells that bind to the modified gp42 polypeptide, thereby identifying B cells that express gp42-specific antibodies (and enabling the isolation of monoclonal antibodies specific for gp42).
  • the modified gp42 polypeptide includes a detectable label, such as but not limited to, an affinity tag, an epitope tag, or a fluorophore.
  • the modified gp42 polypeptide is attached to a solid support (e.g., a multi-well plate).
  • the sample containing B cells is a PBMC sample.
  • the sample containing B cells is obtained from a subject previously infected with EBV, such as a subject suspected of having neutralizing antibodies against EBV. Methods of screening for gp42-specific antibodies using the modified gp42 polypeptides disclosed herein are further described in section IX.
  • a modified gp42 polypeptide disclosed herein is incorporated into a self-assembling protein nanoparticle subunit.
  • the self-assembling protein nanoparticle subunit is a monomer of a self- assembling protein nanoparticle, or a fragment of such a monomer that retains the portion of the monomer required for self-assembly.
  • Non-limiting examples of self-assembling protein nanoparticle subunits that can be used to form a self-assembling protein nanoparticle include ferritin nanoparticle subunits, hepatitis B virus (HBV) core antigen (HBcAg) subunits, HBV e-antigen (HBeAg) subunits, African cichlid nackednavirus core antigen (ACNcAg) subunits, lumazine synthase nanoparticle subunits, encapsulin nanoparticle subunits, sulfur oxygenase reductase (SOR) nanoparticle subunits, bacteriophage Q beta capsid protein (qbeta) subunits, dihydrolipoyl transacetylase protein (e2p) subunits, phosphopantetheine adenylyltransferase (6ccq) subunits, glutamate synthase (lf52) subunits, calcium/calmodulin dependent protein kinase Ila
  • the modified gp42 polypeptide is fused to a ferritin subunit to construct a selfassembling protein nanoparticle.
  • Ferritin nanoparticles and their use for immunization purposes have been disclosed, for example, in Kanekiyo et al. (Nature 499:102-106, 2013); WO 2020/061564; WO 2018/005558; and WO 2013/044203.
  • Ferritin is a globular protein that is found in all animals, bacteria, and plants, and which acts primarily to control the rate and location of polynuclear Fe(III)2O3 formation through the transportation of hydrated iron ions and protons to and from a mineralized core.
  • Ferritin nanoparticles are formed from 24 copies of the ferritin subunit.
  • the globular form of the ferritin nanoparticle is made up of monomeric subunits.
  • the ferritin subunits include one or more cysteine substitutions to introduce non-native disulfide bond(s) that stabilize the ferritin nanoparticle formed from the self-assembled subunits.
  • Non-limiting examples of the sequence of self-assembling ferritin subunits for use in the aspects provided herein include those disclosed in WO 2020/061564.
  • the amino acid sequence of the ferritin subunit is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18, or the amino acid sequence includes or consists of SEQ ID NO: 18.
  • the ferritin subunit is an insect ferritin subunit.
  • Insect ferritin protein nanoparticles and their use and production are described, for example, in WO 2018/005558.
  • insect ferritin includes twelve copies of two different subunits (termed heavy and light chains; 24 subunits total).
  • the insect ferritin heavy chains trimerize and the insect ferritin light chains trimerize (forming four trimers of heavy chains and four trimers of light chains) and self-assemble into the globular nanoparticle.
  • the insect ferritin heavy and light chains are from the Lepidoptera order of insects, such as ferritin heavy and light chains from Trichoplusia (such as Trichoplusia m), or ferritin heavy and light chains from manduca.
  • Trichoplusia such as Trichoplusia m
  • ferritin heavy and light chains from manduca Exemplary ferritin heavy and light chain amino acid sequences for Trichoplusia ni and manduca proteins are disclosed in WO 2020/061564.
  • the modified gp42 polypeptide is fused to a hepatitis virus protein to construct a self-assembling protein nanoparticle.
  • HBV nanoparticles and their use in vaccines have been described, such as in WO2019/028266.
  • the hepatitis virus protein is an HBV protein, such as HBV core antigen (HBcAg) or HBV e-antigen (HBeAg).
  • HBV core antigen HBV core antigen
  • HBV eAg HBV e-antigen
  • the hepatitis virus protein is a core antigen from an HBV-related virus that does not infect humans, such as woodchuck hepatitis virus, duck HBV or African cichlid nackednavirus (ACNDV).
  • the modified gp42 polypeptide is fused to a lumazine synthase (LS) protein to construct a self-assembling protein nanoparticle subunit.
  • LS nanoparticles are formed from 60 copies of the lumazine synthase subunit.
  • the globular form of lumazine synthase nanoparticle is made up of monomeric subunits.
  • the LS subunits include one or more cysteine substitutions to introduce nonnative disulfide bond(s) that stabilize the lumazine synthase nanoparticle formed from self-assembled subunits.
  • Non-limiting examples of the sequence of lumazine synthase subunits are disclosed in WO 2020/061564.
  • D. DNA starvation/stationary phase protection protein (DPS) D.
  • the modified gp42 polypeptide is fused to a subunit of a DNA starvation/stationary phase protection protein (DPS) complex, such as a DPS subunit from Thermosynechococcus elongates, Kineococcuc radiotolerans, or Nostoc punctiforme, to construct a self-assembling DPS nanoparticle.
  • DPS DNA starvation/stationary phase protection protein
  • Nonlimiting examples of the sequence of DPS subunits that can be included in the chimeric polypeptides are disclosed in WO 2020/061564.
  • the modified gp42 polypeptide is fused to a subunit of a bacteriophage Q beta capsid protein (qbeta) complex to construct a self-assembling protein nanoparticle.
  • qbeta bacteriophage Q beta capsid protein
  • the modified gp42 polypeptide is fused to a subunit of a dihydrolipoyl transacetylase protein (e2p) complex to construct a self-assembling protein nanoparticle.
  • E2p nanoparticles are formed from 60 copies of the e2p subunit (structural information is deposited at the Protein Data Bank No. 1B5S).
  • the N-terminus of the subunit is surface exposed and the C- terminus of the subunit is inside the globular nanoparticle.
  • a non-limiting example of the sequence of an ep2 subunit is disclosed in WO 2020/061564.
  • the modified gp42 polypeptide is fused to a subunit of a phosphopantetheine adenylyltransferase (6ccq) complex to construct a self-assembling protein nanoparticle.
  • Phosphopantetheine adenylyltransferase nanoparticles are formed from 6 copies of the phosphopantetheine adenylyltransferase subunit (structural information is deposited at the Protein Data Bank No. 6CCQ).
  • 6ccq phosphopantetheine adenylyltransferase
  • the modified gp42 polypeptide is fused to a subunit of a glutamate synthase (lf52) protein complex to construct a self-assembling protein nanoparticle.
  • a glutamate synthase subunit is disclosed in WO 2020/061564.
  • the self-assembling protein nanoparticle subunit is a C-terminal fragment of a calcium/calmodulin dependent protein kinase Ila (CaMKIIa) protein.
  • the CaMKIIa nanoparticle is formed from 12 copies of the C-terminal fragment of CaMKIIa subunit (structural information is deposited at the Protein Data Bank No. 5U6Y).
  • the N-terminus of the C-terminal fragment is surface exposed in the globular nanoparticle.
  • Non-limiting examples of CaMKIIa sequences that can be included in the compositions described herein is disclosed in WO 2020/061564.
  • the modified gp42 polypeptide is fused to a HIV capsid oligomerization domain to construct a self-assembling protein nanoparticle.
  • HIV capsid oligomerization domain sequences that can be included in the compositions described herein are disclosed in WO 2020/061564.
  • the modified gp42 polypeptide is fused to a Hexamer subunit to construct a selfassembling protein nanoparticle.
  • a hexamer sequence that can be included in the fusion proteins and compositions described herein is disclosed in WO 2020/061564.
  • the modified gp42 polypeptide is fused to a T4 fibritin foldon domain to produce the self-assembling protein nanoparticle.
  • T4 fibritin Foldon domain sequence are disclosed in WO 2020/061564.
  • the modified gp42 polypeptide is fused to an encapsulin subunit to construct a selfassembling protein nanoparticle.
  • Encapsulin nanoparticles are formed from 60 copies of the encapsulin subunit.
  • the globular form of the encapsulin nanoparticle is made up of monomeric subunits.
  • Encapsulin proteins are a conserved family of bacterial proteins also known as linocin-like proteins that form large protein assemblies that function as a minimal compartment to package enzymes.
  • the encapsulin assembly is made up of monomeric subunits, which are polypeptides having a molecule weight of approximately 30 kDa.
  • the monomeric subunits self-assemble into the globular encapsulin assembly including 60, or in some cases, 180 monomeric subunits.
  • Methods of constructing encapsulin nanoparticles are described, for example, in Sutter et al. (Nature Struct. Mol. Biol., 15:939-947, 2008, which is incorporated by reference herein in its entirety).
  • the encapsulin polypeptide is bacterial encapsulin, such as Thermotoga maritime or Pyrococcus furiosus or Rhodococcus erythropolis or Myxococcus xanthus encapsulin.
  • a non-limiting example of the sequence of an encapsulin subunit is disclosed in WO 2020/061564.
  • Acinetobacter phage AP205 (AP205)
  • the modified gp42 polypeptide is fused to an Acinetobacter phage AP205 domain to generate the self-assembling protein nanoparticle.
  • the Acinetobacter phage AP205 subunits include one or more cysteine substitutions to introduce non-native disulfide bond(s) that stabilize the Acinetobacter phage AP205 nanoparticle formed from self-assembled subunits.
  • a non-limiting example of an Acinetobacter phage AP205 domain sequence is disclosed in WO 2020/061564.
  • Nucleic acid molecules encoding a modified gp42 polypeptide, fusion protein, or self-assembling nanoparticle disclosed herein are also provided. These nucleic acid molecules include DNA, cDNA and RNA sequences that encode the modified gp42 polypeptide, fusion protein, or self-assembling nanoparticle.
  • the genetic code can be used to construct a variety of functionally equivalent nucleic acids, such as nucleic acids that differ in sequence but which encode the same protein sequence, or encode a conjugate or fusion protein including the nucleic acid sequence.
  • nucleic acid molecule that includes a coding sequence (such as a codon-optimized coding sequence) for a disclosed modified gp42 polypeptide.
  • the nucleic acid molecule is codon-optimized for expression in mammalian cells.
  • the nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.
  • the nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, and encodes one or more of the following substitutions (such as 1, 2, 3, or 4 of such substitutions) R154A, R154E, R154S, N157A, N157E, N157R, N157S, E160A, E160R, E160S, R220A, R220E and R220S.
  • substitutions such as 1, 2, 3, or 4 of such substitutions
  • the nucleic acid molecule includes SEQ ID NO: 3, or a degenerate variant thereof; SEQ ID NO: 5, or a degenerate variant thereof; SEQ ID NO: 7, or a degenerate variant thereof; SEQ ID NO: 9, or a degenerate variant thereof; SEQ ID NO: 11, or a degenerate variant thereof; SEQ ID NO: 13, or a degenerate variant thereof; or SEQ ID NO: 15, or a degenerate variant thereof.
  • the nucleic acid molecule may further include a coding sequence for a heterologous protein, such as a selfassembling protein nanoparticle subunit (for example, a ferritin subunit or any subunit described in section V above), or a protein tag, a fluorescent protein, or a carrier protein.
  • a heterologous protein such as a selfassembling protein nanoparticle subunit (for example, a ferritin subunit or any subunit described in section V above), or a protein tag, a fluorescent protein, or a carrier protein.
  • the nucleic acid molecule further encodes EBV gH and/or gL proteins.
  • the nucleic acid molecule encodes a precursor of a self-assembling protein nanoparticle subunit, that when expressed in cells under appropriate conditions, is processed and selfassembles into the protein nanoparticle.
  • the nucleic acid molecule can encode an N-terminal signal sequence for entry into the cellular secretory system that is proteolytically cleaved during processing of the fusion protein.
  • Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are known (see, e.g.. Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).
  • Nucleic acids can also be prepared by amplification methods.
  • Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self- sustained sequence replication system (3SR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self- sustained sequence replication system
  • the nucleic acid molecules encoding a modified gp42 polypeptide, fusion protein or self-assembling protein nanoparticle subunit can include a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences.
  • the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double stranded forms of DNA.
  • Nucleic acid sequences encoding a disclosed modified gp42 polypeptide, fusion protein or selfassembling protein nanoparticle can be operatively linked to expression control sequences.
  • An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences.
  • the expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • DNA sequences encoding the modified gp42 polypeptide, fusion protein or self-assembling protein nanoparticle disclosed herein can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • Host cells can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are known.
  • suitable host cells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human cells).
  • Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, Cl 29 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines.
  • mammalian host cell lines include Vero cells, HeLa cells, HEK-293F cells, CHO cells, W138 cells and BHK cells.
  • a cell line is selected based on the ability to provide higher expression, desirable glycosylation patterns, or other features.
  • Transformation of a host cell with recombinant DNA or mRNA can be carried out by conventional techniques.
  • the host is prokaryotic, such as, but not limited to, E. coli
  • competent cells which are capable of nucleic acid uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCE method.
  • MgCE or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
  • Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding a disclosed modified gp42 polypeptide, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the gp42 polypeptide (see for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Modifications can be made to a nucleic acid encoding a modified gp42 polypeptide, fusion protein or self-assembling protein nanoparticle disclosed herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning or expression of the modified gp42 polypeptide, fusion protein or self-assembling protein nanoparticle. Non-limiting examples of such modifications include termination codons, a methionine added at the amino terminus to provide an initiation site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps.
  • the nucleic acid molecule encoding the modified gp42 polypeptide, fusion protein or self-assembling protein nanoparticle is an RNA molecule.
  • the modified gp42 polypeptide, fusion protein or self-assembling protein nanoparticle is administered as an mRNA vaccine or a self-amplifying RNA vaccine (see, e.g., Fuller and Berglund, N Engl J Med 382:2469-2471, 2020).
  • the RNA molecule encodes the modified gp42 polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.
  • Immunogenic compositions that include a modified gp42 polypeptide, fusion protein or selfassembling protein nanoparticle disclosed herein (or a nucleic acid molecule or vector encoding a modified gp42 polypeptide, fusion protein or self-assembling protein nanoparticle) and a pharmaceutically acceptable carrier (such as water or saline) are also provided.
  • the immunogenic composition further includes an adjuvant and/or one or more different immunogens, such as an EBV gp350 immunogen and/or gH/gL immunogens.
  • the immunogenic composition is lyophilized.
  • compositions can be administered to subjects by a variety of administration modes, for example, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes.
  • administration modes for example, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes.
  • Actual methods for preparing administrable compositions are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013.
  • a modified gp42 polypeptide, fusion protein or self- assembling protein nanoparticle described herein can be formulated with pharmaceutically acceptable carriers to help retain biological activity while also promoting increased stability during storage within an acceptable temperature range.
  • Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffered saline solution, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents.
  • the resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing.
  • Formulated compositions may contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually ⁇ 1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben.
  • a bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.
  • the immunogenic compositions of the disclosure can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.
  • pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.
  • the immunogenic composition may optionally include an adjuvant to enhance an immune response in the subject.
  • the adjuvant is or includes AS03, which is composed of a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion (see, e.g., Garcon et al., Expert Rev Vaccines 11 (3):349-366, 2012).
  • the adjuvant includes a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity.
  • suitable adjuvants include, immunostimulatory oligonucleotides (such as those including a CpG motif) and biological molecules, such as costimulatory molecules.
  • the adjuvant includes AlPOi. alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the immunogen and cytokines, non-ionic block copolymers, and chemokines.
  • Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPLTM (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA) may also be used as an adjuvant (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product.
  • the immunogenic composition is provided as a sterile composition.
  • the immunogenic composition typically contains an effective amount (such as therapeutically effective amount or prophylactically effective amount) of a modified gp42 polypeptide, fusion protein or self-assembling protein nanoparticle, and can be prepared by conventional techniques.
  • the amount of gp42 polypeptide, fusion protein or self-assembling protein nanoparticle in each dose of the immunogenic composition is selected as an amount which elicits or primes an immune response without significant, adverse side effects.
  • the immunogenic composition can be provided in unit dosage form for use to elicit or prime an immune response in a subject, for example, to prevent or treat EBV infection in the subject.
  • a unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and/or a metering mechanism for administering the unit dose or multiples thereof.
  • the disclosed chimeric polypeptides, self-assembling protein nanoparticles, nucleic acid molecules or vectors encodings a chimeric polypeptide, and compositions including same can be administered to a subject to induce an immune response to EBV to prevent, inhibit, and/or treat an EBV infection.
  • the immune response can be a protective immune response, for example a response that prevents or reduces subsequent infection with EBV. Elicitation of the immune response can also be used to treat or inhibit infection and illnesses associated with EBV infection.
  • the disclosed immunogenic compositions can be used in methods of preventing, inhibiting, or treating an EBV infection.
  • the methods can be used either to avoid infection in an EBV seronegative subject (e.g., by inducing an immune response that protects against EBV infection), or to treat existing infection in an EBV seropositive subject.
  • the EBV seropositive subject may or may not carry a diagnosis of lymphoproliferative disease (such as a B cell lymphoma).
  • the methods involve selecting a subject at risk for contracting an EBV infection, or a subject at risk of developing a B cell lymphoma (such as a subject with EBV infection), and administering a disclosed immunogenic composition to the subject to elicit an immune response to EBV in the subject.
  • Treatment of EBV by inhibiting EBV replication or infection can include delaying the development of a lymphoproliferative disease, such as a B cell lymphoma, in a subject.
  • Treatment of EBV can also include reducing signs or symptoms associated with the presence of EBV (for example, by reducing or inhibiting EBV replication).
  • treatment using the methods disclosed herein prolongs the time of survival of the subject, such as an increase of at least 3 months, at least 6 months, at least 9 months at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years, for example as compared to the absence of such treatment.
  • Typical subjects intended for treatment with the immunogenic compositions and methods of the present disclosure include humans, as well as non-human primates and other animals.
  • accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject.
  • These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods to detect and/or characterize EBV infection.
  • the disclosed immunogenic compositions can be used in coordinate (or prime-boost) immunization protocols or combinatorial formulations.
  • combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogenic compositions or formulations, each directed toward eliciting an anti-EBV immune response, such as an immune response to EBV gp42.
  • Separate immunogenic compositions that elicit the anti-EBV immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate immunization protocol.
  • the immunization protocol includes administration of a separate composition that includes an EBVgp350 immunogen, such as an EBV gp35O immunogen described in WO 2018/200742.
  • a suitable immunization regimen includes at least two separate inoculations with one or more immunogenic compositions including a disclosed gp42 polypeptide or self-assembling protein nanoparticle, with a second inoculation being administered at least about two, about three, about four, about five, about six or about eight weeks following the first inoculation.
  • a third inoculation can be administered several months after the second inoculation, and in specific aspects, more than about three months, more than about five months or more than about six months after the first inoculation, such about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation. Periodic inoculations beyond the third may also be desirable to enhance the subject's immune memory.
  • the adequacy of the immunization parameters chosen can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program.
  • the T cell populations can be monitored by conventional methods.
  • the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of EBV infection or progression to B cell lymphoma, or improvement in a disease state (e.g., reduction in viral load). If such monitoring indicates that immunization is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the immunization parameters can be modified in a fashion expected to potentiate the immune response.
  • each boost can be a different immunogenic composition. It is also contemplated in some examples that the boost may be the same immunogenic composition as another boost, or the prime.
  • the prime and the boost can be administered as a single dose or multiple doses, for example, two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. Multiple boosts can also be given, such as one to five, or more. Different dosages can be used in a series of sequential inoculations. For example, a relatively large dose in a primary inoculation and then a boost with relatively smaller doses.
  • the immune response against the selected antigenic surface can be generated by one or more inoculations of a subject.
  • the immunogenic composition can be administered to the subject simultaneously with the administration of an adjuvant.
  • the immunogenic composition can be administered to the subject after the administration of an adjuvant and within a sufficient amount of time to elicit the immune response.
  • an effective dosage or effective dose of the immunogenic composition may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes.
  • Dosage can be varied by the attending clinician to maintain a desired concentration at a target site. Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery.
  • the actual dosage of a disclosed immunogenic composition will vary according to factors such as the disease indication and particular status of the subject (for example, the subject’s age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the composition for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response.
  • a non-limiting range for a therapeutically effective amount of a disclosed modified gp42 polypeptide, fusion protein or protein nanoparticle composition to be administered according to the claimed methods is about 0.0001 mg/kg body weight to about 10 mg/kg body weight, such as about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, or about 10 mg/kg, for example, 0.01 mg/kg to about 1 mg/kg body weight
  • the dosage includes a set amount of a disclosed immunogenic composition such as from about 1-300 pg, for example, a dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 pg.
  • the dosage and number of doses can depend on the setting, for example, in an adult or anyone primed by prior EBV infection or immunization, a single dose may be a sufficient booster. In naive subjects, in some examples, at least two doses would be given, for example, at least three doses. In some aspects, an annual boost is given, for example, along with an annual influenza vaccination.
  • assay for neutralization activity include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry based assays, and single-cycle infection.
  • PRNT plaque reduction neutralization
  • immunization with a disclosed immunogenic composition can be combined with anti-viral therapy.
  • immunization with a disclosed immunogenic composition can be combined with anti-EBV therapy, such as additional treatment with one or more of acyclovir, desciclovir, ganciclovir, valgancyclovir, omaciclovir, valomaciclovir, maribavir, cidofovir, vidarabine, adenine arabinoside, phosphonoacetic acid, a thymidine derivative (e.g., l-[(25;4S-2-( hydroxymelhyl)-l,3-dioxolan- 4-yl]5-vjnylpyrimidtne-2,4(l H,3H)-dione; KAY-2-41; and KAH-39-149), interferon (IFN)-oc and IFN-fJ.
  • acyclovir desciclovir, ganciclovir,
  • EBV infection does not need to be completely eliminated or reduced or prevented for the methods to be effective.
  • elicitation of an immune response to EBV with one or more of the disclosed immunogenic compositions can reduce or inhibit EBV infection by, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable EBV infected cells), as compared to EBV infection in the absence of the therapeutic agent.
  • EBV replication can be reduced or inhibited by the disclosed methods.
  • EBV replication does not need to be completely eliminated for the method to be effective.
  • the immune response elicited using one or more of the disclosed immunogens can reduce EBV replication by, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable EBV replication), as compared to EBV replication in the absence of the immune response.
  • immunization of a subject with an immunogenic composition disclosed herein does not require complete (100%) protection against subsequent infection by EBV.
  • immunization reduces the risk of an immunized subject from being infected with EBV by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%, compared to the absence of immunization (or compared to a non-immunized population).
  • modified gp42 polypeptides disclosed herein can also be used as probes to enable screening of B cells for expression of gp42-specific monoclonal antibodies.
  • a gp42 polypeptide Prior to the present disclosure, it was not possible to use a gp42 polypeptide as a probe to select for gp42-specific antibodies in human blood samples because WT gp42 binds HLA class II present on all B cells.
  • a modified gp42 polypeptide also referred to herein as “probe”
  • the modified gp42 polypeptide includes a detectable label.
  • the detectable label is or includes an affinity tag, an epitope tag, or a fluorophore.
  • the modified gp42 polypeptide is attached to a solid support, such as a multi-well plate, tissue culture flask, or bead (e.g., a magnetic bead or fluorescent bead).
  • the sample containing B cells is a peripheral blood mononuclear cell (PBMC) sample, such as from a human, non-human primate, mouse or other non-human animal.
  • PBMC peripheral blood mononuclear cell
  • the sample containing B cells is obtained from a subject previously infected with EBV, such as a subject that has neutralizing antibodies against EBV. Methods of screening biological samples for antigen-specific B cells and/or antigen-specific antibodies are known (see, e.g., WO 2018/200742).
  • the modified gp42 polypeptide includes at least one amino acid substitution at any one of residues 102, 107, 109-111, 115, 118, 150-160, 162, 197 and 220 of a wildtype gp42 amino acid sequence set forth as SEQ ID NO: 2, wherein the modified gp42 polypeptide exhibits a decrease in binding to a human leukocyte antigen (HLA) class II protein compared to the wild-type gp42 of SEQ ID NO: 2.
  • HLA human leukocyte antigen
  • the modified gp42 polypeptides includes at least two, at least three or at least four amino acid substitutions at residues selected from 102, 107, 109, 110, 111, 115, 118, 150, 151, 152,
  • the gp42 polypeptide includes amino acid substitutions at least at residues 154, 157, 160 and 220.
  • the gp42 polypeptide as exactly four amino acid substitutions, which are at residues
  • the amino acid substitution replaces a native amino acid with an alanine, arginine, glutamic acid, serine, methionine, leucine, isoleucine, glycine, valine, phenylalanine, tyrosine, tryptophan, lysine, histidine or aspartic acid.
  • the amino acid substitution replaces a native amino acid with an alanine, arginine or glutamic acid.
  • the modified gp42 polypeptide includes at least one amino acid substitution selected from the group consisting of R154A, R154E, R154S, N157A, N157E, N157R, N157S, E160A, E160R, E160S, R220A, R220E and R220S.
  • the modified gp42 polypeptides includes R154E, N157A, E160R and R220A substitutions; R154E, N157A, E160R and R220E substitutions; an R220A substitution; an R220E substitution; R154E, N157A and E160R substitutions; R154A, N157A and E160A substitutions; or R154A, N157A, E160A and R220A substitutions.
  • the amino acid sequence of the modified gp42 polypeptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.
  • the amino acid sequence of the modified gp42 polypeptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16, and includes one or more of the following substitutions (such as 1, 2, 3, or 4 of such substitutions) R154A, R154E, R154S, N157A, N157E, N157R, N157S, E160A, E160R, E160S, R220A, R220E and R220S.
  • substitutions such as 1, 2, 3, or 4 of such substitutions
  • the amino acid sequence of the modified gp42 polypeptide includes or consists of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.
  • the probe used in the disclosed method is a fusion protein that includes a modified gp42 polypeptide disclosed herein and a heterologous protein, such as a self-assembling protein nanoparticle subunit (for example, a ferritin subunit), as described herein.
  • the fusion protein further includes EBV gH and gL proteins.
  • the heterologous protein can also include a protein tag, a fluorescent protein, or a carrier protein.
  • the probe used in the disclosed method is a self-assembling protein nanoparticle that includes a modified gp42 polypeptide or fusion protein disclosed herein.
  • EBV gp42 which binds HLA class II, is required for virus entry into B cells (FIG. 1 A), and is thus a target for neutralizing antibodies.
  • mAbs monoclonal antibodies that bind gp42 (A10, F-2-1 and 4C12) were used in the structural studies described herein.
  • Humanized mice are mice that have been reconstituted with human lymphocytes, including human B cells. When humanized mice are infected with EBV, the animals become viremic and EBV DNA can be detected in the blood of all animals. Antibodies A10 and F-2-1 were tested in humanized mice. Animals pretreated with mAb A10 did not become viremic after EBV infection, while most of the animals pretreated with mAb F-2-1 did become viremic (FIG. IB). Thus, EBV gp42 mAb A10 protected the animals from EBV infection.
  • FIG. 2A shows the binding of gp42 to HLA class II DR (PDB: 1KG0), mAb F-2-1, and mAb A10.
  • EBV gp42 is shown in complex with EBV gH/gL.
  • FIG. 2B shows the footprint of the HLA class II DR binding site (PDB: 1KG0) on gp42; FIG.
  • FIG. 2C shows the footprint of mAb F-2-1 heavy chain (HC) and light chain (LC) binding sites on gp42; and FIG. 2D shows the footprint of mAb A10 HC and LC binding sites on gp42.
  • EBV gp42 residues involved in the binding interface between gp42 and HLA class II DR, mAb F-2-1 or mAb A 10 are shown in FIG. 2E.
  • Residues contacting HLA class II are distributed in three segments (highlighted in the shaded boxes of FIG. 2E), corresponding to residues 104-111, residues 150-160 and residue 220, respectively, of SEQ ID NO: 2.
  • Residues involved in salt bridge formation are underlined in FIG. 2E (residues 160 and 220 of SEQ ID NO: 2).
  • gp42 with R154E/N157A/E160R substitutions (“EAR”) - SEQ ID NO: 4 gp42 with an R220E substitution - SEQ ID NO: 6 gp42 with R154E/N157A/E160R/R220E substitutions (“EARE”) - SEQ ID NO: 8 gp42 with R154A/N157A/E160A substitutions (“AAA”) - SEQ ID NO: 10 gp42 with an R220A substitution - SEQ ID NO: 12 gp42 with R154A/N157A/E160A/R220A substitutions (“AAAA”) - SEQ ID NO:
  • FIG. 3A Amino acids of EBV gp42 that have close contacts on HLA class II DR1, mAb A10 and/or mAb F- 2-1 are shown in FIG. 3A.
  • gp42 mutants were tested for binding to HLA class II DR1, mAb A10, mAb F-2- 1 and mAb 4C12 using biolayer interferometry (Octet).
  • FIG. 3B shows the percentage of binding of the substitution mutants to HLA class II DR1, mAb A 10, mAb F-2-1, and mAb 4C12.
  • EARA and EARE at amino acid positions 154, 157, 160, and 220
  • PBMCs peripheral blood mononuclear cells
  • Binding of WT gH/gL/gp42 trimer and mutant gH/gL/gp42 trimers to HLA class II on PBMCs was also tested by flow cytometry.
  • PBMCs obtained from four human donors were used in two separate experiments.
  • Biotinylated WT gH/gL/gp42 trimer or mutant (EARA and EARE) gH/gL/gp42 timers were incubated with the human donor PBMCs and then SA was added.
  • Ferritin nanoparticles with WT, EARA and EARE gp42 in trimer format were produced according to previously described methods (Kanekiyo et al., Cell 162:1090-1100, 2015; Wei et al., Sei Transl Med 14(643):eabf3685, 2022).
  • Balb/c mice were administered two doses (at week 0 and week 3) of the nanoparticles at a dose of 0.5 tig. Blood from immunized mice was collected two weeks after the second dose of immunogen.
  • mice immunized with either WT gH/gL/gp42 trimer or gH/gL/gp42 trimer mutants produced similar levels of B cell (FIG. 6A) and epithelial cell (FIG. 6B) neutralizing antibodies to EBV.
  • FIGS. 6A-6B mice immunized with either WT gH/gL/gp42 trimer or gH/gL/gp42 trimer mutants produced similar levels of B cell (FIG. 6A) and epithelial cell (FIG. 6B) neutralizing antibodies to EBV.
  • amino acid residues were identified that are capable of modulating binding of gp42 to HLA class II molecules based on their close proximity to gp42 residues that were determined to make contact with HLA class II molecules (see Example 1). Specifically, residues positioned within 4 Angstroms of gp42 residues R220, V151, N155, L156 or N157 were identified.
  • the phenylalanine at position 109 is within 4 Angstroms of the valine at position 151 (V151).
  • the cysteines at positions 102 (C102) and 115 (Cl 15) and the serine at residue 110 (SI 10) are within 4 Angstroms of the arginine at position 220 (R220).
  • Other residues located within 4 Angstroms of one of the contact residues include residues Y107, Y111, Fl 18, L162 and K197 of gp42.
  • amino acid substitution at any one of residues C102, Y107, F109, S 110, Y111, C115, F118, L162 and K197 can alter the position of any one of contact residues R220, V151, N155, L156 and/or N157, thereby impacting the ability of gp42 to bind HLA class II molecules.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La divulgation concerne des polypeptides de glycoprotéine 42 (gp42) du virus Epstein-Barr (EBV) modifiés qui conservent l'immunogénicité, mais qui sont altérés pour se lier à des molécules HLA de classe II. La divulgation concerne également l'utilisation des polypeptides gp42 modifiés dans des compositions immunogènes, telles que dans des compositions de nanoparticules de protéines à auto-assemblage. La divulgation concerne également des procédés de criblage de lymphocytes B qui expriment des anticorps monoclonaux spécifiques aux gp42 à l'aide des polypeptides gp42 modifiés en tant que sonde.
PCT/US2024/013311 2023-01-30 2024-01-29 Immunogènes de la glycoprotéine 42 du virus epstein-barr pour la vaccination et la découverte d'anticorps Pending WO2024163327A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363482143P 2023-01-30 2023-01-30
US63/482,143 2023-01-30

Publications (1)

Publication Number Publication Date
WO2024163327A1 true WO2024163327A1 (fr) 2024-08-08

Family

ID=90059518

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/013311 Pending WO2024163327A1 (fr) 2023-01-30 2024-01-29 Immunogènes de la glycoprotéine 42 du virus epstein-barr pour la vaccination et la découverte d'anticorps

Country Status (1)

Country Link
WO (1) WO2024163327A1 (fr)

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6194388B1 (en) 1994-07-15 2001-02-27 The University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6214806B1 (en) 1997-02-28 2001-04-10 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders
US6218371B1 (en) 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6339068B1 (en) 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
US6406705B1 (en) 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
WO2013044203A2 (fr) 2011-09-23 2013-03-28 THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPTARTMENT OF HEALTH & HUMAN SERVICES Nouveaux vaccins à base de protéine hémagglutinine de la grippe
WO2015054639A1 (fr) * 2013-10-11 2015-04-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vaccins contre le virus d'epstein-barr
WO2018005558A1 (fr) 2016-06-27 2018-01-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Nanoparticules de ferritine d'insecte à auto-assemblage pour la présentation d'antigènes trimériques co-assemblés
WO2018200742A1 (fr) 2017-04-25 2018-11-01 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Anticorps et procédés de diagnostic et de traitement d'infection par le virus d'epstein barr
WO2019028266A1 (fr) 2017-08-02 2019-02-07 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Vaccin à base de nanoparticules d'hépatite b pour le virus de la grippe
WO2020061564A1 (fr) 2018-09-23 2020-03-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Conjugués de vecteur à nanoparticules de peptide de fusion env du vih-1 et utilisation de ces derniers
WO2021168292A1 (fr) * 2020-02-20 2021-08-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps monoclonaux de virus d'epstein-barr et leurs utilisations

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6194388B1 (en) 1994-07-15 2001-02-27 The University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6214806B1 (en) 1997-02-28 2001-04-10 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders
US6406705B1 (en) 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US6339068B1 (en) 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
US6218371B1 (en) 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
WO2013044203A2 (fr) 2011-09-23 2013-03-28 THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPTARTMENT OF HEALTH & HUMAN SERVICES Nouveaux vaccins à base de protéine hémagglutinine de la grippe
WO2015054639A1 (fr) * 2013-10-11 2015-04-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vaccins contre le virus d'epstein-barr
WO2018005558A1 (fr) 2016-06-27 2018-01-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Nanoparticules de ferritine d'insecte à auto-assemblage pour la présentation d'antigènes trimériques co-assemblés
WO2018200742A1 (fr) 2017-04-25 2018-11-01 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Anticorps et procédés de diagnostic et de traitement d'infection par le virus d'epstein barr
WO2019028266A1 (fr) 2017-08-02 2019-02-07 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Vaccin à base de nanoparticules d'hépatite b pour le virus de la grippe
WO2020061564A1 (fr) 2018-09-23 2020-03-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Conjugués de vecteur à nanoparticules de peptide de fusion env du vih-1 et utilisation de ces derniers
WO2021168292A1 (fr) * 2020-02-20 2021-08-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps monoclonaux de virus d'epstein-barr et leurs utilisations

Non-Patent Citations (49)

* Cited by examiner, † Cited by third party
Title
"GENBAKK", Database accession no. P03205.1
"GENBANK", Database accession no. YP 401667.1
"Lewira's genes XII", 2017
"Vaccine Adjuvants and Delivery Systems", 2007, WILEY-INTERSCIENCE
"Viral Expression Vectors", 2011, SPRINGER PRESS
ALTSCHUL ET AL., J. MOL. BIOL, vol. 215, 1990, pages 403
ALTSCHUL ET AL., NATURE GENET, vol. 6, 1994, pages 119
ALVING ET AL., EXPERT REV VACCINES, vol. 19, no. 3, 2020, pages 279 - 292
AUSUBEL ET AL.: "Remington: The Science and Practice of Pharmacy", 2013, PHARMACEUTICAL PRESS
BORZAHUTT-FLETCHER, NAT MED, vol. 8, 2002, pages 594 - 599
CHEN ET AL., IMMUNITY, vol. 55, no. 11, 2022, pages 2135 - 2148
COHEN ET AL., SCI. TRANSL. MED, vol. 3, no. 107, 2011, pages 107fs7
CORPET ET AL., NUCLEIC ACIDS RESEARCH, vol. 16, 1988, pages 10881
FRANKEL ET AL., MOL. IMMUNOL., vol. 16, 1979, pages 101 - 106
FULLERBERGLUND, N ENGL J MED, vol. 382, 2020, pages 2469 - 2471
GARQON ET AL., EXPERT REV VACCINES, vol. 11, no. 3, 2012, pages 349 - 366
HIGGINSSHARP, CABIOS, vol. 5, 1989, pages 151
HIGGINSSHARP, GENE, vol. 73, 1988, pages 237
IZARD ET AL., PNAS, vol. 96, 1999, pages 1240 - 1245
KANEKIYO ET AL., CELL, vol. 162, 2015, pages 1090 - 1100
KANEKIYO ET AL., NATURE, vol. 499, 2013, pages 102 - 106
KIRSCHNER ET AL., J VIROL, vol. 80, 2006, pages 9444 - 9454
KIRSCHNER ET AL., STRUCTURE, vol. 17, no. 2, 2009, pages 223 - 233
MAUREEN M. MULLEN: "Structure of the Epstein-Barr Virus gp42 Protein Bound to the MHC Class II Receptor HLA-DR1", MOLECULAR CELL, vol. 9, no. 2, 1 February 2002 (2002-02-01), AMSTERDAM, NL, pages 375 - 385, XP093157791, ISSN: 1097-2765, DOI: 10.1016/S1097-2765(02)00465-3 *
MULLEN ET AL., MOL CELL, vol. 9, no. 2, 2002, pages 375 - 385
NEEDLEMANWUNSCH, J, MOL. BIOL, vol. 48, 1970, pages 443
NEWMA ET AL., CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS, vol. 15, 1998, pages 89 - 142
OU ET AL., SCIENTIFIC REPORTS, vol. 10, 2020, pages 3032
PAMELA L SHAW ET AL: "Characteristics of Epstein-Barr virus envelope protein gp42", VIRUS GENES, KLUWER ACADEMIC PUBLISHERS, BO, vol. 40, no. 3, 17 February 2010 (2010-02-17), pages 307 - 319, XP019795982, ISSN: 1572-994X *
PEARSONLIPMAN, PROC. NATL. ACAD. SCI. U.S.A, vol. 85, 1988, pages 2444
PICHICHERO, HUM VACCIN IMMUNOTHER, vol. 9, 2013, pages 2505 - 2523
RESSING ET AL., J. VIROL, vol. 79, 2005, pages 841 - 852
SAMBROOK ET AL.: "Basic Cell Culture Protocols (Methods in Molecular Biology", 2012, COLD SPRING HARBOR
SATHIYAMOORTHY ET AL., PLOS PATHOG, vol. 10, no. 8, 2014, pages e1004309
SATHIYAMOORTHY ET AL., PROC NATL ACAD SCI USA, vol. 114, no. 41, 2017, pages 8703 - 8710
SMITHWATERMAN, ADV. APPL. MATH, vol. 2, 1981, pages 482
SNIJDER ET AL., IMMUNITY, vol. 48, no. 4, 2018, pages 799 - 811
SPRIGGS ET AL., J. VIROL, vol. 70, 1996, pages 5557 - 5563
SUTTER ET AL., NATURE STRUCT. MOL. BIOL, vol. 15, 2008, pages 939 - 947
SUTTER ET AL., NATURE STRUCT. MOL. BIOL., vol. 15, 2008, pages 939 - 947
URICH ET AL., SCIENCE, vol. 311, 2006, pages 996 - 1000
WANG ET AL., J VIROL, vol. 72, 1998, pages 5552 - 5558
WANGHUTT-FLETCHER, J. VIROL, vol. 72, no. 1, 1998, pages 158 - 163
WEI BU: "Epstein-Barr virus gp42 antibodies reveal sites of vulnerability for receptor binding and fusion to B cells", IMMUNITY, vol. 57, no. 3, 12 March 2024 (2024-03-12), AMSTERDAM, NL, pages 559 - 573.e6, XP093157720, ISSN: 1074-7613, DOI: 10.1016/j.immuni.2024.02.008 *
WEI ET AL., SCI TRANSL MED, vol. 14, no. 643, 2022, pages eabf3685
ZHANG ET AL., J. MOL. BIOL., vol. 306, 2001, pages 1099 - 1114
ZHANG, Y, INT. J. MOL. SCI., vol. 12, 2011, pages 5406 - 5421
ZHANG, Y. INT. J. MOL. SCI., vol. 12, 2011, pages 5406 - 5421
ZHU ET AL., NAT COMMUN, vol. 12, no. 1, 2021, pages 6624

Similar Documents

Publication Publication Date Title
US20240408195A1 (en) Epstein-barr virus vaccines
KR102044948B1 (ko) 동종이합체 단백질 구조물
US11136356B2 (en) Recombinant HIV-1 envelope proteins and their use
US20240247033A1 (en) Sars-cov-2 rbd constructs
JP5138844B2 (ja) 抗原およびＥＢＶＧｐ３５０／２２０の受容体の同時刺激によるＢ細胞活性化および免疫グロブリン分泌の増強
WO2020061564A1 (fr) Conjugués de vecteur à nanoparticules de peptide de fusion env du vih-1 et utilisation de ces derniers
WO2022207645A1 (fr) Vaccin sous-unitaire contre le sars-cov-2
US20250109175A1 (en) Recombinant gp120 protein with v1-loop deletion
KR20230084478A (ko) 면역원성 코로나 바이러스 융합 단백질 및 관련 방법
US20240228549A1 (en) Prefusion-stabilized lassa virus glycoprotein complex and its use
WO2023109835A1 (fr) Vaccin recombinant à protéine de fusion vegf-crm197 et procédé de préparation associé et son utilisation
CN115038713A (zh) 流感病毒疫苗及其用途
WO2024163327A1 (fr) Immunogènes de la glycoprotéine 42 du virus epstein-barr pour la vaccination et la découverte d'anticorps
CN118984833A (zh) 表位的组合及其用途、疫苗构建体、诱导免疫反应的方法、表位的鉴定方法
US20230310584A1 (en) Nucleic acids encoding a polypeptide comprising a modified fc region of a human igg1 and at least one heterologous antigen
CN118076646A (zh) 一种可诱导广谱中和活性重组多组分新冠病毒三聚体蛋白疫苗的制备及应用
US20240115674A1 (en) Vaccine and methods for preventing filariasis and dirofilariasis
US20220401546A1 (en) HIV Immunogens, Vaccines, and Methods Related Thereto
KR20250143785A (ko) 수두-대상포진 바이러스의 ge 및 gi 융합 단백질 및 이의 용도
WO2024153235A1 (fr) Protéine de fusion de ge et gi du virus varicelle-zona et utilisation
CN119264267A (zh) 稳定的融合前rsv f蛋白及其应用
CA2886189A1 (fr) Polypeptides et leur utilisation dans le traitement de l'infection a metaneumovirus (mpv)
KR20240168483A (ko) 리노바이러스 백신
BR112016007868B1 (pt) Nanopartícula, seu uso, proteína de fusão, molécula de ácido nucleico, vacina contra o vírus do epstein-barr e método para a sua produção
BR122024015865A2 (pt) Nanopartícula, seu uso, proteína de fusão, molécula de ácido nucleico, vacina contra o vírus do epstein-barr e método para a sua produção

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24708108

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE