Engineered orthopoxvirus proteins and related methods

Abstract

Disclosed herein are engineered Orthopoxvirus proteins and polynucleotides encoding the same. Also disclosed are vaccine compositions comprising any of the engineered Orthopoxvirus proteins and polynucleotides encoding the same. Also disclosed are methods of preventing an Orthopoxvirus infection or a disease associated with an Orthopoxvirus infection in a subject comprising administering to the subject an effect amount of any one of said engineered Orthopoxvirus proteins or polynucleotides encoding the same.

Description

ENGINEERED ORTHOPOXVIRUS PROTEINS AND RELATED METHODSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority and benefits of U.S. Provisional Patent Application No. 63 / 639,520, filed April 26, 2024, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (VCCN_008_01WO_SeqList_ST26.xml; Size: 103,025 bytes; and Date of Creation: April 25, 2025) are herein incorporated by reference in their entirety.BACKGROUND

[0003] Orthopoxviruses are a large genus of viruses belonging to the family Poxviridae.Members of the Orthopoxvirus genus include monkeypox (“MPXV”), variola virus (“VARV”) which causes smallpox, cowpox virus (“CPXV”) and vaccinia virus (“VACV”), a component of the modern smallpox vaccine. Members of the Orthopoxvirus genus comprise large complex, enveloped, deoxyribonucleic acid (DNA) viruses that can cause fever, head and body aches and vomiting in infected individuals, and may result in permanent lesions. Orthopoxviruses are highly contagious and can be transmitted from human to human with worldwide outbreaks documented throughout human history. There is a public health need for rapid development of durable vaccine interventions to address Orthopoxviruses infections and prevention of the same. Disclosed herein are compositions comprising engineered Orthopoxvirus proteins, fusion proteins, and methods and uses that address the same.SUMMARY

[0004] Disclosed herein are engineered Orthopoxvirus proteins and related fusion proteins, ferritin nanoparticles, and eVLPs, and polynucleotides encoding the same, and related compositions.

[0005] In one aspect, disclosed herein are compositions comprising at least two engineered Orthopoxvirus proteins, wherein the at least two engineered Orthopoxvirus proteins comprises: at least one intracellular mature viral (IMV) surface membrane protein; andat least one extracellular enveloped virus (EEV) envelope glycoprotein.

[0006] In some embodiments of the compositions of the disclosure, the IMV surface membrane protein comprises at least one modification relative to a wild type IMV surface membrane protein and the IMV surface membrane protein is selected from the group consisting of: LI and Ml .

[0007] In some embodiments of the compositions of the disclosure, the EEV envelope glycoprotein comprises at least one modification relative to a wild type EEV envelope glycoprotein and the EEV envelope glycoprotein is selected from the group consisting of: A33, A35, A36, B5, B6, and B7.

[0008] In some embodiments of the compositions of the disclosure, the composition comprises one or more linkers.

[0009] In some embodiments of the compositions of the disclosure, the IMV surface membrane protein is selected from the group consisting of LI and Ml; and the IMV surface membrane protein comprises the amino acid sequence of any one of SEQ ID NOs: 6-7, 14, 21, 31-32, 41-42, or 48-50, or an amino acid sequence having at least 70% sequence identity thereto.

[0010] In some embodiments of the compositions of the disclosure, the EEV envelope glycoprotein is selected from the group consisting of B5, B6, and B7; and the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 5, 11, 13, 15, 16, 20, 22, or an amino acid sequence having at least 70% sequence identity thereto.

[0011] In some embodiments of the compositions of the disclosure, the EEV envelope glycoprotein is selected from the group consisting of A33, A35, and A36; and the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, 8-10, 12, 45, or 51-52, or an amino acid sequence having at least about 70% sequence identity thereto.

[0012] In some embodiments of the compositions of the disclosure, the EEV envelope glycoprotein forms a dimer. In certain embodiments, the dimer is a single chain dimer.

[0013] In some embodiments of the compositions of the disclosure, the at least one modification comprises a substitution, a deletion, or an insertion.

[0014] In some embodiments of the compositions of the disclosure, the at least one modification comprises a substitution or deletion, wherein the substitution or deletion removes one or more N-linked glycosylation sites. In some embodiments, the EEV envelope glycoprotein is B6, and the at least one modification is a substitution. In certain embodiments, the substitution is in the B6 glycoprotein at a position corresponding to position 139 relative to SEQ ID NO: 39.In specific embodiments, the substitution comprises substitution of cysteine (C) for isoleucine (I) at position 139 relative to SEQ ID NO: 39.

[0015] In some embodiments of the compositions of the disclosure, the composition comprises at least three engineered Orthopoxvirus proteins. In some embodiments, the at least three engineered Orthopoxvirus proteins comprises: at least one intracellular mature viral (IMV) surface membrane protein; and at least two extracellular enveloped virus (EEV) envelope glycoprotein.

[0016] In some embodiments, the IMV surface membrane protein comprises at least one modification relative to a wild type IMV surface membrane protein; and wherein the IMV surface membrane protein is selected from the group consisting of: LI and Ml.

[0017] In some embodiments, the EEV envelope glycoprotein comprises at least one modification relative to a wild type EEV envelope glycoprotein; and wherein the EEV envelope glycoprotein is selected from the group consisting of: A33, A35, A36, B5, B6, and B7. In certain embodiments, the engineered Orthopoxvirus proteins comprise A35, B6, and Ml. In certain embodiments, the engineered Orthopoxvirus proteins comprise A33, B5, and LI.

[0018] In some embodiments of the compositions of the disclosure, the composition comprises a ferritin nanoparticle.

[0019] In some embodiments of the compositions of the disclosure, at least one engineered Orthopoxvirus protein is fused to a binding partner. In certain embodiments, the binding partner is a ferritin subunit. In certain embodiments, the ferritin subunit comprises a H. Pylori ferritin or a H. Pylori hybrid bullfrog ferritin. In specific embodiments, the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 18, or an amino acid sequence having at least 70% sequence identity thereto. In some embodiments, the ferritin subunit is fused to the engineered Orthopoxvirus proteins by a linker. In certain embodiments, the amino acid sequence of the linker comprises the sequence of SEQ ID NO: 19 or a sequence having at least 70% sequence identity thereto.

[0020] In another aspect, provided herein are fusion proteins comprising: a first Orthopoxvirus protein comprising an extracellular enveloped virus (EEV) envelope glycoprotein; a second Orthopoxvirus protein comprising an intracellular mature viral (IMV) surface membrane protein; anda third Orthopoxvirus protein comprising an extracellular enveloped virus (EEV) envelope glycoprotein.

[0021] In some embodiments of the fusion proteins of the disclosure, the fusion protein comprises one or more linkers.

[0022] In some embodiments of the fusion proteins of the disclosure, the first Orthopoxvirus protein comprises an extracellular enveloped virus (EEV) envelope glycoprotein selected from the group consisting of: B5, B6, and B7; the second Orthopoxvirus protein comprises an intracellular mature viral (IMV) surface membrane protein selected from the group consisting of: Ml and LI; and the third Orthopoxvirus protein comprises an extracellular enveloped virus (EEV) envelope glycoprotein selected from the group consisting of: A33, A35, and A36.

[0023] In some embodiments of the fusion proteins of the disclosure, the third Orthopoxvirus protein comprises a first protein selected from the group consisting of A33, A35, and A36, wherein the first protein is linked to a second protein selected from the group consisting of A33, A35, and A36; and wherein said third Orthopoxvirus protein forms a single chain dimer.

[0024] In some embodiments of the fusion proteins of the disclosure, the first Orthopoxvirus protein comprises B5; the second Orthopoxvirus protein comprises LI; and the third Orthopoxvirus protein comprises A33. In certain embodiments, the first Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 13, 20, or 22 or an amino acid sequence having at least 70% sequence identity thereto; the second Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 14, 21, 48, or 49 or an amino acid sequence having at least 70% sequence identity thereto; and the third Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 12, 51, or 52 or an amino acid sequence having at least 70% sequence identity thereto.

[0025] In some embodiments of the fusion proteins of the disclosure, the first Orthopoxvirus protein comprises B6; the second Orthopoxvirus protein comprises Ml; and the third Orthopoxvirus protein comprises A35. In certain embodiments, the first Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 5 or 15 or an amino acid sequence having at least 70% sequence identity thereto; the second Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 6, 7, or 50 or an amino acid sequence having at least 70% sequence identity thereto; and the third Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, 51, or 52 or an amino acid sequence having at least 70% sequence identity thereto.

[0026] In some embodiments of the fusion proteins of the disclosure, the fusion protein comprises, from N-terminal to C-terminal, an EEV envelope glycoprotein, an IMV surface membrane protein, and an EEV envelope glycoprotein. In certain embodiments, the fusion protein comprises, from N-terminal to C-terminal: A35, Ml, and B6; B6, Ml, and A35; or B5, LI, and A33.

[0027] In some embodiments of the fusion proteins of the disclosure, the fusion protein comprises, from N-terminal to C-terminal, an IMV surface membrane protein, an EEV envelope glycoprotein, and an EEV envelope glycoprotein. In certain embodiments, the fusion protein comprises, from N-terminal to C-terminal: Ml, A35, and B6; or Ml, B6, and A35.

[0028] The fusion protein of any one claim of claims 28-35, wherein the fusion protein comprises, from N-terminal to C-terminal, an EEV envelope glycoprotein, an EEV envelope glycoprotein, and an IMV surface membrane protein.

[0029] In some embodiments of the fusion proteins of the disclosure, the fusion protein comprises, from N-terminal to C-terminal: A35, B6, and Ml; or B6, A35, and Ml.

[0030] In some embodiments of the fusion proteins of the disclosure, the EEV envelope glycoprotein comprises a single chain dimer.

[0031] In some embodiments of the fusion proteins of the disclosure, each of the Orthopoxvirus proteins comprises at least one modification relative to a wild type Orthopoxvirus protein.

[0032] In some embodiments of the fusion proteins of the disclosure, the fusion protein comprises one or more ferritin subunits. In certain embodiments, the one or more ferritin subunits are selected from the group consisting of H. Pylori ferritin and H. Pylori hybrid bullfrog ferritin.

[0033] In some embodiments of the fusion proteins of the disclosure, the fusion protein comprises the amino acid sequence of SEQ ID NO: 46, 47, 66-70, 75, or 76 or an amino acid sequence having at least 70% sequence identity thereto.

[0034] In some embodiments of the fusion proteins of the disclosure, the fusion protein comprises an ESCRT-recruiting domain (ERD). In certain embodiments, the ERD comprises or is derived from an ESCRT and ALIX binding region (EABR). In certain embodiments, the ERD comprises the amino acid sequence of SEQ ID NO: 77 or 78, or a sequence having at least 70% sequence identity thereto.

[0035] In some embodiments of the fusion proteins of the disclosure, the fusion protein comprises the amino acid sequence of SEQ ID NO: 73 or 74 or an amino acid sequence having at least 70% sequence identity thereto.

[0036] In some embodiments of the fusion proteins of the disclosure, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 23-26, 38, 43-44, 46, 47, or 66- 76 or an amino acid sequence having at least 70% sequence identity thereto.

[0037] In another aspect, provided herein are engineered Orthopoxvirus protein comprising an engineered IMV surface membrane protein, where the engineered IMV surface membrane protein comprises at least one modification relative to a wild type IMV surface membrane protein. In certain embodiments, the IMV surface membrane protein is selected from the group consisting of: LI and Ml. In specific embodiments, the engineered IMV surface membrane protein comprises the amino acid sequence of any one of SEQ ID NOs: 6-7, 14, 21, 31-32, 41- 42, or 48-50, or an amino acid sequence having at least 70% sequence identity thereto

[0038] Also provided herein are engineered Orthopoxvirus proteins comprising an engineered EEV envelope glycoprotein, wherein the engineered EEV envelope glycoprotein comprises at least one modification relative to a wild type EEV envelope glycoprotein. In some embodiments, the EEV envelope glycoprotein is selected from the group consisting of: A33, A35, A36, B5, B6, and B7. In certain embodiments, the EEV envelope glycoprotein is selected from the group consisting of B5, B6, and B7; and the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 5, 11, 13, 15, 16, 20, 22, or an amino acid sequence having at least 70% sequence identity thereto. In certain embodiments, the EEV envelope glycoprotein is selected from the group consisting of A33, A35, and A36; and the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, 8-10, 12, 45, or 51-52, or an amino acid sequence having at least about 70% sequence identity thereto.

[0039] In some embodiments of the engineered Orthopoxvirus protein of the disclosure, the engineered Orthopoxvirus protein comprises a B6 glycoprotein, and the at least one modification is a substitution. In some embodiments, the substitution is in the B6 glycoprotein at a position corresponding to position 139 relative to SEQ ID NO: 39. In certain embodiments, the substitution comprises substitution of cysteine (C) for isoleucine (I) at position 139 relative to SEQ ID NO: 39.

[0040] In some embodiments of the engineered Orthopoxvirus protein of the disclosure, the engineered Orthopoxvirus protein comprises two EEV envelope glycoproteins, and the two EEVenvelope glycoproteins form a dimer. In certain embodiments, the two EEV envelope glycoproteins are the same. In specific embodiments, the two EEV envelope glycoproteins are A33. In certain embodiments, the two EEV envelope glycoproteins are not the same. In specific embodiments, the two EEV envelope glycoproteins comprise: A33 and A35; A33 and A36; or A35 and A36. In some embodiments, the dimer is a single chain dimer.

[0041] In some embodiments of the engineered Orthopoxvirus protein of the disclosure, the engineered Orthopoxvirus protein is fused to a binding partner. In certain embodiments, the binding partner is a ferritin subunit. In certain embodiments, the ferritin subunit comprises a H. Pylori ferritin or a H. Pylori hybrid bullfrog ferritin. In specific embodiments, the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 1, 18, or 82, or an amino acid sequence having at least 70% sequence identity thereto. In some embodiments, the ferritin subunit is fused to the engineered Orthopoxvirus protein by a linker.

[0042] In certain embodiments, the linker is a flexible linker. In certain embodiments, the linker is a rigid linker. In specific embodiments, the amino acid sequence of the linker comprises the sequence of SEQ ID NO: 19 or a sequence having at least 70% sequence identity thereto.

[0043] In another aspect, provided herein are ferritin nanoparticles comprising a composition of the disclosure, a fusion protein of the disclosure, or an engineered Orthopoxvirus protein of the disclosure.

[0044] In another aspect, provided herein are eVLPs comprising a fusion protein of the disclosure or an engineered Orthopoxvirus protein of the disclosure.

[0045] In another aspect, provided herein are polynucleotides encoding the amino acid sequence of: an engineered Orthopoxvirus protein of a composition of the disclosure, a fusion protein disclosure; or an engineered Orthopoxvirus protein disclosure. In certain embodiments, the polynucleotide comprises DNA. In certain embodiments, the polynucleotide comprises RNA. In specific embodiments, the RNA is mRNA.

[0046] In another aspect, provided herein are vaccine compositions comprising one or more of: a composition of the disclosure; a fusion protein of the disclosure; an engineered Orthopoxvirus protein of the disclosure; a ferritin nanoparticle of the disclosure; an eVLP of the disclosure; or a polynucleotide of the disclosure; and one or more adjuvants.

[0047] In some embodiments of the vaccine compositions of the disclosure, the one or more adjuvants comprises alum and / or CpG.

[0048] In another aspect, provided herein are pharmaceutical compositions comprising: a composition of the disclosure; a fusion protein of the disclosure; an engineered Orthopoxvirus protein of the disclosure; a ferritin nanoparticle of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; or a vaccine composition of the disclosure.

[0049] In another aspect, provided herein are methods of preventing an Orthopoxvirus infection or a disease or symptom associated with an Orthopoxvirus infection in a subject, comprising administering to the subject an effective amount of: a composition of the disclosure; a fusion protein of the disclosure; an engineered Orthopoxvirus protein of the disclosure; a ferritin nanoparticle of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; a vaccine composition of the disclosure, or a pharmaceutical composition of the disclosure.

[0050] In another aspect, provided herein are methods of vaccinating a subject against an Orthopoxvirus infection or a disease associated with an Orthopoxvirus infection, comprising administering to the subject an effective amount of: a composition of the disclosure; a fusion protein of the disclosure; an engineered Orthopoxvirus protein of the disclosure; a ferritin nanoparticle of the disclosure; an eVLP of the disclosure; a polynucleotide of the disclosure; a vaccine composition of the disclosure, or a pharmaceutical composition of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1A depicts a schematic of a model of a cross section of an Orthopoxvirus.

[0052] FIG. IB depicts a schematic of a model of a cross section of an Extracellular Enveloped Virus (EEV) particle and an Intracellular Mature Virus (IMV) particle.

[0053] FIGs. 2A-2B depict a schematic of various exemplary engineered Orthopox protein antigens of the disclosure.

[0054] FIG. 3A depicts an SDS-PAGE gel of expression of various exemplary engineered antigens of the disclosure in either monomer form or coupled to an H. Pylori ferritin nanoparticle subunit. FIG. 3B depicts an SDS-PAGE gel of expression of various antigens of the disclosure in either monomer form or coupled to an H. Pylori ferritin subunit.

[0055] FIGs. 4A-4B depict representative images of transmission electron microscopy of the exemplary antigens of the disclosure coupled to an H. Pylori ferritin subunit.

[0056] FIGs. 4C-4D depict representative images of transmission electron microscopy of the exemplary antigens of the disclosure coupled to an H. Pylori ferritin subunit.

[0057] FIG. 5A depicts a graph of intracellular mature virus neutralization titers. Neutralization was performed with mouse serum collected at study day 35 (following a prime dose at day 0 and a boost dose at day 21).

[0058] FIG. 5B. depicts a representative image of a comet spread assay for exemplary engineered Vaccinia virus proteins of the disclosure.

[0059] FIGs. 6A-6C depict graphs of Biolayer Interferometry (BLI) of engineered Monkey pox (MPXV) proteins of the disclosure, demonstrating binding of neutralizing antibodies to the engineered MPXV proteins of the disclosure.

[0060] FIGs. 7A-7B depict graphs of BLI of binding of A35 targeting antibodies to 2 different engineered Orthopoxvirus fusion proteins comprising 2 EEV envelope glycoproteins (e.g., A35, and B6) and 1 IMV surface membrane protein (e.g., Ml), as a 3-in-l fusion protein.

[0061] FIGs. 7C-7D depict graphs of BLI of binding of B6 targeting antibodies to 2 different engineered Orthopoxvirus fusion proteins comprising 2 EEV envelope glycoproteins (e.g., A35, and B6) and 1 IMV surface membrane protein (e.g., Ml), as a 3-in-l fusion protein.

[0062] FIGs. 7E-7H depict graphs of BLI of binding of Ml targeting antibodies to 2 different engineered Orthopoxvirus fusion proteins comprising 2 EEV envelope glycoproteins (e.g., A35 and B6) and 1 IMV surface membrane protein (e.g., Ml), as a 3-in-l fusion protein.

[0063] FIG. 8 depicts a schematic of a vaccinia virus (VACV) challenge study in mice and a carton depiction of the components of the vaccine compositions administered to the mice.

[0064] FIG. 9A depicts a schematic of the protein antigens with which mice were immunized in the study outlined in FIG. 8. FIG. 9B depicts graphs of mouse serum binding titers to EEV envelope glycoproteins A35 and B6 and IMV surface membrane protein Ml from mice administered the indicated vaccine compositions or controls. FIG. 9C depicts a graph of VACV neutralization by mouse serum collected from mice administered the indicated vaccine compositions or controls. FIG. 9D depicts a graph of body weights of mice administered the indicated vaccine compositions or controls.

[0065] FIG. 10A depicts a schematic of a VACV challenge study in mice. FIG. 10B depicts a graph of VACV neutralization by mouse serum collected from mice administered the indicated vaccine compositions or controls. FIG. 10C depicts a graph of VACV neutralization by mouse serum collected from mice administered the indicated vaccine compositions or controls. FIG.10D depicts a graph of body weights of mice administered the indicated vaccine compositions or controls.

[0066] FIG. 11 depict graphs of BLI assessment of the binding of A35, B6, and Ml targeting antibodies to an A35 monomer, a B6 monomer, a Ml monomer, and a fusion protein comprising A35, B6, Ml, and a ferritin (ABM 12L Fer).

[0067] FIG. 12A depicts a schematic of four fusion protein constructs. FIG. 12B depicts a dot blot showing binding of A35, B6, and Ml targeting antibodies to cell supernatant from cells transfected with the indicated fusion protein constructs.

[0068] FIG. 13A depicts a schematic of two fusion protein constructs. FIG. 13B depicts flow cytometry histograms showing the binding of A35, B6, and Ml targeting antibodies to cells transfected with the indicated fusion protein construct.

[0069] FIG. 14 depicts a dot blot showing the binding of A35, B6, and Ml targeting antibodies to cell lysate from cells transfected with the indicated constructs.

[0070] FIG. 15 depicts a dot blot showing the binding of A35, B6, and Ml targeting antibodies to supernatant from cells transfected with constructs expressing A35, B6, and M in different N-terminal to C-terminal orders.

[0071] FIG. 16A depicts a schematic of a VACV challenge study in mice. FIG. 16B depicts a graph of VACV neutralization by mouse serum collected from mice administered the indicated vaccine compositions or controls. FIG. 16C depicts a graph of body weights of mice administered the indicated protein vaccine compositions or controls. FIG. 16D depicts a graph of body weights of mice administered the indicated mRNA vaccine compositions or controls.DETAILED DESCRIPTIONTerms and Concepts

[0072] A number of terms and concepts are discussed below. They are intended to facilitate the understanding of various embodiments of the invention in conjunction with the rest of the present document and the accompanying figures. These terms and concepts may be further clarified and understood based on the accepted conventions in the fields of the present invention, as well as the description provided throughout the present document and / or the accompanying figures. Some other terms can be explicitly or implicitly defined in other sections of this document and in the accompanying figures and may be used and understood based on the accepted conventions in the fields of the present invention, the description provided throughout the present document and / or the accompanying figures. The terms not explicitly defined can also be defined and understood based on the accepted conventions in the fields of the presentinvention and interpreted in the context of the present document and / or the accompanying figures.

[0073] Unless otherwise dictated by context, singular terms shall include pluralities, and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry are those well-known and commonly used. Known methods and techniques are generally performed according to conventional methods well-known and as described in various general and more specific references, unless otherwise indicated. The nomenclatures used in connection with the laboratory procedures and techniques described in the present disclosure are those well-known and commonly used.

[0074] As used herein, the terms "a", "an", and "the" can refer to one or more unless specifically noted otherwise. The use of the term "or" is used to mean "and / or," unless explicitly indicated to refer to alternatives only, or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." As used herein "another" can mean at least a second or more.

[0075] The terms "about" and "approximately" as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); e.g., within 10%; or e.g., within 5% of a given value or range of values. Any reference to "about X" or "approximately X" specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.0IX, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions "about X" or "approximately X" are intended to teach and provide written support for a claim limitation of, for example, "0.98X." Alternatively, in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, within 5- fold, e.g. within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated. When "about" is applied to the beginning of a numerical range, it applies to both ends of the range.

[0076] The terms "protein," "peptide," and "polypeptide" are used interchangeably to refer to a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers and non-natural amino acid polymers, as well as to amino acid polymers in which one (or more) amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, or a non-naturally occurring amino acid. The terms encompass amino acid chains ofany length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0077] The terms "sequence identity," and the related terms and expressions used in the context of describing nucleic acid or amino acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of nucleic acid or amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence (subject sequence) to which test sequences (query sequence) are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well-known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection. Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.

[0078] Depending on the algorithm, the calculated percent sequence identity may differ. For example, there are at least three ways in which to calculate a percent sequence identity.% Query sequence identity: = (Number of alignment identities) / (Length of Query sequence);% Subject sequence identity: = (Number of alignment identities) / (Length of Subject sequence);%Alignment sequence identity = (Number of alignment identities) / (Length of Alignment)

[0079] Accordingly, when the term “sequence identity” is used herein, it can include any of the above non-limiting methodologies provided above to calculate.

[0080] The expression "neutralizing antibody" can refer to an antibody capable of reducing the ability of an infectious agent, such as a virus, from infecting a cell by neutralizing or inhibiting one or more parts of the life cycle of the infectious agent. In the context of the present disclosure, in some embodiments, neutralizing antibodies can prevent an Orthopoxvirus, such as, but not limited to, monkeypox virus (MPXV), vaccinia virus (VACV), and variola virus (VARV), from completing its life cycle in a host cell. The life cycle of an Orthopoxvirus starts with attachment of the virus to a host cell and ends with budding of newly formed virus from the host cell. This life cycle includes, but is not limited to, the steps of attaching to a cell, entering a cell, fusion of the viral membrane with the host cell membrane, release of viral contents, including DNA, mRNA, proteins into the cytoplasm, formation of new viral particles, and budding of viral particles from the host cell membrane. Without being held to theory or mechanism, Orthopoxviruses exist in two main forms outside of the cell, the intracellular mature viral (IMV) form the extracellular enveloped virus (EEV) form. The EEV form has an additional membrane separating the viral contents from the extracellular environment, with a variety of different surface proteins compared to the IMV form. While both forms remain functionally infectious, they have different functional roles during the viral life cycle.

[0081] As used herein, designations for EEV envelope glycoproteins (such as A33, A35, A36, B5, B6, or B7) are meant to convey both naturally occurring (e.g. wild type) and modified versions of the protein (modified versions may also be referred to herein as variants or engineered).

[0082] As used herein, designations for IMV surface membrane proteins (such as Ml and LI) are meant to convey both naturally occurring (e.g. wild type) and modified versions of the protein (modified versions may also be referred to herein as variants or engineered).Overview

[0083] Provided herein are engineered extracellular enveloped virus (EEV) envelope proteins, engineered intracellular mature viral (IMV) surface membrane proteins, fusion proteins comprising an EEV envelope glycoprotein and / or an IMV surface membrane protein, ferritin nanoparticles expressing EEV envelope glycoproteins and / or IMV surface membrane proteins, and eVLPs expressing EEV envelope glycoproteins and / or IMV surface membrane proteins. In some embodiments, said engineered EEV envelope glycoproteins, engineered IMV surfacemembrane proteins, fusion proteins, ferritin nanoparticles, and eVLPs are used for protein-based vaccines. In some embodiments, said engineered EEV envelope glycoproteins, engineered IMV surface membrane proteins, fusion proteins, ferritin nanoparticles, and eVLPs are used for nucleic acid-based vaccines.

[0084] Members of the Orthopoxvirus genus are large complex viruses that generally comprise a core wall surrounding a core comprising a nucleocapsid that comprises the DNA genome that encodes viral enzymes necessary for viral infection. Orthopoxviruses may also comprise lateral bodies, an inner membrane, and an outer membrane, as seen in FIG. 1A.

[0085] Without being held by theory or mechanism, members of the Orthopoxvirus genus can generate at least two types of infectious viral particles, IMV and EEV, as shown in FIG. IB.

[0086] The IMV particle comprises a core comprising a core wall that contains the nucleocapsid and genomic DNA therein. The IMV comprises lateral bodies which are encapsulated by a membrane comprising a plurality of IMV surface membrane proteins. IMV surface membrane proteins may include proteins such as Ml (from Variola “VARV”) and LI (from Vaccinia “VACV”). These IMV cell surface membrane proteins may bind to glycosaminoglycans on cells to induce IMV entry into cells. These IMV cell surface membrane proteins may be expressed on the cell surface as monomers.

[0087] The EEV particle comprises structures similar to that of the IMV with an additional lipid envelope that surrounds the IMV, including the IMV cell surface membrane proteins. The additional lipid envelope includes a plurality of external proteins thereon, including glycoproteins (also referred to as EEV envelope glycoproteins). The EEV envelope glycoproteins may mediate viral dissemination and induction of key components from EEV particles into cells. Examples of EEV envelope glycoproteins include, but are not limited to, proteins such as VARV A36 (“A36”), VACV A33 (“A33”), monkeypox A35 (“A35”), VARV B7 (“B7”), VACV B5 (“B5”), monkeypox B6 (“B6”). Some EEV envelope glycoproteins may be expressed on the EEV envelope surface as monomers (e.g., B7, B6, B5), may dimerize with adjacent EEV envelope glycoproteins, and / or be expressed as dimers (e.g., A33, A35, or A37).Engineered Orthopoxvirus Proteins

[0088] Provided herein are engineered Extracellular Enveloped Virus (EEV) envelope glycoproteins comprising at least one modification relative to a wild type (e.g. naturally occurring, or native) EEV envelope glycoprotein and engineered Intracellular Mature Virus (IMV) surface membrane proteins comprising at least one modification relative to a wild type(e.g. naturally occurring, or native) IMV surface membrane protein. These engineered Orthopoxvirus proteins, including engineered EEV envelope glycoproteins and / or engineered IMV surface membrane proteins, can be used in protein-based vaccines or nucleic acid-based vaccines (also referred to herein as polynucleotide-based vaccines).

[0089] In some embodiments, the engineered Orthopoxvirus proteins described herein comprise an amino acid sequence of one or more EEV envelope glycoproteins. In some embodiments, the engineered Orthopoxvirus proteins described herein comprise an amino acid sequence of a monomer of one or more EEV envelope glycoproteins. In some embodiments, the engineered Orthopoxvirus proteins described herein comprise an amino acid sequence of a dimer of one or more EEV envelope glycoproteins.

[0090] In some embodiments, the engineered Orthopoxvirus proteins described herein comprise an amino acid sequence of one or more IMV surface membrane proteins. In some embodiments, the engineered Orthopoxvirus proteins described herein comprise an amino acid sequence of a monomer of one or more IMV surface membrane proteins. In some embodiments, the engineered Orthopoxvirus proteins described herein comprise an amino acid sequence of an EEV envelope glycoprotein and an amino acid sequence of an IMV surface membrane protein. In some embodiments, the engineered Orthopoxvirus proteins described herein or polynucleotides encoding said engineered Orthopoxvirus proteins are used for vaccinating a subject against an infection by an Orthopoxvirus.

[0091] In some embodiments, an engineered IMV surface membrane protein described herein comprises a VACV LI protein, referred to herein as “LI In certain embodiments, the VACV LI protein is a naturally occurring (e.g., wild type, or native) IMV LI surface membrane protein. An exemplary amino acid sequence of a wild type IMV LI surface membrane protein is provided below as SEQ ID NO: 14.

[0092] MGAAASIQTTVNTLSERIS SKLEQEANAS AQTKCDIEIGNF YIRQNHGCNL AVK NMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENY VKQTCNS S AVVDNKLKIQNVIIDEC YGAPGSPTNLEFINTGS SKGNC AIKALMQLTTKAT TQIAPRQVAGTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLILANKENVHWTT YMDTFFRTSPMVIATTDMQN (SEQ ID NO: 14).

[0093] In some embodiments, an engineered IMV surface membrane protein is a modified (engineered version) of SEQ ID NO: 14.

[0094] In some embodiments, an engineered IMV surface membrane protein described herein comprises the sequence of SEQ ID NO: 14 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an engineered IMV surface membrane protein described herein comprises the sequence of SEQ ID NO: 14 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0095] In some embodiments, an engineered IMV surface membrane protein described herein comprises a MPXV Ml protein, referred to herein as “Ml.” In certain embodiments, the MPXV Ml protein is a naturally occurring (e.g., wild type, or native) IMV Ml surface membrane protein. An exemplary amino acid sequence of a wild type IMV Ml surface membrane protein is provided below as SEQ ID NO: 6.

[0096] MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVK NMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENY VKQTCNS S AVVDNKLKIQNVIIDEC YGAPGSPTNLEFINTGS SKGNC AIKALMQLTTKAT TQIAPRQVAGTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLILANKENVHWTT YMDTFFRTSPMIIATTDIQN (SEQ ID NO: 6)

[0097] In some embodiments, an engineered IMV surface membrane protein is a modified (engineered version) of SEQ ID NO: 6.

[0098] In some embodiments, an engineered IMV surface membrane protein described herein comprises the sequence of SEQ ID NO: 6 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an engineered IMV surface membrane protein described herein comprises the sequence of SEQ ID NO: 6 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto

[0099] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises a VACV B5 protein, referred to herein as “B5.” In certain embodiments, the VACV B5 protein is a naturally occurring (e.g., wild type, or native) EEV B5 envelope glycoprotein. Anexemplary amino acid sequence of a wild type EEV B5 envelope glycoprotein is provided below as SEQ ID NO: 13.

[0100] MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNNNQKVTFTCDQGYH SSDPNAVCETDKWKYENPCKKMCTVSDYISELYNKPLYEVNSTMTLSCNGETKYFRCE EKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGASYIS CTANSWNVIPSCQQKCDMPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWN PILPTCVRSNEKFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIVALTIMGVIFL ISVIVLVCSCDKNNDQYKFHKLLP (SEQ ID NO: 13)

[0101] In some embodiments, an engineered EEV envelope glycoprotein is a modified (engineered version) of SEQ ID NO: 13.

[0102] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 13 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 13 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0103] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises a monkeypox virus (MPXV) B6 protein, referred to herein as “B6.” In certain embodiments, the MPXV B6 protein is a naturally occurring (e.g., wild type, or native) EEV B6 envelope glycoprotein. An exemplary amino acid sequence of a wild type EEV B6 envelope glycoprotein is provided below as SEQ ID NO: 5.

[0104] MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYH SLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRC EEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYI SCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWN PILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIMALTIMGVIFL ISIIVLVCSCDKNNDQYKFHKLLP (SEQ ID NO: 5)

[0105] In some embodiments, an engineered EEV envelope glycoprotein is a modified (engineered version) of SEQ ID NO: 5.

[0106] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 5 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 5 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0107] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises a VACV B7 protein, referred to herein as “B7.” In certain embodiments, the VACV B7 protein is a naturally occurring (e.g., wild type, or native) EEV B7 envelope glycoprotein. An exemplary amino acid sequence of a wild type EEV B7 envelope glycoprotein is provided below as SEQ ID NO: 11.

[0108] TCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYYSLDPNAVCETDKWKYENP CKKMCTVSDYVSELYNKPLYEVNAIITLICKDETKYFRCEEKNGNTSWNDTVTCPNAEC QSLQLDHGSCQPVKEKYSFGEHITINCDVGYEVIGASYITCTANSWNVIPSCQQKCDIPSL SNGLISGSTFSIGGVIHLSCKSGFILTGSPSSTCIDGKWNPVLPICIRSNEEFDPVEDGPDDE TDLSKLSKDVVQYEQEIESLE (SEQ ID NO: 11)

[0109] In some embodiments, an engineered EEV envelope glycoprotein is a modified (engineered version) of SEQ ID NO: 11.

[0110] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 11 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 11 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.[oni] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises a VACV A33 protein, referred to herein as “A33.” In certain embodiments, the VACV A33 protein is a naturally occurring (e.g., wild type, or native) EEV A33 envelopeglycoprotein. An exemplary amino acid sequence of a wild type EEV A33 envelope glycoprotein is provided below as SEQ ID NO: 53.

[0112] MMTPENDEEQTSVFS ATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAF LIVRLNQCMSANEAAITDAAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSCYIL HSDYQLFSDAKANCTAESSTLPNKSDVLITWLIDYVEDTWGSDGNPITKTTSDYQDSDV SQEVRKYFCVKTMN (SEQ ID NO: 53)

[0113] In some embodiments, an engineered EEV envelope glycoprotein is a modified (engineered version) of SEQ ID NO: 53.

[0114] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 53 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 53 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0115] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises a VACV A35 protein, referred to herein as “A35.” In certain embodiments, the VACV A35 protein is a naturally occurring (e.g., wild type, or native) EEV A35 envelope glycoprotein. An exemplary amino acid sequence of a wild type EEV A35 envelope glycoprotein is provided below as SEQ ID NO: 54.

[0116] MDAAFVITPMGVLTITDTLYDDLDISIMDFIGPYIIGNIKTVQIDVRDIKYSDMQ KCYFSYKGKIVPQDSNDLARFNIYSICAAYRSKNTIIIACDYDIMLDIEDKHQPFYLFPSID VFNATIIEAYNLYTAGDYHLIINPSDNLKMKLLFNSSFCISDGNGWIIIDGKCNSNFLS (SEQ ID NO: 54)

[0117] In some embodiments, an engineered EEV envelope glycoprotein is a modified (engineered version) of SEQ ID NO: 54.

[0118] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 54 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity thereto. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 54 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0119] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises a VACV A36 protein, referred to herein as “A36.” In certain embodiments, the VACV A36 protein is a naturally occurring (e.g., wild type, or native) EEV A36 envelope glycoprotein. An exemplary amino acid sequence of a wild type EEV A36 envelope glycoprotein is provided below as SEQ ID NO: 8.

[0120] MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGICIRISMVISLLSMITMSAFL IVRLNQCMSANEAAITDATAVAAALSTHRKVASSTTQYKHQESCNGLYYQGSCYIFHS DYQLFSDAKANCATESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTTDYQDSDVS QEVRKYFCVKTMN (SEQ ID NO: 8)

[0121] In some embodiments, an engineered EEV envelope glycoprotein is a modified (engineered version) of SEQ ID NO: 8.

[0122] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 8 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the sequence of SEQ ID NO: 8 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0123] In some embodiments, the engineered Orthopoxvirus proteins described herein comprises an amino acid sequence of a naturally occurring EEV envelope glycoprotein and / or IMV surface membrane protein. In some embodiments, the engineered Orthopoxvirus proteins described herein comprise an amino acid sequence of an EEV envelope glycoprotein having at least one modification relative to a naturally occurring EEV envelope glycoprotein. In some embodiments, the engineered Orthopoxvirus proteins described herein comprise an amino acid sequence of an IMV surface membrane protein having at least one modification relative to a naturally occurring IMV surface membrane protein. In some embodiments, the engineered Orthopoxvirus proteins described herein comprise the amino acid sequence of any one of SEQ ID NOs: 2-16, 20-22, or 48-49, or a sequence having at least 70%, at least 75%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the engineered Orthopoxvirus proteins described herein comprise the amino acid sequence of any one of SEQ ID NOs: 2-16, 20-22, or 48-49, or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0124] As described above, in some embodiments, an engineered EEV envelope protein or engineered IMV surface membrane protein comprises at least one modification relative to a naturally occurring EEV envelope protein or naturally occurring IMV surface membrane protein, respectively. In certain embodiments, the at least one modification comprises a substitution, a deletion, an insertion, and / or a truncation.

[0125] In some embodiments, an engineered IMV surface membrane protein comprises at least one modification relative to a wild-type IMV surface membrane protein. In some embodiments, the at least one modification comprises a substitution, a deletion, an insertion (an addition), a truncation, or a combination thereof. In certain embodiments, the at least one modification comprises a substitution. In certain embodiments, the at least one modification comprises an insertion (an addition). In certain embodiments, the at least one modification comprises a truncation.

[0126] In some embodiments of the engineered IMV surface membrane proteins of the disclosure, the at least one modification comprises a deletion. In certain embodiments, the deletion comprises deletion of one or more amino acid residues at the C-terminal, N-terminal, and / or a middle portion of an IMV surface membrane protein. The deletion of one or more amino acids may include a deletion of one or more consecutive amino acid residues or one or more non-consecutive amino acid residues. In some embodiments, the deletion comprises deletion of about 2 consecutive amino acids, about 3 consecutive amino acids, about 4 consecutive amino acids, about 5 consecutive amino acids, about 6 consecutive amino acids, about 7 consecutive amino acids, about 8 consecutive amino acids, about 9 consecutive amino acids, about 10 consecutive amino acids, about 15 consecutive amino acids, about 20 consecutive amino acids, about 25 consecutive amino acids, about 30 consecutive amino acids, about 35 consecutive amino acids, about 40 consecutive amino acids, about 45 consecutive amino acids, about 50 consecutive amino acids, about 55 consecutive amino acids, about 60 consecutive amino acids, about 65 consecutive amino acids, about 70 consecutive amino acids,about 75 consecutive amino acids, about 80 consecutive amino acids, about 85 consecutive amino acids, about 90 consecutive amino acids, about 95 consecutive amino acids, or about 100 consecutive amino acids.

[0127] In some embodiments of the engineered IMV surface membrane proteins of the disclosure, the at least one modification comprises a truncation. In some embodiments, the truncation comprises truncation of one or more amino acids at the N-terminal or C-terminal of an IMV surface membrane protein. In certain embodiments, the truncation at the N-terminal or C-terminal of an IMV surface membrane protein comprises a truncation of at least 5 amino acids relative to a wild-type IMV surface membrane protein. In some embodiments, the truncation at the N-terminal or C-terminal of an IMV surface membrane protein comprises a truncation of at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, at least about 45 amino acids, at least about 50 amino acids, at least about 55 amino acids, at least about 60 amino acids, at least about 65 amino acids, at least about 70 amino acids, at least about 75 amino acids, at least about 80 amino acids, at least about 85 amino acids, at least about 90 amino acids, at least about 95 amino acids, at least about 100 amino acids, at least about 105 amino acids, at least about 110 amino acids, at least about 115 amino acids, at least about 120 amino acids, at least about 125 amino acids, at least about 130 amino acids, at least about 135 amino acids, at least about 140 amino acids, at least about 145 amino acids, or at least about 150 amino acids relative to a wild-type IMV surface membrane protein.

[0128] In some embodiments, an engineered EEV envelope glycoprotein comprises at least one modification relative to a wild-type EEV envelope glycoprotein. In some embodiments, the at least one modification comprises a substitution, a deletion, an insertion (an addition), a truncation, or a combination thereof. In certain embodiments, the at least one modification comprises a substitution. In certain embodiments, the at least one modification comprises an insertion (an addition). In certain embodiments, the at least one modification comprises a truncation.

[0129] In some embodiments of the engineered EEV envelope glycoproteins of the disclosure, the at least one modification comprises a deletion. In certain embodiments, the deletion comprises deletion of one or more amino acid residues at the C-terminal, N-terminal, and / or in a middle portion of an EEV envelope glycoprotein. The deletion of one or more amino acids may include a deletion of one or more consecutive amino acid residues or one or more non-consecutive amino acid residues. In some embodiments, the deletion comprises deletion of about 2 consecutive amino acids, about 3 consecutive amino acids, about 4 consecutive amino acids, about 5 consecutive amino acids, about 6 consecutive amino acids, about 7 consecutive amino acids, about 8 consecutive amino acids, about 9 consecutive amino acids, about 10 consecutive amino acids, about 15 consecutive amino acids, about 20 consecutive amino acids, about 25 consecutive amino acids, about 30 consecutive amino acids, about 35 consecutive amino acids, about 40 consecutive amino acids, about 45 consecutive amino acids, about 50 consecutive amino acids, about 55 consecutive amino acids, about 60 consecutive amino acids, about 65 consecutive amino acids, about 70 consecutive amino acids, about 75 consecutive amino acids, about 80 consecutive amino acids, about 85 consecutive amino acids, about 90 consecutive amino acids, about 95 consecutive amino acids, or about 100 consecutive amino acids.

[0130] In some embodiments of the engineered EEV envelope glycoproteins of the disclosure, the at least one modification comprises a truncation. In some embodiments, the truncation comprises truncation of one or more amino acids at the N-terminal or C-terminal of an EEV envelope glycoprotein. In certain embodiments, the truncation at the N-terminal or C-terminal of an EEV envelope glycoprotein comprises a truncation of at least 5 amino acids relative to a wild-type EEV envelope glycoprotein. In some embodiments, the truncation at the N-terminal or C-terminal of an EEV envelope glycoprotein comprises a truncation of at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, at least about 45 amino acids, at least about 50 amino acids, at least about 55 amino acids, at least about 60 amino acids, at least about 65 amino acids, at least about 70 amino acids, at least about 75 amino acids, at least about 80 amino acids, at least about 85 amino acids, at least about 90 amino acids, at least about 95 amino acids, at least about 100 amino acids, at least about 105 amino acids, at least about 110 amino acids, at least about 115 amino acids, at least about 120 amino acids, at least about 125 amino acids, at least about 130 amino acids, at least about 135 amino acids, at least about 140 amino acids, at least about 145 amino acids, or at least about 150 amino acids relative to a wild-type EEV envelope glycoprotein.

[0131] In some embodiments, a N-terminal or C-terminal truncation relative to a wild-type IMV surface membrane protein removes a domain of a portion thereof of the wild-type IMV surface membrane protein. Domains which may be removed include, but are not limited to, atransmembrane (TM) domain and a C-terminal domain. In certain embodiments, a truncation removes a TM domain. In certain embodiments, a truncation removes a C-terminal domain.

[0132] In some embodiments, a N-terminal or C-terminal truncation relative to a wild-type EEV envelope glycoprotein removes a domain of a portion thereof of the wild-type EEV envelope glycoprotein. Domains which may be removed include, but are not limited to, a transmembrane (TM) domain and a C-terminal domain. In certain embodiments, a truncation removes a TM domain. In certain embodiments, a truncation removes a C-terminal domain.

[0133] C-terminal truncations of various lengths may provide an unexpected benefit, such as increased expression, better folding, or increased immunogenicity, compared to full length IMV surface membrane proteins and / or full length EEV envelope glycoproteins. In some embodiments, the engineered IMV surface membrane proteins described herein comprising a C- terminal truncation exhibits increased protein expression, better folding, increased immunogenicity, or a combination thereof relative to a wild type IMV surface membrane protein. In some embodiments, the engineered EEV envelope glycoproteins described herein comprising a C-terminal truncation exhibits increased protein expression, better folding, increased immunogenicity, or a combination thereof relative to a wild type EEV envelope glycoprotein.

[0134] In some embodiments, an engineered IMV surface membrane protein or an EEV envelope glycoprotein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 20, 22, 48, or 49, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 20, 22, 48, or 49. In some embodiments, an engineered IMV surface membrane protein or an EEV envelope glycoprotein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 20, 22, 48, or 49, or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0135] In some embodiments of the engineered IMV surface membrane proteins of the disclosure, the at least one modification comprises a substitution. The substitution may be within any one of the domains of an IMV surface membrane protein. In some embodiments, a substitution leads to increased protein expression, better folding, increased immunogenicity, or a combination thereof, compared to a wild-type IMV surface membrane protein. In someembodiments, the engineered IMV surface membrane proteins described herein comprises a substitution, wherein the substitution comprises a point mutation which removes a N-linked glycosylation site.

[0136] In some embodiments of the engineered EEV envelope glycoproteins of the disclosure, the at least one modification comprises a substitution. The substitution may be within any one of the domains of an EEV envelope glycoprotein. In some embodiments, a substitution leads to increased protein expression, better folding, increased immunogenicity, or a combination thereof, compared to a wild-type EEV envelope glycoprotein. In some embodiments, the engineered EEV envelope glycoproteins described herein comprises a substitution, wherein the substitution comprises a point mutation which removes a N-linked glycosylation site.

[0137] In some embodiments of the engineered EEV envelope glycoproteins of the disclosure, the engineered EEV envelope glycoprotein comprises a B6 envelope protein comprising an amino acid substitution at a position corresponding to position 139 of SEQ ID NO: 39. In certain embodiments, said amino acid substitution comprises substitution of a cysteine at a position corresponding to position 139 of SEQ ID NO: 39 for another amino acid. In certain embodiments, said amino acid substitution comprises substitution of a cysteine for an isoleucine at a position corresponding to position 139 of SEQ ID NO: 39. This substitution of cysteine for isoleucine at position 139 relative to SEQ ID NO: 39 is referred to as C139I herein. An exemplary amino acid sequence comprising a C139I substitution is provided in Table 1 below as SEQ ID NO: 40. In some embodiments, a C139I substitution improves expression of an EEV envelope glycoprotein comprising said substitution compared to a wild-type EEV envelope glycoprotein. In certain embodiments, a C139I substitution in a B6 protein improves expression of the B6 protein comprising said substitution compared to a wild-type B6 protein. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises the amino acid sequence of SEQ ID NO: 40 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0138] In some embodiments, an IMV Ml surface membrane protein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 6, 7, or 50, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an IMV Ml surface membrane protein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 6, 7, or 50, or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0139] In some embodiments, an IMV LI surface membrane protein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 14, 21, 48, or 49, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an IMV LI surface membrane protein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 14, 21, 48, or 49, or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0140] In some embodiments, an EEV A33 envelope glycoprotein described herein comprises or consists of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an EEV A33 envelope glycoprotein described herein comprises or consists of the amino acid sequence of SEQ ID NO: 12 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0141] In some embodiments, an EEV A35 envelope glycoprotein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 2-4 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an EEV A35envelope glycoprotein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 2-4 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0142] In some embodiments, an EEV A36 envelope glycoprotein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8-10 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an EEV A36 envelope glycoprotein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8-10 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0143] In some embodiments, an EEV B5 envelope glycoprotein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 13, 20, or 22, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an EEV B5 envelope glycoprotein described herein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 13, 20, or 22, or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0144] In some embodiments, an EEV B6 envelope glycoprotein described herein comprises or consists of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 15 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an EEV B6 envelope glycoprotein described herein comprises or consists of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 15 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0145] In some embodiments, an EEV B7 envelope glycoprotein described herein comprises or consists of the amino acid sequence of SEQ ID NOs: 11 or SEQ ID NO: 16 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, an EEV B7 envelope glycoprotein described herein comprises or consists of the amino acid sequence of SEQ ID NOs: 11 or SEQ ID NO: 16 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0146] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises dimers of monomers selected from the group consisting of: A33, A35, and A36. In some embodiments, the dimers of monomers selected from the group consisting of A33, A35, and A36 are expressed as a single polypeptide chain, wherein the monomers are joined together by a linker. The dimers may be either heterodimers or homodimers of A33, A35, and A36. In some embodiments, the dimers are heterodimers of A33, A35, and / or A36. In some embodiments, the dimers are homodimers of A33, A35, or A36. In some embodiments, the dimers are joined by a linker.

[0147] In some embodiments, an engineered EEV envelope glycoprotein described herein comprises an A33 monomer joined to an A33 monomer by a linker, thereby resulting in a homodimer of A33. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises an A33 monomer joined to an A35 monomer by a linker, thereby resulting in a heterodimer of A33 and A35. In some embodiments, an engineered EEV envelope glycoprotein described herein comprises an A35 monomer joined to an A36 monomer, or an A36 monomer joined to an A33 monomer, or the like. In some embodiments, the engineered EEV envelope glycoproteins described herein comprise the amino acid sequence of any one of SEQ ID NOs: 45, 51, or 52, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0148] In some embodiments, the engineered EEV envelope glycoprotein or the engineered IMV surface membrane protein described herein do not comprise a signal sequence. In some embodiments, the engineered EEV envelope glycoproteins or the engineered IMV surface membrane proteins described herein comprise a signal sequence. In some embodiments, the signal sequence comprises or consists of the amino acid sequence MKTISVVTLLCVLPAVVYS (SEQ ID NO: 57) or the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 58), or an amino acid sequence having at least 70%,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58.

[0149] In some embodiments, the engineered EEV envelope glycoproteins or the engineered IMV surface membrane proteins described herein comprise a tag. In some embodiments, the tag is a histidine tag. In certain embodiments, the histidine tag comprises or consists of the amino acid sequence HHHHHHHH (SEQ ID NO: 66), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0150] In some embodiments, the tag comprises or consists of the amino acid sequence NHGLYLADQYVKGIAKSRKS (SEQ ID NO: 67), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0151] In some embodiments, an engineered IMV surface membrane protein or an EEV envelope glycoprotein described herein comprises or consists of any one of the amino acid sequences in Table 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0152] For all amino acid sequences provided herein, any signal sequence or tag sequence (including the amino acid sequence of any one of SEQ ID NOs: 57-58, 66, or 67 or sequences comprising 70% sequence identity thereto) are optional and may be removed or replaced. It is noted that sequences which do not comprise a signal sequence and / or a tag sequence as described above are within the disclosure provided herein, even if an independent sequence identifier is not provided.Table 1: Exemplary Amino Acid Sequences of Engineered EEV envelope glycoproteins and Engineered IMV Surface Membrane Proteins of the DisclosureFusion Proteins

[0153] In some embodiments, one or more of the engineered Orthopoxvirus proteins of the disclosure are presented as individual protein antigens. In some embodiments, one or more of the engineered Orthopoxvirus proteins of the disclosure are coupled together and expressed as a fusion protein.

[0154] Provided herein are fusion proteins comprising at least one engineered Orthopoxvirus proteins described herein, or a fragment thereof. In some embodiments, the fusion proteins of the disclosure comprise one engineered Orthopoxvirus protein or a fragment thereof. In some embodiments, the fusion proteins of the disclosure comprise at least two engineered Orthopoxvirus proteins or fragments thereof. In some embodiments, the fusion proteins of the disclosure comprise two engineered Orthopoxvirus proteins or fragments thereof. In some embodiments, the fusion proteins of the disclosure comprise at least three engineered Orthopoxvirus proteins described herein or fragments thereof. In some embodiments, the fusion proteins of the disclosure comprise three engineered Orthopoxvirus proteins described herein or fragments thereof. In some embodiments, the fusion proteins of the disclosure comprise at least four or more engineered Orthopoxvirus proteins or fragments thereof. In some embodiments, the fusion proteins of the disclosure comprise four engineered Orthopoxvirus proteins described herein or fragments thereof. In some embodiments, the fusion proteins of the disclosure comprise at least five, at least six, at least seven, at least eight, at least nine, or at least ten engineered Orthopoxvirus proteins described herein or fragments thereof. In some embodiments, the fusion proteins of the disclosure comprise five, six, seven, eight, nine, or ten engineered Orthopoxvirus proteins described herein or fragments thereof.

[0155] In some embodiments, the fusion proteins of the disclosure comprise at least two amino acid sequences derived from one or more engineered Orthopoxvirus proteins. For example, the fusion proteins of the disclosure may comprise two amino acid sequences derived from the same EEV envelope glycoprotein or two amino acid sequences derived from the same IMV surface membrane protein. In certain embodiments, the two amino acid sequences derived from the same EEV envelope glycoprotein are identical. In certain embodiments, the two amino acid sequences derived from the same EEV envelope glycoprotein are not the same. In certain embodiments, the two amino acid sequences derived from the same IMV surface membrane protein are identical. In certain embodiments, the two amino acid sequences derived from the same IMV surface membrane protein are not the same.

[0156] Also provided herein are fusion proteins comprising at least one IMV surface membrane protein and at least one EEV envelope glycoprotein. In some embodiments, the fusion proteins of the disclosure comprise at least two engineered Orthopoxvirus proteins described herein, wherein the at least two engineered Orthopoxvirus proteins comprises at least one engineered EEV envelope glycoprotein and at least one engineered IMV surface membrane protein. In some embodiments, a fusion protein described herein comprises two EEV envelope glycoproteins and one IMV surface membrane protein. In some embodiments, a fusion protein of the disclosure comprises a monomer of an EEV envelope glycoprotein, a monomer of an EEV envelope glycoprotein, and a monomer of an IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise at least two engineered Orthopoxvirus proteins described herein, wherein the at least two engineered Orthopoxvirus proteins comprise a monomer of an IMV surface membrane protein and a monomer or a dimer of an EEV envelope glycoprotein. In some embodiments, a fusion protein comprising at least two engineered Orthopoxvirus proteins described herein comprises a dimer of an EEV envelope glycoprotein or two monomers of an IMV surface membrane protein.

[0157] In some embodiments, the fusion proteins of the disclosure comprise one EEV envelope glycoprotein and one IMV surface membrane protein. In certain embodiments, the fusion proteins of the disclosure comprise one IMV surface membrane protein selected from the group consisting of LI and Ml and at least one EEV envelope glycoprotein selected from the group consisting of: A33, A35, A36, B5, B6, and B7. In some embodiments, the fusion proteins of the disclosure comprise a LI IMV surface membrane protein and a monomer or a dimer of an A33 EEV envelope glycoprotein. In some embodiments, the fusion proteins of the disclosure comprise a LI IMV surface membrane protein and an EEV envelope glycoprotein or a dimer of an A35 EEV envelope glycoprotein. In some embodiments, the fusion proteins of the disclosure comprise a LI IMV surface membrane protein and an A36 EEV envelope glycoprotein or a dimer of an A36 EEV envelope glycoprotein. In some embodiments, the fusion proteins of the disclosure comprise a B5 EEV envelope glycoprotein and a LI IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise a B6 EEV envelope glycoprotein and a LI IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure a B7 EEV envelope glycoprotein and a LI IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A33 EEV envelope glycoprotein, or a dimer of an A33 EEV envelope glycoprotein, and a Ml IMV surfacemembrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A35 EEV envelope glycoprotein, or a dimer of a A35 EEV envelope glycoprotein, and a Ml IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A36 EEV envelope glycoprotein. In some embodiments, the fusion proteins of the disclosure comprise an A36 EEV envelope glycoprotein and a Ml IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise a B5 EEV envelope glycoprotein and a Ml IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise a B6 EEV envelope glycoprotein and a Ml IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise a B7 EEV envelope glycoprotein and a Ml IMV surface membrane protein.

[0158] . In some embodiments, the fusion proteins of the disclosure comprise at least two EEV envelope glycoproteins and at least one IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise two EEV envelope glycoproteins and one IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise no more than two EEV envelope glycoproteins and one IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise one EEV envelope glycoprotein selected from the group consisting of: A33, A35, and A36, one EEV envelope glycoprotein selected from the group consisting of: B5, B6, and B7, and one IMV surface membrane protein selected from the group consisting of LI and Ml, or combinations thereof. In some embodiments, the fusion proteins of the disclosure comprise one IMV surface membrane protein and two EEV envelope glycoproteins, wherein one EEV envelope glycoproteins is a monomer and the other EEV envelope glycoprotein is a dimer. In some embodiments, the monomer of the EEV envelope glycoprotein is selected from the group consisting of B5, B6, and B7. In some embodiments, the dimer of the EEV envelope glycoprotein is selected from the group consisting of A33, A35, and A36. In certain embodiments, the dimer of the EEV envelope glycoprotein is a homodimer. In certain embodiments, the dimer of the EEV envelope glycoprotein is a heterodimer. In some embodiments, the monomer of the IMV surface membrane protein is selected from the group consisting of Ml and LI.

[0159] In some embodiments, the fusion proteins of the disclosure comprise an A33 envelope glycoprotein or a dimer of an A33 envelope glycoprotein, a B5 envelope glycoprotein, and a LI surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprisean A35 envelope glycoprotein or a dimer of an A35 envelope glycoprotein, a B6 envelope glycoprotein, and a Ml surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A36 envelope glycoprotein or a dimer of an A36 envelope glycoprotein, a B7 envelope glycoprotein, and a Ml surface member protein.

[0160] In some embodiments, the fusion proteins of the disclosure comprise an A33 EEV envelope protein or a dimer of an A33 EEV envelope protein, a B5 EEV envelope protein, and a Ml IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A33 EEV envelope protein or a dimer of an A33 EEV envelope protein, a B6 EEV envelope protein, and a Ml IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A33 EEV envelope protein or a dimer of an A33 EEV envelope protein, a B7 EEV envelope protein, and a Ml IMV surface membrane protein.

[0161] In some embodiments, the fusion proteins of the disclosure comprise an A33 EEV envelope protein or a dimer of an A33 EEV envelope protein, a B5 EEV envelope protein, and a LI IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A33 EEV envelope protein or a dimer of an A33 EEV envelope protein, a B6 EEV envelope protein, and a LI IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A33 EEV envelope protein or a dimer of an A33 EEV envelope protein, a B7 EEV envelope protein, and a LI IMV surface membrane protein.

[0162] In some embodiments, the fusion proteins of the disclosure comprise an A35 EEV envelope protein or a dimer of an A35 EEV envelope protein, a B5 EEV envelope protein, and a Ml IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A35 EEV envelope protein or a dimer of an A35 EEV envelope protein, an B6 EEV envelope protein, and a Ml IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A35 EEV envelope protein or a dimer of an A35 EEV envelope protein, a B7 EEV envelope protein, and an Ml IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A35 EEV envelope protein or a dimer of an A35 EEV envelope protein, a B5 EEV envelope protein, and a LI IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A35 EEV envelope protein or a dimer of an A35 EEV envelope protein, an B6 EEV envelope protein, and a LI IMV surface membrane protein. In some embodiments, the fusion proteins of the disclosure comprise an A35 EEV envelope protein or a dimer of an A35 EEV envelope protein, a B7 EEV envelope protein, and an LI IMV surface membrane protein.

[0163] In some embodiments, the fusion proteins of the disclosure comprise the EEV envelope protein A36 or a dimer of the EEV envelope protein A36, the EEV envelope protein B5, and the IMV surface membrane protein Ml. In some embodiments, the fusion proteins of the disclosure comprise the EEV envelope protein A36 or a dimer of the EEV envelope protein A36, the EEV envelope protein B6, and the IMV surface membrane protein Ml. In some embodiments, the fusion proteins of the disclosure comprise the EEV envelope protein A36 or a dimer of the EEV envelope protein A36, the EEV envelope protein B7, and the IMV surface membrane protein Ml. In some embodiments, the fusion proteins of the disclosure comprise the EEV envelope protein A36 or a dimer of the EEV envelope protein A36, the EEV envelope protein B5, and the IMV surface membrane protein LI. In some embodiments, the fusion proteins of the disclosure comprise the EEV envelope protein A36 or a dimer of the EEV envelope protein A36, the EEV envelope protein B6, and the IMV surface membrane protein LI. In some embodiments, the fusion proteins of the disclosure comprise the EEV envelope protein A36 or a dimer of the EEV envelope protein A36, the EEV envelope protein B7, and the IMV surface membrane protein LI.

[0164] In some embodiments, the engineered Orthopoxvirus proteins or fusion proteins described herein are fused to a binding partner. In some embodiments, the engineered Orthopoxvirus proteins or fusion proteins described herein are fused to a binding partner and form a nanoparticle. In some embodiments, the binding partner is a ferritin subunit. In some embodiments, the ferritin subunit comprises a H. Pylori ferritin or a hybrid bullfrog ferritin. In some embodiments, the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0165] In some embodiments, the engineered Orthopoxvirus proteins or fusion proteins described herein comprise a tag. In some embodiments, the fusion proteins described herein comprise a tag. In some embodiments, the tag is a histidine tag. In certain embodiments, the histidine tag comprises or consists of the amino acid sequence HHHHHHHH (SEQ ID NO: 66), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0166] In some embodiments, the tag comprises or consists of the amino acid sequence NHGLYLADQYVKGIAKSRKS (SEQ ID NO: 67), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0167] In some embodiments, the fusion proteins of the disclosure comprise an EEV envelope glycoprotein coupled to a ferritin nanoparticle, or an IMV surface membrane protein coupled to a ferritin nanoparticle. For example, in some embodiments, the EEV envelope protein selected from the group consisting of A33, A35, and A36 may be coupled to a ferritin nanoparticle. In some embodiments, the EEV envelope protein selected from the group consisting of B5, B6, and B7 may be coupled to a ferritin nanoparticle. In some embodiments, the IMV surface membrane protein selected from the group consisting of LI and Ml may be coupled to a ferritin nanoparticle. In some embodiments, the fusion proteins comprising an EEV envelope glycoprotein coupled to a ferritin subunit, or an IMV surface membrane protein coupled to a ferritin subunit, comprise amino acid sequences selected from the group consisting of SEQ ID NOS: 25-37, 40-42, or any amino acid sequence having a 70% sequence identity thereto. In some embodiments, the fusion proteins comprising an EEV envelope glycoprotein coupled to a ferritin subunit, or an IMV surface membrane protein coupled to a ferritin subunit may be selected from the amino acid sequences of Table 2.Table 2: Exemplary Amino Acid Sequences of Fusion Protein comprising one or more EEV Envelope Proteins and one or more IMV Surface membrane proteins

[0168] In some embodiments, the fusion proteins disclosed herein are useful for vaccinating a subject against an infection by an Orthopoxvirus, thereby preventing or reducing the severity of a disease or a symptom thereof associated with an Orthopoxvirus infection in a subject.Orientation of EEV Envelope Glycoproteins and IMV Surface Membrane Proteins with Fusion Proteins

[0169] In some embodiments, the fusion proteins of the disclosure comprising the two or more engineered proteins may have a specific orientation. In some embodiments, the specific orientation may provide an added benefit, wherein, by way of example, the specific orientation may demonstrates increased immunogenicity in vivo or in vitro. The specific orientation may reference the orientation of the two or more engineered proteins within the fusion protein from N-terminal to C-terminal. For example, wherein the fusion protein comprises a monomer or a dimer of an EEV envelope glycoprotein and a IMV surface membrane protein, the fusion protein may comprise N-terminal to C-terminal: EEV envelope glycoprotein (monomer or dimer) and IMV surface membrane protein or IMV surface membrane protein and EEV envelope glycoprotein (monomer or dimer), wherein the EEV envelope glycoproteins are selected from the group consisting of: A33, A35, and A36 or B5, B6, and B7, and the IMV surface membrane protein is selected from the group consisting of LI and Ml.

[0170] In some embodiments, wherein the fusion protein comprises a first EEV envelope glycoprotein (monomer or dimer), a second EEV envelope glycoprotein (monomer or dimer), and a IMV surface membrane protein, the fusion protein may comprise N-terminal to C- terminal: first EEV envelope glycoprotein (monomer or dimer), second EEV envelope glycoprotein (monomer or dimer), and IMV surface membrane protein. In some embodiments,the fusion protein may comprise N-terminal to C-terminal: second EEV envelope glycoprotein (monomer or dimer), first EEV envelope glycoprotein (monomer or dimer), and IMV surface membrane protein.

[0171] In some embodiments, the fusion protein may comprise N-terminal to C-terminal: first EEV envelope glycoprotein, (monomer or dimer) IMV surface membrane protein, and second EEV envelope glycoprotein (monomer or dimer). In some embodiments, the fusion protein may comprise N-terminal to C-terminal: second EEV envelope glycoprotein (monomer or dimer), IMV surface membrane protein, and first EEV envelope glycoprotein (monomer or dimer). In some embodiments, the fusion protein may comprise N-terminal to C-terminal: IMV surface membrane protein, first EEV envelope glycoprotein (monomer or dimer), and second EEV envelope glycoprotein (monomer or dimer). In some embodiments, the fusion protein may comprise N-terminal to C-terminal: IMV surface membrane protein, second EEV envelope glycoprotein (monomer or dimer), and first EEV envelope glycoprotein (monomer or dimer). In some embodiments, the first EEV envelope glycoprotein, as a monomer or a dimer, is selected from the group consisting of: A33, A35, and A36. In some embodiments, the second EEV envelope glycoprotein is selected from the group consisting of: B5, B6, and B7. In some embodiments, the IMV surface membrane protein is selected from the group consisting of: LI and Ml.

[0172] Table 3 provides exemplary fusion proteins with one or more EEV Envelope Proteins and an IMV Surface Membrane proteins of the disclosure. As noted above these sequences listed in Table 3 may include signal peptides and / or tag sequences, which are optional.Table 3: Fusion proteins with one or more EEV Envelope Proteins and an IMV Surface Membrane Proteins

[0173] Also provided herein are polynucleotides that encode any one or more of the engineered proteins of Table 1, Table 2, or Table 3, including any of the EEV envelope proteins, the IMV surface membrane proteins, or the fusion proteins described herein. In some embodiments, the polynucleotides comprise DNA or RNA (e.g., mRNA). In exemplary embodiments, provided herein are mRNA that encode any one or more of the engineered proteins of Table 1, Table 2, or Table 3, including any of the EEV envelope proteins, the IMV surface membrane proteins, or the fusion proteins described herein. The mRNA may be modified.Ferritin Nanoparticles

[0174] Ferritin is globular protein found in animals, bacteria, and plants that acts primarily to control the rate and location of polynuclear Fe(III)2O3 formation through transportation of hydrated iron ions and protons to and from a mineralized core. The globular form of ferritin is made up of monomeric subunit proteins (also referred to as monomeric ferritin subunits), which are polypeptides having a molecular weight of approximately 17-20 kDa. An example of a sequence of a monomeric ferritin subunit is represented by SEQ ID NO: 1 below.

[0175] DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKK LIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFN FLQWYVAEQHEEEVLFNDILDI<IELIGNENHGLYLADQYVI<GIAI<SRI<S (SEQ ID NO: 1)

[0176] Without being bound to theory or mechanism, in some embodiments, each monomeric ferritin subunit has the topology of a helix bundle. A helix bundle comprises a four antiparallel helix motif, with a fifth shorter helix (the C-terminal helix) lying roughly perpendicular to the long axis of the 4-helix bundle. According to convention, the helices are labeled ‘A, B, C, and D and E, from the N-terminus respectively. The N-terminal sequence lies adjacent to the capsid three-fold axis and extends to the surface, while the E helices pack together at the four-fold axis with the C-terminus extending into the particle core. The consequence of this packing creates two pores on the capsid surface. It is expected that one or both of these pores represent the point by which the hydrated iron diffuses into and out of the capsid. In some embodiments, following production, these monomeric ferritin subunit proteins self-assemble into the globular ferritin protein. Accordingly, in some embodiments, the globular form of ferritin comprises 24 monomeric ferritin subunit proteins and has a capsid-like structure with 432 symmetry.

[0177] Provided herein are fusion proteins comprising a ferritin subunit. In some embodiments, a fusion protein described herein comprises a ferritin subunit. In some embodiments of the fusion proteins comprising an engineered Orthopovirus protein provided herein, the engineered Orthopoxvirus protein is fused to a binding partner. In some embodiments, the binding partner comprises a ferritin subunit. In some embodiments, the fusion proteins described herein comprising a ferritin subunit self-assemble into a ferritin nanoparticle.

[0178] In some embodiments, the fusion proteins of the disclosure comprise a ferritin subunit. The ferritin subunit may comprise an amino acid sequence of a full length, single ferritin polypeptide, or any portion thereof that is capable of directing self-assembly of monomeric ferritin subunits into oligomers. Fusion proteins comprising a ferritin subunit polypeptide are described, for example, in U.S. Patent No, 7,097,841, the contents of which are incorporated by reference in their entirety. The amino acid sequences of monomeric ferritin subunits, or portions thereof, of any ferritin protein can be used in the fusion proteins of the disclosure to produce ferritin nanoparticles, so long as the monomeric ferritin subunits are capable of self-assembling into an oligomer or a nanoparticle.

[0179] Variations can be made in the amino acid sequence of a ferritin protein without affecting its ability to self-assembly into an oligomer or a nanoparticle. Such variations include insertion of amino acid residues, deletions of amino acid residues, or substitutions of amino acid residues. In some embodiments, the fusion proteins described herein comprises a ferritin subunit, wherein the ferritin subunit comprises an amino acid sequence derived from a mammalianferritin. In some embodiments, the fusion proteins described herein comprises a modified ferritin subunit, wherein the modified ferritin subunit comprises a modification relative to a naturally occurring ferritin. In certain embodiments, the modified ferritin subunit is derived from a naturally occurring mammalian ferritin. Without being held to theory or mechanism, in some embodiments, relative to a naturally occurring ferritin, a modified ferritin subunit exhibits reduced immunogenicity when it is administered to a subject of the species in which the naturally occurring ferritin is found. In some embodiments, a modified ferritin subunit, when administered to a subject, does not result in the production of antibodies that react with the natural ferritin protein of the subject.

[0180] A ferritin subunit described herein may comprise an amino acid sequence derived from a bacterial ferritin protein, a plant ferritin protein, an algal ferritin protein, an insect ferritin protein, a fungal ferritin protein, a mammalian ferritin protein, or a combination thereof. As discussed above, fusion proteins of the disclosure need not comprise a full-length sequence of a ferritin subunit polypeptide. Portions, regions, or fragments of a full-length ferritin subunit (e.g. a H. Pylori or bullfrog ferritin subunit) polypeptide that are capable of directing self-assembly of monomeric ferritin subunits into oligomers can be used.

[0181] In some embodiments of the fusion proteins of the disclosure where the fusion protein comprises a ferritin subunit, the ferritin subunit comprises an amino acid sequence derived from H. Pylori. In some embodiments, the ferritin subunit comprises an amino acid sequence derived from SEQ ID NO: 1 or 82, or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 or 82. In some embodiments, the ferritin subunit comprises an amino acid sequence derived from SEQ ID NO: 1 or 82, a sequence having at least 1, 2, 3, 4, or 5 modifications thereto.

[0182] In some embodiments, the ferritin subunit comprises a fragment of a full-length H. Pylori ferritin. In some embodiments, the ferritin subunit comprises a fragment of a full-length H. Pylori ferritin, wherein the fragment comprises the amino acids at positions 5-168 of an amino acid sequence of a full-length H. Pylori ferritin, or any fragment thereof. In certain embodiments, the ferritin subunit comprises a fragment of a full-length H. Pylori ferritin, wherein the fragment comprises the amino acids at positions 5-168 relative to SEQ ID NO: 1, or any fragment thereof. In specific embodiments, the fragment comprises amino acids 5-71relative to SEQ ID NO: 1. In specific embodiments, the fragment comprises amino acids 6-72 relative to SEQ ID NO: 1. In specific embodiments, the fragment comprises amino acids 7-73 relative to SEQ ID NO: 1. In specific embodiments, the fragment comprises amino acids 72-168 relative to SEQ ID NO: 1. In specific embodiments, the fragment comprises amino acids 73-168 relative to SEQ ID NO: 1. In specific embodiments, the fragment comprises amino acids 74-168 relative to SEQ ID NO: 1.

[0183] In certain embodiments, the ferritin subunit comprises a fragment of a full-length H. Pylori ferritin, wherein the fragment comprises the amino acids at positions 5-168 relative to SEQ ID NO: 82, or any fragment thereof. In specific embodiments, the fragment comprises amino acids 5-71 relative to SEQ ID NO: 82. In specific embodiments, the fragment comprises amino acids 6-72 relative to SEQ ID NO: 82. In specific embodiments, the fragment comprises amino acids 7-73 relative to SEQ ID NO: 82. In specific embodiments, the fragment comprises amino acids 72-168 relative to SEQ ID NO: 82. In specific embodiments, the fragment comprises amino acids 73-168 relative to SEQ ID NO: 82. In specific embodiments, the fragment comprises amino acids 74-168 relative to SEQ ID NO: 82.

[0184] In some embodiments of the fusion proteins of the disclosure where the fusion protein comprises a ferritin subunit, the ferritin subunit comprises an amino acid sequence of a bullfrog ferritin. In some embodiments, the ferritin subunit comprises a hybrid bullfrog ferritin. A hybrid bullfrog ferritin comprises (i) a bullfrog ferritin or portion thereof and (ii) a ferritin derived from a bacterium. For example, a hybrid bullfrog ferritin may comprise a bullfrog ferritin and a ferritin derived from H. Pylori, referred to as a hybrid H. Pylori bullfrog ferritin. In some embodiments, the ferritin subunit comprises a hybrid H. Pylori bullfrog ferritin. In some embodiments, the ferritin subunit comprises an amino acid sequence derived from SEQ ID NO: 18 or 19 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18 or 19. In some embodiments, the ferritin subunit comprises an amino acid sequence derived from SEQ ID NO: 18 or 19 or a sequence having at least 1, 2, 3, 4, or 5 modifications thereto. In some embodiments, the ferritin subunit comprises a fragment of a full-length hybrid bullfrog ferritin.

[0185] A ferritin subunit in a fusion protein of the disclosure may comprise artificial glycosylation sites, for example, artificial (engineered) N-glycosylation sites. In someembodiments, a substitution is made to an amino acid of a ferritin subunit amino acid sequence to create a consensus glycosylation sequence. For example, an artificial N-glycosylation site may be created by introducing a consensus sequence N-X-S / T, wherein X is any amino acid except proline, in a ferritin amino acid sequence. A consensus glycosylation sequence can be created by substitutions of amino acid residues in a ferritin amino acid sequence. In some embodiments, amino acid substitutions are made to SEQ ID NO: 1 to introduce an artificial N- glycosylation site. In certain embodiments, the amino acid substitutions made to SEQ ID NO: 1 comprise substitution of lysine (K) to asparagine (N) at a position corresponding to position 75 of SEQ ID NO: 1 and substitution of glutamate (E) to threonine (T) at a position corresponding to position 77 of SEQ ID NO: 1. In certain embodiments, the amino acid substitutions made to SEQ ID NO: 1 comprise substitution of T to N at a position corresponding to position 67 of SEQ ID NO: 1 and substitution of isoleucine (I) to T at a position corresponding to position 69 of SEQ ID NO: 1. In certain embodiments, the amino acid substitutions made to SEQ ID NO: 1 comprise substitution of histidine (H) to N at a position corresponding to position 74 of SEQ ID NO: 1 and substitution of phenylalanine (F) to T at a position corresponding to position 76 of SEQ ID NO: 1. In certain embodiments, the amino acid substitutions made to SEQ ID NO: 1 comprise substitution of E to N at a position corresponding to position 143 of SEQ ID NO: 1 and substitution of H to T at a position corresponding to position 145 of SEQ ID NO: 1.

[0186] In some embodiments, amino acid substitutions are made to SEQ ID NO: 82 to introduce an artificial N-glycosylation site. In certain embodiments, the amino acid substitutions made to SEQ ID NO: 82 comprise substitution of lysine (K) to asparagine (N) at a position corresponding to position 75 of SEQ ID NO: 82 and substitution of glutamate (E) to threonine (T) at a position corresponding to position 77 of SEQ ID NO: 82. In certain embodiments, the amino acid substitutions made to SEQ ID NO: 82 comprise substitution of T to N at a position corresponding to position 67 of SEQ ID NO: 82 and substitution of isoleucine (I) to T at a position corresponding to position 69 of SEQ ID NO: 82. In certain embodiments, the amino acid substitutions made to SEQ ID NO: 82 comprise substitution of histidine (H) to N at a position corresponding to position 74 of SEQ ID NO: 82 and substitution of phenylalanine (F) to T at a position corresponding to position 76 of SEQ ID NO: 82. In certain embodiments, the amino acid substitutions made to SEQ ID NO: 82 comprise substitution of E to N at a position corresponding to position 143 of SEQ ID NO: 82 and substitution of H to T at a position corresponding to position 145 of SEQ ID NO: 82.

[0187] In some embodiments, a ferritin subunit in a fusion protein described herein comprises a deletion or a substitution, wherein the deletion or substitution eliminates a glycosylation site. In some embodiments, a substitution is made to an amino acid of a ferritin subunit amino acid sequence to eliminate a glycosylation site. In certain embodiments, without being held to theory or mechanism, elimination of a glycosylation site in a ferritin subunit allows for more efficient production. In some embodiments, an amino acid substitution is made to SEQ ID NO: 1 to eliminate a glycosylation site. In some embodiments, an amino acid substitution made to SEQ ID NO: 1 to eliminate a glycosylation site comprises substitution of N to Q at a position corresponding to position 9 of SEQ ID NO: 1. In some embodiments, an amino acid substitution made to SEQ ID NO: 1 to eliminate a glycosylation site comprises substitution of K to N at a position corresponding to position 131 of SEQ ID NO: 1.

[0188] In some embodiments, an amino acid substitution is made to SEQ ID NO: 82 to eliminate a glycosylation site. In some embodiments, an amino acid substitution made to SEQ ID NO: 82 to eliminate a glycosylation site comprises substitution of N to Q at a position corresponding to position 9 of SEQ ID NO: 82. In some embodiments, an amino acid substitution made to SEQ ID NO: 82 to eliminate a glycosylation site comprises substitution of K to N at a position corresponding to position 131 of SEQ ID NO: 82.

[0189] A ferritin subunit may be positioned at the N-terminus or the C-terminus of the fusion proteins described herein. In some embodiments, the ferritin subunit is at the N-terminus of a fusion protein described herein. In some embodiments, the ferritin subunit is at the C-terminus of a fusion protein described herein. In some embodiments, the amino acid sequence of the ferritin subunit is internally integrated into the amino acid sequence of the fusion protein. In some embodiments, the fusion proteins of the disclosure comprise more than one ferritin subunit or fragments thereof. For example, in some embodiments, the fusion proteins described herein comprise three engineered Orthopoxvirus proteins and two ferritin subunits or fragments thereof.

[0190] In some embodiments, the fusion proteins of the disclosure comprise one or more linkers. The one or more linkers may comprise one or more amino acid sequences. In some embodiments, the linker fuses a EEV envelope glycoprotein to a IMV surface membrane protein. In some embodiments, the linker fuses a EEV envelope glycoprotein to a ferritin subunit. In some embodiments, the linker fuses a IMV surface membrane protein to a ferritin subunit. In some embodiments, the linker fuses a first EEV envelope glycoprotein to a second EEV envelope glycoprotein. In some embodiments, the linker fuses a first EEV envelope proteinto a IMV surface membrane protein or a second EEV envelope protein to a IMV surface membrane protein.

[0191] In some embodiments, a ferritin subunit or fragment thereof may be fused to an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, or a fusion protein described herein by one or more linkers. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a glycine-serine (GS) linker. In certain embodiments, the length of the linker is about 2 amino acids to about 5 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 30 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 50 amino acids, or about 2 amino acids to about 60 amino acids, or more amino acids in length, for example, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 25 amino acids, about 35 amino acids, about 45 amino acids, about 50 amino acids, or about 60 amino acids.

[0192] Depending on length, one or more linker sequences may have various conformations in secondary structure, such as helical, -strand, coil / bend, and turns. In some embodiments, a linker sequence has an extended conformation and functions as an independent domain that does not interact with the adjacent protein domains. A linker sequence may be rigid or flexible. A flexible linker sequence may increase the range of orientations that can be adopted by the domains of the fusion proteins described herein. A rigid linker can be used to keep a fixed distance between the domains and to help maintain their independent functions.

[0193] In some embodiments, a fusion protein described herein comprises a flexible linker. In some embodiments, a fusion protein described herein comprises a rigid linker. Linker sequences for fusion proteins are described, for example, in Chen et al, 2013. In some embodiments, a linker comprises an amino acid sequence isolated or derived from a naturally occurring or engineered ferritin sequence. For example, in some embodiments, the linker comprises a sequence isolated or derived from a hybrid bullfrog ferritin sequence. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, a linker comprises or consists of SGG, GSG, GG, GSGG, NGTGGSG, GGGGS, or SGG. Linker sequences of the disclosure may comprise any one of the sequences in Table 4A below.Table 4A: Exemplary Linker Sequences

[0194] Provided herein are ferritin nanoparticles comprising a fusion protein described herein, wherein the fusion protein comprises a ferritin subunit. In some embodiments, of the ferritin nanoparticles described herein, the fusion protein comprises one or more EEV envelope glycoproteins, a IMV surface membrane protein, and a ferritin subunit. Such fusion proteins can self-assemble into oligomers. An oligomeric structure, or supramolecule, resulting from such self-assembly is referred to herein as a ferritin nanoparticle. An exemplary embodiment of the present disclosure is a nanoparticle comprising an oligomer of a fusion protein described herein.

[0195] When the nanoparticles comprising one or more EEV envelope glycoproteins and / or a IMV surface membrane protein and a ferritin subunit are administered to a subject, the surface- exposed EEV envelope glycoproteins and IMV surface membrane protein are accessible to the immune system of the subject and thus can elicit an immune response to an Orthopoxvirus. Immunogenic nanoparticles composed of fusion proteins incorporating ferritin subunits are described, for example, in US Patent Nos. 9,441,019 and 10,137,190, and Kanekiyo et al. 2013, Kanekiyo et al., 2015, and He et al 2016.

[0196] Without being held to theory or mechanism, in some embodiments, expression of multiple engineered Orthopoxvirus proteins from a single chain fusion protein coupled to a ferritin subunit leads to impediment of exposure to epitopes upon self-assembly of a ferritin nanoparticle. In some embodiments, Orthopoxvirus proteins are engineered to be displayed in multiple axes for increased access to epitopes upon self-assembly of a ferritin nanoparticle. In some embodiments, the amino acid sequence of a ferritin subunit is divided into at least twofragments. In some embodiments, the amino acid sequence of a ferritin subunit is divided into at least two fragments and one fragment of the amino acid sequence of the ferritin subunit is coupled to a first engineered Orthopoxvirus protein and one fragment is coupled to a second engineered Orthopoxvirus protein. The divided amino acid sequence of the ferritin subunit may place a first engineered Orthopoxivurs proteins in a three-fold axis of symmetry of the assembled ferritin nanoparticle while placing a second engineered Orthopoxvirus proteins in a two-fold axis of symmetry of the assembled ferritin nanoparticle. Exemplary amino acid sequences that comprise a divided ferritin subunit amino acid sequence are provided herein, including SEQ ID NOs: 23, 24, 43, 44, 46, or 47.

[0197] In some embodiments, wherein the fusion proteins of the disclosure are fused to one or more ferritin subunits or fragments thereof, one or more of the engineered Orthopoxvirus proteins are displayed on a three-fold axis of symmetry, while one or more of the engineered Orthopoxvirus proteins are displayed on an internal two-fold axis of symmetry. Without being held to theory of mechanism, displaying one or more engineered Orthopoxvirus proteins on the three-fold axis of symmetry and one or more engineered Orthopoxvirus proteins on the two-fold axis of symmetry may lead to increased immunogenicity of the fusion protein. In some embodiments, the one or more engineered Orthopoxvirus protein displayed on the two-fold axis of symmetry dimerize with an adjacent engineered Orthopoxvirus protein coupled to a different ferritin subunit or fragment thereof.

[0198] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B6, the second EEV envelope glycoprotein is A35, and the IMV surface membrane protein is Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, B6, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, Ml, and B6. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, A35, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, Ml, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B6, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A35, and B6.

[0199] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B5, the second EEV envelope glycoprotein is A35, and the IMV surface membrane protein is Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, B5, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, Ml, and B5. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, A35, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, Ml, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B5, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A35, and B5.

[0200] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B7, the second EEV envelope glycoprotein is A35, and the IMV surface membrane protein is ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, B7, and ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, Ml, and B7. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, A35, and ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, Ml, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B7, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A35, and B7.

[0201] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B6, the second EEV envelope glycoprotein is A33, and the IMV surface membrane protein is ML In some embodiments, the fusion proteincomprises, from N-terminus to C-terminus, A33, B6, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, Ml, and B6. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, A33, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, Ml, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B6, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A33, and B6.

[0202] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B5, the second EEV envelope glycoprotein is A33, and the IMV surface membrane protein is Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, B5, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, Ml, and B5. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, A33, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, Ml, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B5, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A33, and B5.

[0203] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B7, the second EEV envelope glycoprotein is A33, and the IMV surface membrane protein is ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, B7, and ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, Ml, and B7. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, A33, and ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, Ml, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B7,and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A33, and B7.

[0204] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B6, the second EEV envelope glycoprotein is A36, and the IMV surface membrane protein is Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, B6, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, Ml, and B6. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, A36, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, Ml, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B6, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A36, and B6.

[0205] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B5, the second EEV envelope glycoprotein is A36, and the IMV surface membrane protein is ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, B5, and ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, Ml, and B5. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, A36, and ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, Ml, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B5, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A36, and B5.

[0206] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certainembodiments, the first EEV envelope glycoprotein is B7, the second EEV envelope glycoprotein is A36, and the IMV surface membrane protein is Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, B7, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, Ml, and B7. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, A36, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, Ml, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B7, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A36, and B7.

[0207] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B6, the second EEV envelope glycoprotein is A35, and the IMV surface membrane protein is LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, B6, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, LI, and B6. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, A35, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, LI, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, B6, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, A35, and B6.

[0208] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B5, the second EEV envelope glycoprotein is A35, and the IMV surface membrane protein is LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, B5, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, LI, and B5. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, A35, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, LI, and A35. Insome embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, B5, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, A35, and B5.

[0209] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B7, the second EEV envelope glycoprotein is A35, and the IMV surface membrane protein is LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, B7, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A35, LI, and B7. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, A35, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, LI, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, B7, and A35. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, A35, and B7.

[0210] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B6, the second EEV envelope glycoprotein is A33, and the IMV surface membrane protein is LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, B6, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, LI, and B6. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, A33, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, LI, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, B6, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, A33, and B6.

[0211] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMVsurface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B5, the second EEV envelope glycoprotein is A33, and the IMV surface membrane protein is LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, B5, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, LI, and B5. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, A33, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, LI, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, B5, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, A33, and B5.

[0212] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B7, the second EEV envelope glycoprotein is A33, and the IMV surface membrane protein is LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, B7, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A33, LI, and B7. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, A33, and LI. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, LI, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, B7, and A33. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, LI, A33, and B7.

[0213] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B6, the second EEV envelope glycoprotein is A36, and the IMV surface membrane protein is ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, B6, and ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, Ml, and B6. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, A36, and ML In someembodiments, the fusion protein comprises, from N-terminus to C-terminus, B6, Ml, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B6, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A36, and B6.

[0214] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B5, the second EEV envelope glycoprotein is A36, and the IMV surface membrane protein is Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, B5, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, Ml, and B5. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, A36, and Ml. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B5, Ml, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B5, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A36, and B5.

[0215] In some embodiments, the fusion proteins described herein comprise a first EEV envelope glycoprotein selected from the group consisting of B5, B6, and B7, a second EEV envelope glycoprotein selected from the group consisting of A33, A35, and A36, and an IMV surface membrane protein selected from the group consisting of Ml and LI. In certain embodiments, the first EEV envelope glycoprotein is B7, the second EEV envelope glycoprotein is A36, and the IMV surface membrane protein is ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, B7, and ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, A36, Ml, and B7. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, A36, and ML In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, B7, Ml, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, B7, and A36. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, Ml, A36, and B7.

[0216] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a B6 EEV envelope glycoprotein, a Ml IMV surface membrane protein, and a ferritin subunit fused to an A35 EEV envelope glycoprotein, wherein the A35 glycoprotein is presented at a 2- fold axis of symmetry upon self-assembly of a ferritin nanoparticle. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, an A35 EEV envelope glycoprotein, a B6 EEV envelope glycoprotein, and a ferritin subunit fused to a Ml IMV surface membrane protein, wherein the Ml protein is presented at a 2-fold axis of symmetry upon self-assembly of a ferritin nanoparticle. In some embodiments, the fusion proteins described herein comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 23, 24, 43, 44, 46, 47, or 66-76 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0217] Also provided herein are polynucleotides that encode any one or more of the fusion proteins of Table 3. In some embodiments, the polynucleotides comprise DNA or RNA (e.g., mRNA), as will be described in more detail herein.Enveloped Virus like Particles (eVLPs)

[0218] Extracellular vesicles can be produced from host cells. For example, the EndosomalSorting Complex Required for Transport (ESCRT) is a component of the cellular machinery facilitating the transit of proteins and cargo within cells. It plays a role in shuttling proteins from the endoplasmic reticulum (ER) to the cell surface, as well as in the generation of extracellular vesicles, which can originate from within the ER or at the cell surface, before being extruded from the cell. Incorporation of ESCRT -recruiting domains (ERDs) may enhance immunogenicity by inducing antigen expression on cell surfaces and promoting the formation of extracellular vesicles presenting antigens on their cell surface, also referred to as enveloped virus like particles (eVLPs).

[0219] Provided herein are eVLPs presenting one or more EEV envelope glycoproteins or IMV surface membrane proteins of the disclosure. Also provided herein are eVLPs presenting one or more fusion proteins of the disclosure. The eVLPs of the present disclosure are non- infectious membraned particles whose production does not require a viral capsid protein and is instead driven by addition (insertion) of one or more polypeptides. The one or more polypeptides which drive eVLP production can be dependent on host ESCRT machinery (such as ERDs) orcan be entirely independent of host ESCRT machinery. For example, polypeptides that enable transit and extrusion of proteins in extracellular vesicles without the use of ESCRT machinery can be suitable for eVLP production.

[0220] Examples of polypeptides that depend on host ESCRT machinery to drive eVLPs formation (such as ERDs) and the use of these polypeptides for vaccine design are disclosed in International Pub. No. WO2022261230A1, the contents of which are herein incorporated in its entirety by reference. Without being held to theory or mechanism, it is expected that the inclusion of an ERD sequence in an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, or a fusion protein described herein would recruit host ESCRT proteins to the engineered protein at the plasma membrane and would result in the self-assembly and budding of eVLPs bearing the engineered EEV envelope glycoprotein, the engineered IMV surface membrane protein, or the fusion protein described herein. Exemplary ERD sequences are provided in Table 4B below.Table 4B: Exemplary ERD Sequences

[0221] In some embodiments, the engineered Orthopoxvirus proteins, the engineered EEV envelope glycoproteins, engineered IMV surface membrane proteins, or fusion proteins described herein comprise an addition (insertion) of one or more ERDs at the C-terminal, N- terminal, and / or middle portions of the protein. In some embodiments, the one or more ERDs is inserted at the C-terminal. An ERD may comprise or may be derived from the amino acid sequence of a human protein, a nonhuman protein, a nonmammalian protein, or a viral protein. In some embodiments, an ERD comprises or is derived from the amino acid sequence of a nonhuman protein. In some embodiments, an ERD comprises or is derived from the amino acid sequence of a nonmammalian protein.

[0222] In some embodiments, an ERD comprises or is derived from the amino acid sequence of a chicken protein, a mouse protein, a lizard protein, a reptile protein, a hamster protein, or agoldfish protein. In some embodiments, an ERD comprises the sequence of any one of SEQ ID NOs: 77-78, or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 77-78.

[0223] In some embodiments, an ERD comprises or is derived from the amino acid sequence of a human protein. In some embodiments, an ERD interacts with the ESCRT proteins TSG101, NEDD4, and / or ALIX. In some embodiments, an ERD comprises or is derived from the ESCRT and ALIX binding region (EABR) of the human CEP55 protein. In certain embodiments, an ERD comprises or is derived from residues 170-213 of the human CEP55 protein, relative to SEQ ID NO: 77. An EABR sequence recruits host ESCRT proteins to the engineered EEV envelope glycoproteins, engineered IMV surface membrane proteins, or fusion proteins at the plasma membrane and results in the self-assembly and budding of eVLPs bearing the engineered EEV envelope glycoproteins, engineered IMV surface membrane proteins, or fusion proteins described herein.

[0224] In some embodiments, an ERD comprises or is derived from a viral protein or fragment thereof. Viral proteins include, but are not limited to, a retroviral protein, a herpes simplex viral protein, a vaccinia viral protein, a hepadnaviral protein, a togaviral protein, a flavivirus protein, a arenaviral protein, a coronaviral protein, an orthomyxoviral protein, a paramyxoviral protein, a bunyaviral protein, a bomaviral protein, a rhabdoviral protein, a filoviral protein, an Ebola protein, or proteins from Equine infectious anemia virus (EIAV), Human T-lymphotropic virus 1 (HTLV-1), Murine leukemia virus (MLV), or Mason-Pfizer monkey virus (MPMV).

[0225] In some embodiments, the engineered EEV envelope glycoproteins, engineered IMV surface membrane proteins, or fusion proteins described herein include an addition (insertion) of one or more ESCRT-independent polypeptides that drive eVLP production at the C-terminal, N- terminal, and / or middle portions of the protein. In some embodiments, the one or more ESCRT- independent polypeptides that drive eVLP production is inserted at the C-terminal. For example, polypeptides that enable transit and extrusion of proteins in extracellular vesicles without the use of ESCRT machinery can be suitable for eVLP production.

[0226] Also provided herein are polynucleotides encoding the amino acid sequence of an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, or afusion protein comprising one or more polypeptides which drive eVLP production. In some embodiments, the one or more polypeptides which drive eVLP production comprises an ERD. In some embodiments, the one or more polypeptides which drive eVLP production is ESCRT- independent. In some embodiments, delivery to a cell of polynucleotides encoding an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, or a fusion protein comprising one or more polypeptides which drive eVLP production enables eVLP formation and extrusion from the cell.Polynucleotides, Constructs, and Vectors

[0227] Also disclosed herein are polynucleotides (DNA and / or RNA, e.g. mRNA) encoding one or more of the amino acid sequences disclosed herein, e.g., one or more of the engineered Orthopoxvirus proteins, engineered EEV envelope glycoproteins, engineered IMV surface membrane proteins, or fusion proteins described herein. In some embodiments, the polynucleotides of the disclosure encode one or more fusion proteins described herein.

[0228] In some embodiments, the polynucleotides encode any one of the amino acid sequences of Table 1, any one of the amino acid sequences of Table 2, or any one of the amino acid sequence of Table 3, or an amino acid sequence having at least 70% sequence identity thereto including at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the amino acid sequence of Table 1, Table 2, or Table 3.

[0229] In some embodiments, the polynucleotides encode the amino acid sequence of any one of SEQ ID NOs: 2-76 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2-76. In some embodiments, the polynucleotides encode an amino acid sequence of a fusion protein of the disclosure. In some embodiments, the polynucleotides encode the amino acid sequence of any one of SEQ ID NOs: 23, 24, 43, 44, 46, 47, or 66-76 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 23, 24, 43, 44, 46, 47, or 66-76.

[0230] In some embodiments, the polynucleotides are or comprise DNA. In some embodiments, the polynucleotides are or comprise RNA. In some embodiments, the polynucleotides are or comprise mRNA. Vaccines containing mRNA may trigger the body’s own immune system to attack the mRNA molecule. This immune response may destroy the mRNA before it can have its intended effect. Different modifications to the mRNA molecule have been developed to disguise the mRNA from the body’s immune system, such as those described in U.S. Patent No. 10,898,574, U.S. Patent No. 10,703,789, U.S. Patent No. 10,577,403, and U.S. Patent No. 10, 064,959, the contents of which are herein incorporated by reference in their entirety.

[0231] In some embodiments, where the polynucleotides of the present disclosure comprise mRNA, the mRNA comprises one or more modified nucleotides selected from the group consisting of: pseudouridine, N-l-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C 5 -bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5-propynyl-cytidine, C5 -methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,0(6)-methylguanine, and 2- thiocytidine. In some embodiments, the mRNA comprises a modified nucleotide in place of one or more uridines. In some embodiments, the modified nucleoside is selected from pseudouridine (\| / ), N 1-methyl-pseudouridine (m 1 \|f), and 5-methyl-uridine(m5U). In some embodiments, the mRNA comprises a modified nucleotide in place of one or more uridines. In some embodiments where the polynucleotides comprise mRNA, the mRNA may be formulated in a lipid nanoparticle (LNP). In some embodiments, the mRNA may be complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, optionally encapsulating mRNA.

[0232] In some embodiments, where the polynucleotides of the present disclosure is mRNA, the mRNA molecule can be synthesized through in vitro transcription of a corresponding DNA template molecule. For example, synthetic mRNA can be produced by in vitro transcription of a cDNA template, such as plasmid DNA (pDNA).

[0233] In some embodiments, wherein the polynucleotides of the present disclosure is mRNA, the mRNA molecule is capable of inducing eVLP formation upon delivery to a cell.

[0234] In some embodiments, the polynucleotides described herein are incorporated into an expression cassette for expression of a protein encoded by the polynucleotides in a host cell or an organism of interest. In some embodiments, the polynucleotides described herein are codon- optimized for expression in a host cell or an organism of interest.

[0235] Also provided herein are vectors comprising the polynucleotides described herein. In some embodiments, the vectors further comprise functional elements that direct and regulate transcription of the polynucleotides included in the vector. These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers that can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region that may serve to facilitate the expression of the inserted gene or hybrid. In some embodiments, the vector is a non-viral vector. The vector, for example, can be a plasmid. In some embodiments, the vector is a viral vector.

[0236] In some embodiments, the polynucleotides described herein are delivered into a host cell or host organism using a non-viral vector. Examples of non-viral vectors include, but are not limited to, a plasmid, a transposable element, a naked DNA vector, a lipid nanoparticle (LNP), or any combination thereof.

[0237] Polynucleotides or vectors of the present disclosure may be formulated in a lipid nanoparticle (LNP). Generally, LNP has four components: ionizable cationic lipids, phospholipids, cholesterol, and PEG lipids. Each component contributes to LNP stability, transfection efficacy, and safety. In some embodiments, the vectors may be complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, optionally encapsulating the vector and / or nucleic acids. In some embodiments, the vector is an LNP comprising mRNA encoding a fusion protein described herein.

[0238] Also provided herein are cells comprising the polynucleotides or vectors described herein. Such cells can be referred to as host cells. A host cell can be an in vitro, ex vivo, or in vivo host cell. Populations of any of the host cells and cell cultures comprising one or more hostcells are also provided herein. The host cell can be a prokaryotic cell, including, for example, a bacterial cell. Alternatively, the cell can be a eukaryotic cell. Examples of prokaryotic host cells are cells of E. coli, Pseudomonas, Bacillus or Streptomyces. Examples of eukaryotic cells are yeast cells (such as cells of Saccharomyces yeast, or methylotrophic yeast such as Pichia, Candida, Hansenula, and Torulopsis); animal cells, such as CHO, RI. l, B-W and LM cells, African Green Monkey kidney cells (for example, COS 1, COS 7, BSCI, BSC40, and BMTIO), insect cells (for example, Sf9), human cells (such as human embryonic kidney cells, for instance, HEK293, Expi293F, or HeLa cells).

[0239] In some embodiments, polynucleotides encoding one or more engineered EEV envelope glycoproteins and one or more engineered IMV surface membrane proteins, or a fusion protein of the disclosure are incorporated into a viral vector for delivery into a host cell or host organism. Accordingly, the vectors described herein, in some embodiments, comprise viral vectors that transport the polynucleotides encoding one or more engineered EEV envelope glycoproteins and one or more engineered IMV surface membrane proteins, or fusion proteins of the disclosure into cells without degradation and include a promoter yielding expression of the polynucleotides in the cells into which it is delivered. Suitable viral vectors include adenovirus vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, poxviral vectors, or lentiviral vectors. In some embodiments, the polynucleotides described herein are delivered via a LNP.

[0240] Methods of producing or generating host cells (meaning cells comprising a polynucleotide or a vector described herein) are also provided herein. A polynucleotide or a vector described herein can be transferred or introduced into a host cell by well-known methods, which vary depending on the type of the host cell. "Introducing" and related terms or phrases used in the context of introducing a polynucleotide or a vector into a cell refers to the translocation of a nucleic acid sequence from outside a cell to inside the cell. In some cases, introducing refers to translocation of a polynucleotide from outside the cell to inside the nucleus of an eukaryotic cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, nanoparticle delivery, viral delivery, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, DEAE dextran, lipofectamine, calcium phosphate or any method now known or identified in the future for introduction of polynucleotides into prokaryotic or eukaryotic cellular hosts. A targeted nuclease system (e.g., an RNA-guidednuclease (CRISPR-Cas9), a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a megaT AL (MT) can also be used to introduce a nucleic acid into a cell.

[0241] Methods of producing or generating an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, a fusion protein, or a polynucleotide described herein are also provided herein. In some embodiments, a method of producing an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, or a polynucleotide described herein comprises a step of introducing into a cell a polynucleotide or vector of the disclosure. In some embodiments, a method of producing an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, or a fusion protein described herein comprises a step of incubating the host cell under conditions allowing for expression of the protein.

[0242] In some embodiments, a method of producing a ferritin nanoparticle comprises a step of incubating the host cell under conditions allowing for expression of fusion proteins and selfassembly of fusion proteins into a nanoparticle. After expression in the host cell, a fusion protein or nanoparticle can be isolated or purified using various purification methods. In some embodiments, the fusion protein can be isolated from the host cell and allowed to self-assemble into nanoparticles in vitro.

[0243] In one example illustrating a process of producing or generating an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, a fusion protein, or a polynucleotide described herein, polynucleotides encoding the engineered EEV envelope glycoprotein, the engineered IMV surface membrane protein, or the fusion protein are introduced into a plasmid or other vector, which is then used to transfect or transduce living cells. For instance, in some embodiments, polynucleotides of the disclosure are inserted in a correct orientation into an expression vector that provides the necessary regulatory regions, such as promoters, enhancers, poly A sites and other sequences. In some embodiments, it may be desirable to express the engineered EEV envelope glycoprotein, the engineered IMV surface membrane protein, or the fusion protein under the control of an inducible or tissue-specific promoter. The expression vector may then be transfected into living cells using various methods, such as lipofection or electroporation, thus generating host cells expressing the fusion protein.

[0244] Cells comprising an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, a fusion protein or polynucleotides of the disclosure may be selected by appropriate antibiotic selection or other methods and cultured. Larger amounts of anengineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, a fusion protein, or polynucleotides of the disclosure may be produced by growing the cells in commercially available bioreactors. Once expressed by the host cells, the engineered EEV envelope glycoproteins, the engineered IMV surface membrane proteins, the fusion proteins, or polynucleotides encoding the same may be isolated (purified) according to standard procedures, such as dialysis, filtration and chromatography. Isolation of the proteins or polynucleotides from the cells may comprise lysing the cells.

[0245] In some embodiments, a method of producing or generating an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, a fusion protein, or a polynucleotide of the disclosure comprises one or more steps of: culturing a cell comprising a vector under conditions permitting expression of proteins or polynucleotides, harvesting the cells and / or harvesting the medium from the cultured cells, or isolating the proteins or polynucleotides from the cells and / or the culture medium. Compositions, methods and kits related to the production of an engineered EEV envelope glycoprotein, an engineered IMV surface membrane protein, a fusion protein, or a polynucleotide described herein are within the scope of the present disclosure.

[0246] Also provided herein are compositions comprising an engineered Orthopoxvirus protein described herein, an engineered EEV envelope glycoprotein described herein, an engineered IMV surface membrane protein described herein, a fusion protein described herein, a ferritin nanoparticle described herein, an eVLP described herein, a polynucleotide described herein, or a vector described herein.

[0247] In some embodiments, the compositions of the disclosure comprise at least two engineered Orthopoxvirus proteins, wherein the at least two engineered Orthopoxvirus proteins comprises at least one intracellular mature viral (IMV) surface membrane protein and at least one extracellular enveloped virus (EEV) envelope glycoprotein.

[0248] In some embodiments, the compositions of the disclosure comprise at least three engineered Orthopoxvirus proteins. In some embodiments, the at least three engineered Orthopoxvirus proteins comprise at least one intracellular mature viral (IMV) surface membrane protein and at least two extracellular enveloped virus (EEV) envelope glycoproteins.

[0249] In some embodiments, the IMV surface membrane protein comprises at least one modification relative to a native IMV surface membrane protein. In some embodiments, the IMV surface membrane protein is selected from the group consisting of: LI and Ml. In certainembodiments, the IMV surface membrane protein comprises the amino acid sequence of any one of SEQ ID NOs: 6-7, 14, 21, 31-32, 41-42, or 48-50, or an amino acid sequence having at least70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity thereto.

[0250] In some embodiments, the EEV envelope glycoprotein comprises at least one modification relative to a native EEV envelope glycoprotein. In some embodiments, the EEV envelope glycoprotein is selected from the group consisting of A33, A35, A36, B5, B6, and B7. In some embodiments, the EEV envelope glycoprotein is selected from the group consisting of A33, A35, and A36. In certain embodiments, the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, 8-10, 12, 45, or 51-52, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the EEV envelope glycoprotein is selected from the group consisting of B5, B6, and B7. In certain embodiments, the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 5, 11, 13, 15, 16, 20, 22, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0251] In some embodiments, the EEV envelope glycoprotein forms a dimer. In certain embodiments, the dimer is a single chain dimer.

[0252] In some embodiments of the compositions of the disclosure, the IMV surface membrane protein comprises at least one modification relative to a native IMV surface membrane protein, wherein the at least one modification comprises a substitution, a deletion, or an insertion. In some embodiments of the compositions of the disclosure, the EEV envelope glycoprotein comprises at least one modification relative to a native EEV envelope glycoprotein, wherein the at least one modification comprises a substitution, a deletion, or an insertion. In some embodiments, the at least one modification comprises a substitution or deletion, whereinthe substitution or deletion removes one or more N-linked glycosylation sites. In some embodiments, the EEV envelope glycoprotein is B6, and the at least one modification is a substitution. In certain embodiments, the substitution is in the B6 glycoprotein at a position corresponding to position 139 relative to SEQ ID NO: 39. In specific embodiments, the substitution comprises substitution of cysteine (C) for isoleucine (I) at position 139 relative to SEQ ID NO: 39.

[0253] In some embodiments of the compositions of the disclosure wherein the composition comprises at least three engineered Orthopoxvirus proteins, the engineered Orthopoxvirus proteins comprise A35, B6, and Ml. In some embodiments, the engineered Orthopoxvirus proteins comprise A33, B5, and LI.

[0254] In some embodiments, the compositions of the disclosure further comprise a linker. In some embodiments, the compositions of the disclosure further comprises a ferritin nanoparticle.

[0255] In some embodiments of the compositions of the disclosure, at least one engineered Orthopoxvirus protein is fused to a binding partner. In certain embodiments, the binding partner is a ferritin subunit. In certain embodiments, the ferritin subunit comprises a 7 / . Pylori ferritin or a 7 / . Pylori hybrid bullfrog ferritin. In specific embodiments, the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 18, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the ferritin subunit is fused to the engineered Orthopoxvirus proteins by a linker. In some embodiments, the amino acid sequence of the linker comprises the sequence of SEQ ID NO: 19 or a sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.Vaccine Compositions

[0256] Provided herein are vaccine compositions comprising an engineered Orthopoxvirus protein described herein, an engineered EEV envelope glycoprotein described herein, an engineered IMV surface membrane protein described herein, a fusion protein described herein, a ferritin nanoparticle described herein, an eVLP described herein, a polynucleotide describedherein, a vector described herein, or a composition described herein. In some embodiments, the vaccine composition comprises an engineered Orthopoxvirus protein described herein. In some embodiments, the vaccine composition comprises an engineered EEV envelope glycoprotein described herein. In some embodiments, the vaccine composition comprises an engineered IMV surface membrane protein described herein. In some embodiments, the vaccine composition comprises a fusion protein described herein. In some embodiments, the vaccine composition comprises a ferritin nanoparticle described herein. In some embodiments, the vaccine composition comprises an eVLP described herein. In some embodiments, the vaccine composition comprises a polynucleotide described herein. In some embodiments, the vaccine composition comprises a vector described herein. In some embodiments, the vaccine composition comprises a composition described herein.

[0257] In some embodiments, the vaccine compositions are administered to a subject. In some embodiments, a vaccine composition comprising polynucleotides encoding one or more engineered EEV envelope glycoproteins and / or one or more engineered IMV surface membrane proteins of the disclosure is delivered to a subject as a mRNA-based vaccine. In some embodiments, a vaccine composition comprising one or more engineered EEV envelope glycoproteins and / or one or more engineered IMV surface membrane proteins of the disclosure is delivered to a subject as a protein-based vaccine. In some embodiments, a vaccine composition comprising a fusion protein or polynucleotides encoding the fusion protein (e.g. comprising a ferritin subunit) is delivered to a subject.

[0258] In some embodiments, the vaccine compositions of the disclosure comprise one or more adjuvants. In some embodiments, the adjuvant comprises aluminum hydroxide (“alum”), a synthetic TLR agonist including agonists for TLR-7 / 8 / 9, a synthetic form of DNA, saponin, an oil in water emulsion, or a combination thereof. In some embodiments, the adjuvant comprises Alhydrogel. In some embodiments, the adjuvant comprises Quil-A® + monophosphoryl-Lipid A (MPLA). In some embodiments, the adjuvant comprises AddaVax™. In some embodiments, the adjuvant comprises an aluminum hydroxide wet gel suspension. In some embodiments, the one or more adjuvants comprise aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, or aluminum potassium sulfate. Examples of synthetic TLR-7 / 8 / 9 agonists include, but are not limited to, imidozoquinolines including 3M-052 or cytosine phosphoguanine (CpG). In some embodiments, the adjuvant comprises imidozoquinoline. In some embodiments, the adjuvant comprises 3M-052. Example of a synthetic form of DNA is cytosine phosphoguanine(CpG). In some embodiments, the adjuvant comprises CpG. In some embodiments, the one or more adjuvants comprises monophosphoryl lipid A or one or more of: QS-21 or aluminum salt. In some embodiments, two or more adjuvants may be used. For example, in some embodiments, alum and CpG in combination are used as adjuvants. In some embodiments, the adjuvants comprise Quil-A® and MPLA+Alum. In some embodiments, the adjuvants comprise 3M- 052+ Alum.

[0259] In some embodiments, the vaccine compositions of the disclosure further comprise one or more of a buffer agent, an osmotic agent, or a stabilizer. In some embodiments, the buffer agent comprises Tris-HCl, Trizma Base, phosphate buffered saline, or the like. In some embodiments, the osmotic agent comprises NaCl or the like. In some embodiments, the stabilizer comprises sucrose, lactose, glycine, glutamic acid, potassium, sodium, human serum albumin, gelatin, or the like.Methods of Use

[0260] In some embodiments, the vaccine compositions of the disclosure are administered to a subject. In some embodiments, the vaccine compositions are used in a method of preventing an Orthopox viral infection or a disease or symptom associated with an Orthopox viral infection in a subject, wherein the method includes administering to the subject an effect amount of a vaccine composition described herein. A subject may include a mammal including but not limited to a human, or a non-human primate. In some embodiments, the subject is a human subject. In some embodiments, the human subject is at least about 1 month old. In some embodiments, the human subject may include a human that is at least about 2 years old. In some embodiments, the human subject may include a human that is at least about 13 years old. In some embodiments, the subject may be at least about 18 years old. For example, in some embodiments, the human subject may be within the ages of about 18 years old to about 55 years old or older than about 56 years old.

[0261] In some embodiments, the subject is a mammal. Non-limiting examples of mammals include, a human, a non-human primate, a monkey, a rodent, a dog, a cat, a rabbit, a cow, a horse, a goat, a sheep, a llama, a camel, a donkey, a bat, a deer, a bear, a squirrel, or a pig. In some embodiments, the subject is a human subject. In some embodiments, the subject in need thereof is not a human subject. In some embodiments, the subject is a bird, such as a chicken, a duck, a pheasant, a turkey, or a goose. In some embodiments, the biological sex of the subject is female. In some embodiments, the subject is pregnant. In some embodiments, the subject beingtreated in accordance with the methods described herein has been diagnosed with an Orthopoxvirus infection or is at risk of being infected by an Orthopoxvirus. In some embodiments, the subject has had one or more prior infections with an Orthopoxvirus. In some embodiments, the subject has never been infected with an Orthopoxvirus.

[0262] As contemplated herein, a subject (e.g., a human subject) may receive at least one dose of a vaccine composition described herein. In some embodiments, a vaccine composition is delivered in multiple doses, e.g., at least two, three, four, or more doses, separated by time. In some embodiments, each of the multiple doses comprises an engineered EEV envelope glycoprotein described herein, an engineered IMV surface membrane protein described herein, a fusion protein described herein, a ferritin nanoparticle described herein, an eVLP described herein, a polynucleotide described herein, or a vector described herein. In some embodiments, each of the multiple doses may be different. For example, in some embodiments, the first dose comprises an engineered EEV envelope glycoprotein and an engineered IMV surface membrane protein described herein, and the second dose comprises polynucleotides encoding an engineered EEV envelope glycoprotein and an engineered IMV surface membrane protein. In some embodiments, the first dose comprises a fusion protein, the second dose comprises polynucleotides encoding the fusion protein, the third dose comprises an engineered EEV envelope glycoprotein and an engineered IMV surface membrane protein, and the fourth dose comprises polynucleotides encoding the engineered EEV envelope glycoproteins and the engineered IMV surface membrane proteins.

[0263] Also disclosed herein are methods of inducing antibodies against an Orthopoxvirus, methods of inducing an Orthopoxvirus-specific immune response, preventing an Orthopoxvirus infection, and treating an Orthopoxvirus infection in a subject. In some embodiments, the subject has an Orthopoxvirus infection or is at risk of an Orthopoxvirus infection. In some embodiments, the methods described herein reduce the severity of a symptom of an Orthopoxvirus infection. Also disclosed herein are methods of vaccinating a subject against infection by an Orthopoxvirus, or a method of preventing an Orthopox infection including a disease or symptom associated with an Orthopoxvirus infection in a subject.

[0264] In some embodiments, the methods described herein comprise administering to the subject a composition described herein, a fusion protein described herein, an engineered Orthopoxvirus protein described herein, a ferritin nanoparticle described herein, an eVLPdescribed herein, a polynucleotide described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.

[0265] In some embodiments, the methods described herein comprise administering to the subject at least a first dose of a composition, a fusion protein, an engineered Orthopoxvirus protein, a ferritin nanoparticle, an eVLP, a polynucleotide, a vaccine composition, or a pharmaceutical composition of the disclosure. In some embodiments, the method comprises administering to the subject a first dose of a vaccine composition, wherein the vaccine composition comprises: one or more engineered EEV envelope glycoproteins and an engineered IMV surface membrane protein, polynucleotides encoding one or more engineered EEV envelope glycoproteins and an engineered IMV surface membrane protein, a fusion protein comprising one or more engineered EEV envelope glycoproteins and an engineered IMV surface membrane protein, polynucleotides encoding a fusion protein comprising one or more engineered EEV envelope glycoproteins and one or more engineered IMV surface membrane proteins, a ferritin nanoparticle, or a vector described herein.

[0266] In some embodiments of the methods of vaccinating a subject, the method comprises administering to the subject a first dose of a vaccine composition comprising a fusion protein described herein, wherein the vaccine composition further comprises: one or more subunits of one or more ferritin nanoparticles; and one or more adjuvants. In some embodiments, the fusion protein comprises one or more engineered EEV envelope glycoproteins and an engineered IMV surface membrane protein, wherein the one or more engineered EEV envelope glycoproteins comprise the amino acid sequence of any one of SEQ ID NOs: 2-4, 5, 8-13, 15-16, 20, 22, 27- 30, 33-37, 39, and 40, or an amino acid sequence comprising at least 70% sequence identity thereto, and the engineered IMV surface membrane protein comprises the amino acid sequence of any one of SEQ ID NOs: 6-7, 14, 21, 31-32, 41-42, 48-50, or an amino acid sequence comprising at least 70% sequence identity thereto. In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 23, 24, 43, 44, 46, 47, or 66-76 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0267] In some embodiments of the methods of the disclosure, the method comprises administering a composition, a fusion protein, an engineered Orthopoxvirus protein, a ferritinnanoparticle, an eVLP, a polynucleotide, a vaccine composition, or a pharmaceutical composition described herein to a subject using any suitable route of administration. In some embodiments, the route of administration is mucosal. In some embodiments, the route of administration is intradermal. In some embodiments, the route of administration is intravenous. In some embodiments, the route of administration is intramuscular. In some embodiments, the route of administration is subcutaneous.

[0268] A variety of routes of administration are contemplated. In some embodiments, a composition, a fusion protein, an engineered Orthopoxvirus protein, a ferritin nanoparticle, an eVLP, a polynucleotide, a vaccine composition, or a pharmaceutical composition described herein is administered to a subject through mucosal, intradermal, intravenous, intramuscular, or subcutaneous delivery and / or by any other known methods of physical delivery. In some embodiments, for intramuscular, subcutaneous, or intradermal delivery, a delivery apparatus is used. In some embodiments, the delivery apparatus is a single use delivery apparatus or a multiuse delivery apparatus. In some embodiments, the delivery apparatus is a syringe with an appropriately sized needle for the route of administration.

[0269] In some embodiments, the methods of the disclosure comprise administering to the subject one or more additional doses of a composition, a fusion protein, an engineered Orthopoxvirus protein, a ferritin nanoparticle, an eVLP, a polynucleotide, a vaccine composition, or a pharmaceutical composition described herein. In some embodiments, administering to the subject an additional dose comprises administering to the subject at least a second dose of a composition, a fusion protein, an engineered Orthopoxvirus protein, a ferritin nanoparticle, an eVLP, a polynucleotide, a vaccine composition, or a pharmaceutical composition. In some embodiments, administering to the subject an additional dose comprises administering to the subject a second dose of the vaccine composition described herein. For example, in some embodiments, the vaccine composition of the second dose comprises one or more engineered EEV envelope glycoproteins and one or more engineered IMV surface membrane proteins, or polynucleotides that encode one or more engineered EEV envelope glycoproteins and one or more engineered IMV surface membrane proteins. In some embodiments, the vaccine composition of the second dose comprises one or more fusion proteins of the disclosure or polynucleotides that encode the one or more fusion proteins of the disclosure, wherein the one or more fusion proteins comprise the amino acid sequence of any one of SEQ ID NOs: 23, 24, 43, 44, or 66-76, or a sequence having at least 70%, at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0270] In some embodiments, the second dose or additional doses are administered to the subject using the same route of administration as the first dose administered to the subject. In some embodiments, the second dose or additional doses are administered to the subject using a different route of administration as the first dose administered to the subject. For example, in some embodiments, the first dose is administered to the subject intramuscularly and the second dose is administered to the subject intravenously.

[0271] The disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference for all purposes.EXEMPLARY EMBODIMENTS

[0272] Embodiment 1-1. A composition comprising at least two engineered Orthopoxvirus proteins, wherein the at least two engineered Orthopoxvirus proteins comprises: a. at least one intracellular mature viral (IMV) surface membrane protein; and b. at least one extracellular enveloped virus (EEV) envelope glycoprotein.

[0273] Embodiment 1-2. The composition of embodiment 1-1, wherein the IMV surface membrane protein comprises at least one modification relative to a wild type IMV surface membrane protein; and wherein the IMV surface membrane protein is selected from the group consisting of LI and ML

[0274] Embodiment 1-3. The composition of embodiment 1-1 or 1-2, wherein the EEV envelope glycoprotein comprises at least one modification relative to a wild type EEV envelope glycoprotein; and wherein the EEV envelope glycoprotein is selected from the group consisting of A33, A35, A36, B5, B6, and B7.

[0275] Embodiment 1-4. The composition of any one of embodiments 1-1 to 1-3, wherein the composition comprises one or more linkers.

[0276] Embodiment 1-5. The composition of any one of embodiments 1-2 to 1-4, wherein the IMV surface membrane protein is selected from the group consisting of LI and Ml; and wherein the IMV surface membrane protein comprises the amino acid sequence of any one of SEQ IDNOs: 6-7, 14, 21, 31-32, 41-42, or 48-50, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0277] Embodiment 1-6. The composition of any one of embodiments 1-3 to 1-5, wherein the EEV envelope glycoprotein is selected from the group consisting of B5, B6, and B7; and wherein the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 5, 11, 13, 15, 16, 20, 22, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0278] Embodiment 1-7. The composition of any one embodiment of embodiments 1-3 to 1-5, wherein the EEV envelope glycoprotein is selected from the group consisting of A33, A35, and A36; and wherein the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, 8-10, 12, 45, or 51-52, or an amino acid sequence having at least about 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0279] Embodiment 1-8. The composition of embodiment 1-7, wherein the EEV envelope glycoprotein forms a dimer.

[0280] Embodiment 1-9. The composition of embodiment 1-8, wherein the dimer is a single chain dimer.

[0281] Embodiment 1-10. The composition of any one embodiment of embodiments 1-2 to I- 9, wherein the at least one modification comprises a substitution, a deletion, or an insertion.

[0282] Embodiment 1-11. The composition of any one embodiment of embodiments 1-2 to I- 9, wherein the at least one modification comprises a substitution or deletion, wherein the substitution or deletion removes one or more N-linked glycosylation sites.

[0283] Embodiment 1-12. The composition of embodiment 1-11, wherein the EEV envelope glycoprotein is B6, and wherein the at least one modification is a substitution.

[0284] Embodiment 1-13. The composition of embodiment 1-12, wherein the substitution is in the B6 glycoprotein at a position corresponding to position 139 relative to SEQ ID NO: 39.

[0285] Embodiment 1-14. The composition of embodiment 1-13, wherein the substitution comprises substitution of cysteine (C) for isoleucine (I) at position 139 relative to SEQ ID NO: 39.

[0286] Embodiment 1-15. The composition of any one embodiment of embodiments 1-1 to I- 14, wherein the composition comprises at least three engineered Orthopoxvirus proteins.

[0287] Embodiment 1-16. The composition of embodiment 1-15, wherein the at least three engineered Orthopoxvirus proteins comprises: a. at least one intracellular mature viral (IMV) surface membrane protein; and b. at least two extracellular enveloped virus (EEV) envelope glycoprotein.

[0288] Embodiment 1-17. The composition of embodiment 1-16, wherein the IMV surface membrane protein comprises at least one modification relative to a wild type IMV surface membrane protein; and wherein the IMV surface membrane protein is selected from the group consisting of: LI and Ml.

[0289] Embodiment 1-18. The composition of embodiment 1-16 or 1-17, wherein the EEV envelope glycoprotein comprises at least one modification relative to a wild type EEV envelope glycoprotein; and wherein the EEV envelope glycoprotein is selected from the group consisting of: A33, A35, A36, B5, B6, and B7.

[0290] Embodiment 1-19. The composition of any one embodiment of embodiments 1-1 to I- 18, wherein the engineered Orthopoxvirus proteins comprise A35, B6, and Ml.

[0291] Embodiment 1-20. The composition of any one embodiment of embodiments 1-1 to I- 18, wherein the engineered Orthopoxvirus proteins comprise A33, B5, and LI.

[0292] Embodiment 1-21. The composition of any embodiment of embodiments 1-1 to 1-20, wherein the composition comprises a ferritin nanoparticle.

[0293] Embodiment 1-22. The composition of embodiment 1-21, wherein at least one engineered Orthopoxvirus protein is fused to a binding partner.

[0294] Embodiment 1-23. The composition of embodiment 1-22, wherein the binding partner is a ferritin subunit.

[0295] Embodiment 1-24. The composition of embodiment 1-23, wherein the ferritin subunit comprises a H. Pylori ferritin or a H. Pylori hybrid bullfrog ferritin.

[0296] Embodiment 1-25. The composition of embodiment 1-24, wherein the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 1, 18, or 82, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0297] Embodiment 1-26. The composition of any one of embodiments 1-23 to 1-25, wherein the ferritin subunit is fused to the engineered Orthopoxvirus proteins by a linker.

[0298] Embodiment 1-27. The composition of embodiment 1-26, wherein the amino acid sequence of the linker comprises the sequence of SEQ ID NO: 19 or a sequence having at least70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0299] Embodiment 1-28. A fusion protein comprising: a. a first Orthopoxvirus protein comprising an extracellular enveloped virus (EEV) envelope glycoprotein; b. a second Orthopoxvirus protein comprising an intracellular mature viral (IMV) surface membrane protein; and c. a third Orthopoxvirus protein comprising an extracellular enveloped virus (EEV) envelope glycoprotein.

[0300] Embodiment 1-29. The fusion protein of embodiment 1-28, wherein the fusion protein comprises one or more linkers.

[0301] Embodiment 1-30. The fusion protein of embodiment 1-28 or 1-29, wherein a. the first Orthopoxvirus protein comprises an extracellular enveloped virus (EEV) envelope glycoprotein selected from the group consisting of B5, B6, and B7; b. the second Orthopoxvirus protein comprises an intracellular mature viral (IMV) surface membrane protein selected from the group consisting of Ml and LI; and c. the third Orthopoxvirus protein comprises an extracellular enveloped virus (EEV) envelope glycoprotein selected from the group consisting of A33, A35, and A36.

[0302] Embodiment 1-31. The fusion protein of embodiment 1-30, wherein the thirdOrthopoxvirus protein comprises a first protein selected from the group consisting of A33, A35, and A36, wherein the first protein is linked to a second protein selected from the group consisting of A33, A35, and A36; and wherein said third Orthopoxvirus protein forms a single chain dimer.

[0303] Embodiment 1-32. The fusion protein of embodiment 1-30, wherein: a. the first Orthopoxvirus protein comprises B5; b. the second Orthopoxvirus protein comprises LI; and c. the third Orthopoxvirus protein comprises A33.

[0304] Embodiment 1-33. The fusion protein of embodiment 1-32, wherein: a. the first Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 13, 20, or 22 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto;b. the second Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 14, 21, 48, or 49 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto; and c. the third Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 12, 51, or 52 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0305] Embodiment 1-34. The fusion protein of embodiment 1-30, wherein: a. the first Orthopoxvirus protein comprises B6; b. the second Orthopoxvirus protein comprises Ml; and c. the third Orthopoxvirus protein comprises A35.

[0306] Embodiment 1-35. The fusion protein of embodiment 1-34, wherein: a. the first Orthopoxvirus protein comprises the amino acid saequence of any one of SEQ ID NOs: 5 or 15 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto; b. the second Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 6, 7, or 50 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto; and c. the third Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, 51, or 52 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0307] Embodiment 1-36. The fusion protein of any one embodiment of embodiments 1-28 to 1-35, wherein the fusion protein comprises, from N-terminal to C-terminal, an EEV envelope glycoprotein, an IMV surface membrane protein, and an EEV envelope glycoprotein.

[0308] Embodiment 1-37. The fusion protein of embodiment 1-36, wherein the fusion protein comprises, from N-terminal to C-terminal: a. A35, Ml, and B6; b. B6, Ml, and A35; or c. B5, LI, and A33.

[0309] Embodiment 1-38. The fusion protein of any one embodiment of embodiments 1-28 to 1-35, wherein the fusion protein comprises, from N-terminal to C-terminal, an IMV surface membrane protein, an EEV envelope glycoprotein, and an EEV envelope glycoprotein.

[0310] Embodiment 1-39. The fusion protein of embodiment 1-38, wherein the fusion protein comprises, from N-terminal to C-terminal: a. Ml, A35, and B6; or b. Ml, B6, and A35.

[0311] Embodiment 1-40. The fusion protein of any one embodiment of embodiments 1-28 to 1-35, wherein the fusion protein comprises, from N-terminal to C-terminal, an EEV envelope glycoprotein, an EEV envelope glycoprotein, and an IMV surface membrane protein.

[0312] Embodiment 1-41. The fusion protein of embodiment 1-40, wherein the fusion protein comprises, from N-terminal to C-terminal: a. A35, B6, and Ml; or b. B6, A35, and Ml.

[0313] Embodiment 1-42. The fusion protein of any one embodiment of embodiments 1-36 to 1-38, wherein the EEV envelope glycoprotein comprises a single chain dimer.

[0314] Embodiment 1-43. The fusion protein of any one embodiment of embodiments 1-28 to 1-39, wherein each of the Orthopoxvirus proteins comprises at least one modification relative to a wild type Orthopoxvirus protein.

[0315] Embodiment 1-44. The fusion protein of any one embodiment of embodiments 1-28 to 1-40, wherein the fusion protein comprises one or more ferritin subunits.

[0316] Embodiment 1-45. The fusion protein of embodiment 1-41, wherein the one or more ferritin subunits are selected from the group consisting of H. Pylori ferritin and H. Pylori hybrid bullfrog ferritin.

[0317] Embodiment 1-46. The fusion protein of any one embodiment of embodiments 1-28 to 1-42, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 46, 47, 66- 70, 75, or 76 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0318] Embodiment 1-47. The fusion protein of any one embodiment of embodiments 1-28 to 1-40, wherein the fusion protein comprises an ESCRT-recruiting domain (ERD).

[0319] Embodiment 1-48. The fusion protein of embodiment 1-45, wherein the ERD comprises or is derived from an ESCRT and ALIX binding region (EABR).

[0320] Embodiment 1-49. The fusion protein of embodiment 1-46, wherein the ERD comprises the amino acid sequence of SEQ ID NO: 77 or 78, or a sequence having at least 70%sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0321] Embodiment 1-50. The fusion protein of any one embodiment of embodiments 1-47 to 1-49, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 73 or 74 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0322] Embodiment 1-51. The fusion protein of any one of embodiments 1-28 to 1-47, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 23-26, 38, 43- 44, 46, 47, or 66-76 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0323] Embodiment 1-52. An engineered Orthopoxvirus protein comprising an engineered IMV surface membrane protein, wherein the engineered IMV surface membrane protein comprises at least one modification relative to a wild type IMV surface membrane protein.

[0324] Embodiment 1-53. The engineered Orthopoxvirus protein of embodiment 1-52, wherein the IMV surface membrane protein is selected from the group consisting of LI and Ml.

[0325] Embodiment 1-54. The engineered Orthopoxvirus protein of embodiment 1-52 or 1-53, wherein the engineered IMV surface membrane protein comprises the amino acid sequence of any one of SEQ ID NOs: 6-7, 14, 21, 31-32, 41-42, or 48-50, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0326] Embodiment 1-55. An engineered Orthopoxvirus protein comprising an engineered EEV envelope glycoprotein, wherein the engineered EEV envelope glycoprotein comprises at least one modification relative to a wild type EEV envelope glycoprotein.

[0327] Embodiment 1-56. The engineered Orthopoxvirus protein of embodiment 1-55, wherein the EEV envelope glycoprotein is selected from the group consisting of: A33, A35, A36, B5, B6, and B7.

[0328] Embodiment 1-57. The engineered Orthopoxvirus protein of embodiment 1-56, wherein the EEV envelope glycoprotein is selected from the group consisting of B5, B6, and B7; and wherein the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 5, 11, 13, 15, 16, 20, 22, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0329] Embodiment 1-58. The engineered Orthopoxvirus protein of embodiment 1-56, wherein the EEV envelope glycoprotein is selected from the group consisting of A33, A35, and A36; and wherein the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, 8-10, 12, 45, or 51-52, or an amino acid sequence having at least about 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0330] Embodiment 1-59. The engineered Orthopoxvirus protein of any one of embodiments 1-55 to 1-58, wherein the at least one modification comprises a substitution or deletion, wherein the substitution or deletion removes one or more N-linked glycosylation sites.

[0331] Embodiment 1-60. The engineered Orthopoxvirus protein of embodiment 1-59, wherein the engineered Orthopoxvirus protein comprises a B6 glycoprotein, wherein the at least one modification is a substitution.

[0332] Embodiment 1-61. The engineered Orthopoxvirus protein of embodiment 1-60, wherein the substitution is in the B6 glycoprotein at a position corresponding to position 139 relative to SEQ ID NO: 39.

[0333] Embodiment 1-62. The engineered Orthopoxvirus protein of embodiment 1-61, wherein the substitution comprises substitution of cysteine (C) for isoleucine (I) at position 139 relative to SEQ ID NO: 39.

[0334] Embodiment 1-63. The engineered Orthopoxvirus protein of any one of embodiments 1-58 to 1-62, wherein the engineered Orthopoxvirus protein comprises two EEV envelope glycoproteins, wherein the two EEV envelope glycoproteins form a dimer.

[0335] Embodiment 1-64. The engineered Orthopoxvirus protein of embodiment 1-63, wherein the two EEV envelope glycoproteins are the same.

[0336] Embodiment 1-65. The engineered Orthopoxvirus protein of embodiment 1-64, wherein the two EEV envelope glycoproteins are A33.

[0337] Embodiment 1-66. The engineered Orthopoxvirus protein of embodiment 1-63, wherein the two EEV envelope glycoproteins are not the same.

[0338] Embodiment 1-67. The engineered Orthopoxvirus protein of embodiment 1-66, wherein the two EEV envelope glycoproteins comprise: a. A33 and A35; b. A33 and A36; or c. A35 and A36.

[0339] Embodiment 1-68. The engineered Orthopoxvirus protein of any one of embodiments 1-63 to 1-67, wherein the dimer is a single chain dimer.

[0340] Embodiment 1-69. The engineered Orthopoxvirus protein of any one of embodiments 1-52 to 1-68, wherein the engineered Orthopoxvirus protein is fused to a binding partner.

[0341] Embodiment 1-70. The engineered Orthopoxvirus protein of embodiment 1-69, wherein the binding partner is a ferritin subunit.

[0342] Embodiment 1-71. The engineered Orthopoxvirus protein of embodiment 1-70, wherein the ferritin subunit comprises a H. Pylori ferritin or a H. Pylori hybrid bullfrog ferritin.

[0343] Embodiment 1-72. The engineered Orthopoxvirus protein of embodiment 1-71, wherein the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 1, 18, or 82, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0344] Embodiment 1-73. The engineered Orthopoxvirus protein of any one of embodiments 1-70 to 1-72, wherein the ferritin subunit is fused to the engineered Orthopoxvirus protein by a linker.

[0345] Embodiment 1-74. The engineered Orthopoxvirus protein of embodiment 1-73, wherein the linker is a flexible linker.

[0346] Embodiment 1-75. The engineered Orthopoxvirus protein of embodiment 1-73, wherein the linker is a rigid linker.

[0347] Embodiment 1-76. The engineered Orthopoxvirus protein of embodiment 1-73, wherein the amino acid sequence of the linker comprises the sequence of SEQ ID NO: 19 or a sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0348] Embodiment 1-77. A ferritin nanoparticle comprising the composition of any one of embodiments 1-23 to 1-27, the fusion protein of any one of embodiments 1-44 to 1-46, or the engineered Orthopoxvirus protein of any one of embodiments 1-70 to 1-76.

[0349] Embodiment 1-78. An eVLP comprising the fusion protein of any one of embodiments 1-28 to 1-43 or 47-50 or the engineered Orthopoxvirus protein of any one of embodiments 1-52 to 1-69.

[0350] Embodiment 1-79. A polynucleotide encoding the amino acid sequence of: a. the one or more of the engineered Orthopoxvirus proteins of the compositions of any one of embodiments 1-1 to 1-27,b. the fusion protein of any one of embodiments 1-28 to 1-51; or c. the engineered Orthopoxvirus protein of any one of embodiments 1-52 to 1-76.

[0351] Embodiment 1-80. The polynucleotide of embodiment 1-79, wherein the polynucleotide comprises DNA.

[0352] Embodiment 1-81. The polynucleotide of embodiment 1-79, wherein the polynucleotide comprises RNA.

[0353] Embodiment 1-82. The polynucleotide of embodiment 1-81, wherein the RNA is mRNA.

[0354] Embodiment 1-83. A vaccine composition comprising one or more of: a. the compositions of any one of embodiments 1-1 to 1-27, b. the fusion protein of any one of embodiments 1-28 to 1-51; c. the engineered Orthopoxvirus protein of any one of embodiments 1-52 to 1-76; d. the ferritin nanoparticle of embodiment 1-77; e. the eVLP of embodiment 1-78; or f. the polynucleotide of any one of embodiments 1-79 to 1-82; and g. one or more adjuvants.

[0355] Embodiment 1-84. The vaccine composition of embodiment 1-83, wherein the one or more adjuvants comprises alum and / or CpG.

[0356] Embodiment 1-85. A pharmaceutical composition comprising: a. the compositions of any one of embodiments 1-1 to 1-27, b. the fusion protein of any one of embodiments 1-28 to 1-51; c. the engineered Orthopoxvirus protein of any one of embodiments 1-52 to 1-76; d. the ferritin nanoparticle of embodiment 1-77; e. the eVLP of embodiment 1-78; f. the polynucleotide of any one of embodiments 1-79 to 1-82; or g. the vaccine compositions of embodiment 1-83 or 1-84.

[0357] Embodiment 1-86. A method of preventing an Orthopoxvirus infection or a disease or symptom associated with an Orthopoxvirus infection in a subject, comprising administering to the subject an effective amount of: a. the compositions of any one of embodiments 1-1 to 1-27, b. the fusion protein of any one of embodiments 1-28 to 1-51; c. the engineered Orthopoxvirus protein of any one of embodiments 1-52 to 1-76;d. the ferritin nanoparticle of embodiment 1-77; e. the eVLP of embodiment 1-78; f. the polynucleotide of any one of embodiments 1-79 to 1-82; g. the vaccine compositions of embodiment 1-83 or 1-84; or h. the pharmaceutical composition of embodiment 1-85.

[0358] Embodiment 1-87. A method of vaccinating a subject against an Orthopoxvirus infection or a disease associated with an Orthopoxvirus infection, comprising administering to the subject an effective amount of: a. the compositions of any one of embodiments 1-1 to 1-27, b. the fusion protein of any one of embodiments 1-28 to 1-51; c. the engineered Orthopoxvirus protein of any one of embodiments 1-52 to 1-76; d. the ferritin nanoparticle of embodiment 1-77; e. the eVLP of embodiment 1-78; f. the polynucleotide of any one of embodiments 1-79 to 1-82; g. the vaccine compositions of embodiment 1-83 or 1-84; or h. the pharmaceutical composition of embodiment 1-85.EXAMPLESExample 1: Constructs of Engineered EEV Envelope Proteins and Engineered IMV Surface membrane proteins of Vaccinia and MPOX

[0359] EEV envelope proteins and IMV surface membrane proteins from Vaccinia virus and Mpox virus were designed from full-length amino acid sequences that come from Vaccinia (VACV), Variola (VARV), and / or mpox (MPXV) including A33 (VACV) / A35 (MPXV) / A36 (VARV), B5 (VACV) / B6 (MPXV) / B7 (VARV), and LI (VACV) / M1(VARV / MPXV). The constructs encoding the engineered EEV envelope proteins or engineered IMV surface membrane proteins were designed as monomers, monomers coupled to other monomers, dimers, or monomers coupled to dimers. As described above the engineered EEV envelope proteins or engineered IMV surface membrane proteins may comprise one or more modifications relative to native (i.e., naturally occurring) EEV envelope proteins or native IMV surface membrane proteins, wherein the modifications include but are limited to one or more substitutions, one or more truncations, one or more insertions, and / or one or more deletions.

[0360] The constructs encoding full-length and C-terminally truncated engineered EEV envelope proteins (in monomer or dimer form) or engineered IMV surface membrane proteins were transferred into an expression vector and were cloned. For some constructs, cloning was followed by stitching PCR in which the constructs were annealed to an amplicon encoding a SGG linker followed by a hybrid bullfrog linker (e.g., SEQ ID NO: 19) followed by residues 5- 168 of a H. Pylori ferritin sequence (SEQ ID NO: 1) or the amino acid sequence of SEQ ID NO: 18, thus generating the fusion proteins of the disclosure. The gene encoding Helicobacter pylori- bullfrog hybrid ferritin was constructed by fusing residues 2-9 of bullfrog (Rana catesbeiana) ferritin lower subunit (UniProt: P07797 with a N8Q mutation to abolish a potential N- glycosylation site) to H. Pylori nonheme ferritin (UniProt: Q9ZLI1, residues 3—167) with an I7E mutation to preserve the conserved salt bridge found in human and bullfrog ferritins (6R and 14E in both human light chain and bullfrog lower-subunit ferritins) with 6R of bullfrog ferritin. Mutation N19Q was introduced in H. Pylori ferritin to abolish a potential N-glycosylation site. The sequences were confirmed using Sanger Sequencing.

[0361] All proteins were expressed in ExpiCHO cells. ExpiCHO cells and were cultured using ExpiCHO media (ThermoFisher) and grown in polycarbonate shaking flasks at 37 degrees C in 5% CO2. The cells were transfected at a density of approximately 6 x 106cells / mL. Transfections were conducted using the ExpiCHO transfection kit (ThermoFisher) following manufacturer’s guidelines. The cells were harvested 5-7 days post-transfection by spinning the cultures at 7,000-15,000 x g for 10-30 minutes. Supernatants were filtered using a 0.22-pm filter.

[0362] Following purification, all proteins were analyzed using SDS-PAGE analysis under reducing (R) or non-reducing (NR) conditions. 1 pg protein antigens from Vaccinia or mpox were diluted using LDS loading buffer + / - betamercaptoethanol, boiled at 95 °C for 5 min and loaded onto a denaturing SDS-PAGE gel and protein bands were visualized using GelCode Blue staining reagent, as shown in FIGs. 3A-3B.

[0363] Denaturing SDS-PAGE analysis was performed by diluting proteins with Laemmli sample buffer (with betamercaptoethanol for reducing conditions and without betamercaptoethanol for non-reduced conditions) and boiling samples for 5 min at 95 °C. Samples were then loaded on a 4-20% MiniPROTEAN TGX precast gel and run for 30 min at 230 V. Gels were stained using GelCode Blue staining reagent and imaged using a Thermo Fisher Scientific imager. FIG. 3A depicts an image of the SDS-PAGE gel, demonstratingexpression each of the A33 dimer (approximately 22.9 kDa in Lane 1), A33 ferritin (approximately 64.3 kDa in Lane 2), B5 monomer (approximately 25.8 kDa in Lane 3), LI monomer (approximately 20.5 kDa in Lane 4), and LI ferritin (approximately 40.7 kDa in Lane 5).

[0364] Denaturing SDS-PAGE analysis was performed by diluting proteins with Laemmli sample buffer (with betamercaptoethanol for reducing conditions and without betamercaptoethanol for non-reduced conditions) and boiling samples for 5 min at 95 °C. Samples were then loaded on a 4-20% MiniPROTEAN TGX precast gel and run for 30 min at 230 V. Gels were stained using GelCode Blue staining reagent and imaged using a Thermo Fisher Scientific imager. FIG. 3B depicts an image of the SDS-PAGE gel, demonstrating expression each of the A35 dimer (in Lane 1), A35 ferritin (in Lane 2), B6 monomer (in Lane 3), B6 ferritin (in Lane 4), Ml monomer (in Lane 5), and Ml ferritin (in Lane 6).Transmission Electron Microscopy (“TEM”) analysis of engineered Vaccinia proteins and engineered MPOX proteins

[0365] For TEM analysis, purified engineered Vaccinia fusion proteins (A33 or LI with ferritin) or purified engineered mpox fusion proteins (A35 or Ml with ferritin) were diluted using 20 mM Tris pH 7.5, 150 mM sodium chloride. 3.5 pL of sample was placed on a carbon- coated, copper, transmission EM grid that had been glow -discharged to make it more hydrophilic. After approximately one minute, filter paper was used to remove excess solution from the grid by touching the side of the grid. This was followed by two steps of brief (approx. 1-2 seconds) wash in 20 mM Tris pH 7.5, 150 mM sodium chloride and blotting with filter paper in the same manner. Next, this process was repeated twice but with the negative stain solution (1% ammonium molybdate). Finally, the grid was incubated for 15-20 seconds in a droplet of 1% ammonium molybdate, blotted again, and allowed to air dry. Negatively stained specimens were viewed on a JEOL JEM1400-Plus transmission electron microscope operated at 120 kV. Images were recorded on a Gatan Orius camera.

[0366] FIGs. 4A-4D depict exemplary transmission electron microscopy images. FIG. 4A depicts assembled ferritin nanoparticles with the engineered EEV envelope glycoprotein VACV A33 and VARV A35 as single-chain dimers on the surface. FIG. 4B depicts assembled ferritin nanoparticles with the engineered IMV surface membrane protein LI as a monomer on the surface. FIG. 4C depicts assembled ferritin nanoparticles with the engineered EEV envelope glycoprotein A35 as a dimer on the surface. FIG. 4D depicts assembled ferritin nanoparticles with the engineered IMV surface membrane protein Ml as a monomer on the surface. For eachof the purified engineered Vaccinia fusion proteins or the engineered MPXV fusion proteins, the nanoparticles exhibit an expected diameter that corresponds to the size of a ferritin nanoparticle with an engineered EEV envelope glycoprotein a dimer on the surface, or an engineered IMV surface membrane protein as a monomer on the surface.Purification of Engineered EEV Envelope Glycoproteins and Engineered IMV Surface membrane proteins Antigens

[0367] Antigens comprising monomer proteins and fusion proteins of Vaccinia were purified from ExpiCHO supernatants obtained 5-7 days following transient transfection. Supernatants were filtered through a 0.22-pm filter. Proteins were purified either via anion exchange chromatography on a HiTrap Q HP column (A33 and A35 ferritins), a tandem purification on a HiTrap CQI followed by HiTrap Q HP (LI and Ml ferritins), or via NiNTA gravity flow purification (B6 ferritin and 3-in-l ferritins). Following these initial purification steps, proteins were purified with a final size-exclusion chromatography step on an S6 (Cytiva) preparativescale column pre-equilibrated in 20 mM Tris pH 7.5, 150 mM NaCl. Protein-containing fractions were assessed using SDS-PAGE or western blot analysis and fractions containing homogenous protein were pooled. Fractions were then supplemented with 5% sucrose for freezing (final formulation 5% sucrose, 20 mM Tris pH 7.5, 150 mM sodium chloride), frozen in liquid nitrogen, and stored at -80° C until use.Antigen Preparation for Immunization

[0368] Antigens comprising monomer proteins (e.g., A33, or LI) or fusion proteins (A33- ferritin, LI ferritin, A33+L1 ferritin), purified as described above, were formulated in Tris pH 7.5, 150 mM NaCl, 5% sucrose, sterile filtered through an 0.22 pm filter, flash frozen on liquid nitrogen, and stored at -80 C. Prior to immunization, some antigens were combined (e.g., A33 + LI or A33+L1+B5) and frozen as either individual component or combinations. On the day of immunization, antigens were thawed and Alhydrogel (aluminum hydroxide) adjuvant was added to a final aluminum concentration of 1.5 mg / mL and CpG (ODN2395, Invivogen) was added to a final concentration of 0.2 mg / mL, bringing the total Orthopox antigen concentration to a final concentration of 0.05 mg / mLDelivery of Vaccine Compositions of Engineered Vaccinia Proteins as Monomers or fused toFerritin to Mice

[0369] To investigate the effectiveness of engineered Vaccinia proteins as monomers or fusion proteins as vaccine compositions, Vaccinia proteins (e.g., A33, LI) alone or in combination (A33+L1 or A33 +L1 +B5) or Vaccinia fusion proteins (e.g., A33 ferritin, LI ferritin, or A33 + LI ferritin) was isolated and administered to mice as described below.

[0370] Just prior to injection, antigen-adjuvant mixtures were inverted 50 times. 50 pL of the adjuvanted vaccine candidates described above were injected intramuscularly into both hind legs of approximately 8-week-old female, vaccine-naive BALB / c mice (100 pL total injection volume, resulting in delivery of 5 pg engineered Vaccinia antigen, and 150 pg of aluminum, as aluminum hydroxide, and 20 pg CpG). Mice were immunized on study day 0 and day 21. 35 days after the first injection (14 days after the second injection), whole blood was collected and centrifuged to isolate serum, which was frozen at -80 C and subsequently thawed, heat inactivated at 56° C for 30 minutes, and used for neutralization assays, described below

[0371] FIG. 5A depicts a graph of IC50 values obtained from mice administered A33, LI monomer, a combination of A33 and LI, a combination of A33, LI and B5, the A33 ferritin nanoparticle, the LI ferritin nanoparticle or the combination of the A33 and LI ferritin nanoparticles. Each point represents the serum IC50 value obtained from an individual mouse.

[0372] These results demonstrate that LI monomer, and the combination of LI and A33 or A33, LI and B5 as monomer groups are immunogenic in mice as determined by an intracellular mature viral neutralization assay using a GFP reporter virus. However, the nanoparticles of LI or LI +A33 demonstrated the most immunogenicity in mice. A33 as a monomer or as a fusion protein with ferritin demonstrated very little IMV neutralizing activity in mice.

[0373] Neutralizing antibody responses demonstrate that these engineered Vaccinia monomers or engineered Vaccinia nanoparticles display the engineered Vaccinia proteins in conformations that elicits functional antibodies capable of blocking viral entry into cells in a live-virus neutralization assay using a GFP-reporter VACV.

[0374] To evaluate the engineered Vaccinia proteins, specifically the EEV envelope glycoproteins (A33 and LI) against a vaccinia virus, a virus comet assay was performed. Cells were infected with a Vaccinia virus and incubated for a period of time, according to standard techniques in the presence of serum from mice immunized with VACV antigens (or alone in the control samples). After an approximate incubation time, the cells were fixed with 4% paraformaldehyde, stained with crystal violet and allowed time to dry. The cells were then photographed and a representative image is depicted in FIG. 5B.Antibody Binding Analysis using BLI to engineered mpox proteins

[0375] Biolayer interferometry (BLI) analysis of engineered mpox Proteins (e.g., A35, B6, or Ml) was performed using an Octet R8 instrument with Protein A functionalized tips. Antibodies were diluted to 10 pg / mL and histidine tagged engineered mpox protein monomers (e.g., A35- His, B6-His, or Ml-His) or engineered mpox fusion protein nanoparticles (e.g., A35-Fer, B6- Fer, or Ml-Fer) were diluted to 5 pg / mL in 10X Kinetics buffer (PBS + 0.1% BSA, 0.02% Tween20 and Kathon) and pipetted into black-walled, black-bottom plates (200 pL per well). Protein A tips were dipped into antibody wells and subsequently dipped into either histidine tagged engineered mpox protein monomer wells or engineered mpox fusion protein nanoparticle wells to assess binding association for 120 sec. Dissociation was measured for 120 sec by moving tips into a buffer only well.

[0376] FIG. 6A depicts a graph of the biolayer interferometry analysis of neutralizing antibodies binding to histidine tagged engineered mpox protein A35 or the the engineered mpox fusion protein nanoparticles A35-Fer. FIG. 6B depicts a graph of the biolayer interferometry analysis of neutralizing antibodies binding to histidine tagged engineered mpox protein B6 or the engineered mpox fusion protein nanoparticles B6-Fer. FIG. 6C depicts a graph of biolayer interferometry analysis of neutralizing antibodies binding to histidine tagged engineered mpox protein Ml or the the engineered mpox fusion protein nanoparticles Ml-Fer. For each of the engineered mpox proteins, off-target binding was low, while binding to the either the histidine tagged engineered mpox proteins or the engineered mpox fusion protein nanoparticles was notably higher at all time points. This may demonstrate that MPXV ferritin antigen constructs show antigenicity to known Orthopoxvirus neutralizing antibodies.Evaluating mpox ferritin antigen 3-in-l constructs using BLI for Neutralizing Antibody Analysis

[0377] Two Mpox fusion protein constructs were generated, as described above, that comprised two mpox EEV envelope proteins (A35 and B6) with an IMV surface membrane protein (Ml) and a ferritin sequences as a “3-in-l” antigen design. The first mpox fusion protein construct comprised an orientation N-terminal to C-terminal of A35, B6, and Ml with a histidine tag and coupled to a ferritin sequence (“ABM His Ferritin”) (e.g., SEQ ID NO: 46). The second mpox fusion protein construct comprised an orientation of N-terminal to C-terminal of a histidine tagged B6, Ml, A35 coupled to a ferritin sequence (“His BMA Ferritin”) (e g., SEQ ID NO: 47).

[0378] FIGs. 7A-7H depict graphs of the BLI analysis of either A35 targeting antibodies binding to fusion proteins (FIGs. 7A-7B), B6 targeting antibodies binding to fusion proteins (FIGs. 7C-7D), or Ml targeting antibodies binding to fusion proteins (FIGs. 7E-7H).

[0379] As shown in FIGs. 7A-7B, A35 targeting antibodies were capable of binding to all constructs including the A35-Fer fusion protein and each of the 3-in-l fusion proteins comprising A35, B6, and Ml. Similarly, as shown in FIGs. 7C-7D, B6 targeting antibodies were capable of binding to all constructs including the B6-HisFer fusion protein and each of the 3-in-l fusion proteins comprising A35, B6, and Ml. The Ml targeting antibodies bound to each of the 3-in-l fusion proteins. This may demonstrate that the mpox “3-in-l” constructs show antigenicity to known Orthopoxvirus neutralizing antibodies.Example 2: Two-dose immunization and VACV challenge

[0380] To test the effectiveness of various vaccine compositions of the disclosure, a Vaccinia (VACV) virus challenge study was performed, as depicted in FIG. 8. BALB / c mice (n = 5) were immunized with either adjuvant only, Modified Vaccinia Ankara (MV A), or protein antigens adjuvanted with Alhydrogel (150 pg) and CpG (20 pg). As depicted in FIG. 9A, protein antigens used for immunization were either soluble proteins (denoted as Subunit), protein nanoparticles (denoted as Nanoparticle), or fusion protein nanoparticles (denoted as ABM fusion or BMA fusion), administered at a dose of either 0.5 pg per antigen or 2 pg per antigen.

[0381] Table 5 below provides the antigen and dose administered to each experimental group of mice in the study, along with graph labels used to refer to each group. ABM refers to a fusion protein comprising, from N-terminal to C-terminal, A35, B6, and Ml. BMA refers to a fusion protein comprising, from N-terminal to C-terminal, B6, Ml, and A35.Table 5: VACV challenge study groups

[0382] Mice were immunized on study Day 0 (“prime”) and Day 21 (“homologous boost”) and serum was collected on Day 0, Day 21, and Day 35. Mice were challenged with a lethal dose of VACV on study Day 35 and either sacrificed when they reached 70% body weight or at 14 days post challenge (Day 49 of study).

[0383] Binding titers were evaluated by ELISA against either MPXV A35, B6, or Ml antigens with serum collected on study Day 35. As shown in FIG. 9B, antigens delivered as protein nanoparticles (Nanoparticle) or fusion protein nanoparticles (ABM fusion or BMA fusion) induced significantly higher serum titers against A35 compared to soluble protein (Subunit) at both 0.5pg and 2pg doses. Antigens delivered as fusion protein nanoparticles also induced higher titers against A35 compared to protein nanoparticles. Antigens delivered as protein nanoparticles (Nanoparticle) or fusion protein nanoparticles (ABM fusion or BMA fusion) induced higher serum titers against Ml compared to soluble protein (Subunit) at the 0.5 pg and 2pg doses. Antigens delivered as protein nanoparticles (Nanoparticle) or fusion protein nanoparticles (BMA fusion) induced higher serum titers against B6 compared to soluble protein (Subunit) at the 0.5 pg dose. Taken together, these results indicate that antigens delivered as protein nanoparticles or fusion protein nanoparticles generally induce significantly higher titers or at least similar titers against A35, B6, and Ml when compared to antigens delivered as soluble proteins.

[0384] VACV neutralization was evaluated using a VACV-WR GFP reporter virus with serum collected on study Day 35. The reciprocal dilution of serum required to inhibit viral infection by 50% (neutralizing antibody titer at 50% inhibition [NT50]) was determined. As shown in FIG. 9C, serum from mice immunized with antigens delivered as protein nanoparticles or fusion protein nanoparticles neutralized VACV more effectively compared to that from mice immunized with antigens delivered as soluble antigens.

[0385] Body weight was recorded for 11 days post challenge for each mouse. As shown in FIG. 9D, body weights for mice treated with adjuvant only decreased significantly post challenge.

[0386] Taken together, these results show that antigens delivered as protein nanoparticles or fusion protein nanoparticles using ferritin nanoparticles can be an effective two-dose vaccine and can be more effective as a vaccine compared to antigens delivered as soluble antigens.I l lExample 3: Single dose immunization and VACV challenge

[0387] To test the effectiveness of various vaccine compositions of the disclosure, a Vaccinia virus (VACV) challenge study was performed, as depicted in FIG. 10A. BALB / c mice (n = 5) were immunized with either adjuvant only, Modified Vaccinia Ankara (MV A), or protein antigens adjuvanted with Alhydrogel (150 pg) and CpG (20 pg). Protein antigens included either soluble protein, protein nanoparticles, or fusion protein nanoparticles administered at a dose of either 0.5 pg per antigen or 2 pg per antigen. Mice were immunized on study Day 0 and serum was collected on Day 0 and Day 21. Mice were challenged with a lethal dose of VACV on study Day 21 and either sacrificed when they reached 70% body weight or at 14 days post challenge (Day 35 of study).

[0388] Binding titers were evaluated by ELISA against either MPXV A35, B6, or Ml antigens with serum collected on study Day 21. As shown in FIG. 10B, antigens delivered as protein nanoparticles (Nanoparticle) or fusion protein nanoparticles (ABM fusion or BMA fusion) induced significantly higher serum titers against A35 compared to soluble protein (Subunit) at both 0.5pg and 2pg doses. Antigens delivered as protein nanoparticles (Nanoparticle) or fusion protein nanoparticles (ABM fusion or BMA fusion) also induced higher serum titers against Ml compared to soluble protein (Subunit) at the 0.5pg and 2pg doses. Antigens delivered as protein nanoparticles (Nanoparticle) or fusion protein nanoparticles (BMA fusion) induced similar serum titers against B6 compared to soluble protein (Subunit) at the 0.5pg dose. Taken together, these results indicate that antigens delivered as protein nanoparticles or fusion protein nanoparticles generally induce significantly higher titers or at least similar titers against A35, B6, and Ml when compared to antigens delivered as soluble proteins.

[0389] VACV neutralization was evaluated using a VACV-WR GFP reporter virus with serum collected on Day 21 and the NT50 was determined. As shown in FIG. 10C, serum from mice immunized with antigens delivered as protein nanoparticles or fusion protein nanoparticles neutralized VACV similarly to that from mice immunized with antigens delivered as soluble antigens at the 0.5pg dose. At the 2pg dose, serum from mice immunized with antigens delivered as protein nanoparticles or fusion protein nanoparticles neutralized VACV more effectively compared to that from mice immunized with antigens delivered as soluble antigens.

[0390] Body weight was recorded for 11 days post challenge for each mouse. As shown in FIG. 10D, body weights for mice treated with adjuvant only decreased significantly post challenge. Body weights for mice treated with high dose (2pg) ABM, high-dose (2 .g) BMA,and high-dose (2pg) soluble antigens also decreased significantly post challenge but body weights recovered by the end of the study.

[0391] Taken together, these results show that antigens delivered as protein nanoparticles or fusion protein nanoparticles using ferritin nanoparticles can be effective as a single dose vaccine.Example 4: Mammalian expression of ABM-12L-Fer fusion protein construct

[0392] ABM-12L-Fer fusion protein was purified from Expi293F cells and analyzed for binding to antigen specific monoclonal antibodies (mAbs) targeting A35 (A27D7), B6 (8AH8AL), and Ml (7D11) via biolayer interferometry. ABM refers to a fusion protein comprising, from N-terminal to C-terminal, A35, B6, and Ml. The mAbs were loaded onto protein A functionalized tips and dipped into diluted antigens. ABM nanoparticles were compared to soluble antigens A35, B6, and Ml (denoted as monomers). As shown in FIG. 11, each mAb targeting A35, B6, or Ml bound to the corresponding soluble antigen as expected. Each mAb also bound to the ABM-12L-Fer fusion protein, suggesting that the structure of A35, B6, and Ml as presented on the ferritin nanoparticle is preserved.Example 4: Mammalian expression of secreted and cell-anchored forms of the A35-B6-M1 antigen

[0393] Plasmids encoding A35-B6-M1 fusion protein as either a ferritin nanoparticle, a soluble monomer, a transmembrane (TM)-anchored form, or an EABR-tagged antigen, as depicted in FIG. 12A, were transfected into Expi293F mammalian cells. Cell supernatants were evaluated via dot blot, in which 2 pL of cell supernatant was blotted onto a nitrocellulose membrane and probed with either an A35-, B6-, or Ml -specific mAb. Truncations to the C- terminal tail of the membrane anchored (TM and EABR) forms were evaluated, denoted by either WT (full-length C-terminal tail), A35 (with the 35 amino acids at the C-terminal tail deleted), or A45 (with the 45 amino acids at the C-terminal tail deleted). As shown in FIG. 12B, fusion protein expressed on a ferritin nanoparticle or as a soluble monomer were readily detected in the cell supernatant. Fusion protein expressed in a transmembrane (TM)-anchored form were detected minimally in the cell supernatant. Addition of an EABR to the TM-anchored fusion protein led to generation of eVLPs displaying the antigens, with A35 and B6 readily detected in the cell supernatant. Truncated forms of the antigens were comparable to the full-length form in expression.

[0394] Cell supernatants from Expi293F cells expressing A35-B6-M1 fusion protein as either a transmembrane (TM)-anchored form or an EABR-tagged antigen, as depicted in FIG. 13A, were also evaluated via flow cytometry by staining with an antigen-specific mAb. As shown in FIG. 13B, a greater proportion of cells transfected with EABR-tagged A35-B6-M1 fusion protein were stained by A35-, B6-, and Ml -targeting antibodies compared to cells transfected with the TM-anchored form of A35-B6-M1 fusion protein. These results show that inclusion of an EABR sequence in a construct can facilitate improved cell-surface display of antigens.Example 5: Evaluation of linker length and antigen positions in fusion constructs

[0395] To evaluate the effects of various linker lengths in fusion constructs, constructs encoding either a 3 amino acid (3L), 6 amino acid (6L), or 12 amino acid (12L) linker between the A35-B6-M1 encoded region were designed as a ferritin nanoparticle, in a TM-anchored form, or as an EABR-tagged antigen. Mammalian cells were transfected with plasmids encoding these constructs under control of a CMV promoter and cell supernatants were collected. Supernatants were blotted on nitrocellulose membranes and probed with either an A35-, B6-, or Ml -targeting antibody. As shown in FIG. 14, linker length did not affect expression of B6 or A35 while a shorter linker length may improve Ml expression for the ferritin nanoparticle design. Linker length also did not affect expression of B6 or A35 for the EABR design, while a longer linker may improve Ml expression for the EABR design.

[0396] To evaluate the effects of the positions of each antigen in a fusion construct, constructs containing A35, B6, and Ml proteins in different orientations, as shown in Table 6 below, were designed as ferritin nanoparticles using a H. Pylori ferritin sequence.Table 6: Fusion constructs with different antigen orientations

[0397] Plasmids encoding the constructs under control of a CMV promoter were transfected into Expi293F mammalian cells and supernatants were collected. Supernatants were blotted ontonitrocellulose membranes and probed with either A35-, B6-, or Ml -targeting antibodies. As shown in FIG. 15, A35, B6, and Ml were all detected in the supernatant, regardless of the N- terminal to C-terminal order of the three antigens. This result indicates that antigen order does not largely impact expression of the fusion nanoparticle.Example 6: Evaluation of immunization with protein or mRNA-encoded antigens

[0398] To test the effectiveness of various vaccine compositions of the disclosure, a Vaccinia virus (VACV) challenge study was performed, as depicted in FIG. 16A. BALB / c mice (n = 5) were immunized with either adjuvant only, Modified Vaccinia Ankara (MV A), or protein antigens adjuvanted with Alhydrogel (150 pg) and CpG (20 pg) or mRNA constructs encoding various antigen designs. Protein antigens included protein nanoparticles (NP combo, with each antigen expressed on different nanoparticles) or fusion protein nanoparticles (Fusion NP, with fusion protein comprising A35, B6, and Ml expressed on the same nanoparticle) administered at a dose of 2 pg per antigen. mRNA antigens included an off-target mRNA, a combination of A35, B6, Ml, and A29 encoded on different mRNAs (at 0.5 pg per mRNA), or the A35-B6-M1 antigen encoded as either a soluble, ferritin, membrane-anchored (TM), or EABR-tagged antigen, administered at a dose of 2 pg. Mice were immunized on study Day 0 and serum was collected on Day 0 and Day 21. Mice were challenged with live VACV on study day 21 and either sacrificed when they reached 70% body weight or at 14 days post challenge (Day 35 of study).

[0399] VACV neutralization was evaluated using a VACV-WR GFP reporter virus with serum collected on study Day 21 and the NT50 was determined. As shown in FIG. 16B, serum from mice immunized with protein antigens (NP combo and fusion NP) exhibited the highest neutralizing antibody titers. Serum from mice immunized with mRNA constructs also exhibited detectable levels of neutralizing antibody titers.

[0400] Body weight was recorded for 11 days post challenge for each mouse. As shown in FIG. 16C, within the groups treated with protein antigens, significant weight loss was observed following VACV challenge in mice treated with adjuvant only but not in other treatment groups. As shown in FIG. 16D, within the groups treated with mRNA constructs, significant weight loss following VACV challenge was observed in mice treated with off-target mRNA. Mice treated with a combination of A35, B6, Ml, and A29 encoded on different mRNAs had more weight loss on days 9 and 10 post VACV challenge compared to mice treated with mRNA encoding the A35-B6-M1 antigen as a soluble, ferritin, membrane-anchored (TM), or EABR-tagged antigen.

[0401] Taken together, protein antigens and mRNA constructs encoding said protein antigens in various forms are all effective at inducing neutralizing antibodies.

Claims

CLAIMS1. A composition comprising at least two engineered Orthopoxvirus proteins, wherein the at least two engineered Orthopoxvirus proteins comprises: a. at least one intracellular mature viral (IMV) surface membrane protein; and b. at least one extracellular enveloped virus (EEV) envelope glycoprotein.

2. The composition of claim 1, wherein the IMV surface membrane protein comprises at least one modification relative to a wild type IMV surface membrane protein; and wherein the IMV surface membrane protein is selected from the group consisting of: LI and Ml.

3. The composition of claim 1 or 2, wherein the EEV envelope glycoprotein comprises at least one modification relative to a wild type EEV envelope glycoprotein; and wherein the EEV envelope glycoprotein is selected from the group consisting of: A33, A35, A36, B5, B6, and B7.

4. The composition of any one of claims 1-3, wherein the composition comprises one or more linkers.

5. The composition of any one of claims 2-4, wherein the IMV surface membrane protein is selected from the group consisting of LI and Ml; and wherein the IMV surface membrane protein comprises the amino acid sequence of any one of SEQ ID NOs: 6-7,14, 21, 31-32, 41-42, or 48-50, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

6. The composition of any one of claims 3-5, wherein the EEV envelope glycoprotein is selected from the group consisting of B5, B6, and B7; and wherein the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 5, 11, 13,15, 16, 20, 22, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

7. The composition of any one claim of claims 3-5, wherein the EEV envelope glycoprotein is selected from the group consisting of A33, A35, and A36; and wherein the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 2- 4, 8-10, 12, 45, or 51-52, or an amino acid sequence having at least about 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

8. The composition of claim 7, wherein the EEV envelope glycoprotein forms a dimer.

9. The composition of claim 8, wherein the dimer is a single chain dimer.

10. The composition of any one claim of claims 2-9, wherein the at least one modification comprises a substitution, a deletion, or an insertion.

11. The composition of any one claim of claims 2-9, wherein the at least one modification comprises a substitution or deletion, wherein the substitution or deletion removes one or more N-linked glycosylation sites.

12. The composition of claim 11, wherein the EEV envelope glycoprotein is B6, and wherein the at least one modification is a substitution.

13. The composition of claim 12, wherein the substitution is in the B6 glycoprotein at a position corresponding to position 139 relative to SEQ ID NO: 39.

14. The composition of claim 13, wherein the substitution comprises substitution of cysteine (C) for isoleucine (I) at position 139 relative to SEQ ID NO: 39.

15. The composition of any one claim of claims 1-14, wherein the composition comprises at least three engineered Orthopoxvirus proteins.

16. The composition of claim 15, wherein the at least three engineered Orthopoxvirus proteins comprises:a. at least one intracellular mature viral (IMV) surface membrane protein; and b. at least two extracellular enveloped virus (EEV) envelope glycoprotein.

17. The composition of claim 16, wherein the IMV surface membrane protein comprises at least one modification relative to a wild type IMV surface membrane protein; and wherein the IMV surface membrane protein is selected from the group consisting of: LI and Ml.

18. The composition of claim 16 or 17, wherein the EEV envelope glycoprotein comprises at least one modification relative to a wild type EEV envelope glycoprotein; and wherein the EEV envelope glycoprotein is selected from the group consisting of: A33, A35, A36, B5, B6, and B7.

19. The composition of any one claim of claims 1-18, wherein the engineered Orthopoxvirus proteins comprise A35, B6, and Ml.

20. The composition of any one claim of claims 1-18, wherein the engineered Orthopoxvirus proteins comprise A33, B5, and LI.

21. The composition of any claim of claims 1-20, wherein the composition comprises a ferritin nanoparticle.

22. The composition of claim 21, wherein at least one engineered Orthopoxvirus protein is fused to a binding partner.

23. The composition of claim 22, wherein the binding partner is a ferritin subunit.

24. The composition of claim 23, wherein the ferritin subunit comprises a H. Pylori ferritin or a H. Pylori hybrid bullfrog ferritin.

25. The composition of claim 24, wherein the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 1, 18, or 82, or an amino acid sequence having at least 70%sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

26. The composition of any one of claims 23-25, wherein the ferritin subunit is fused to the engineered Orthopoxvirus proteins by a linker.

27. The composition of claim 26, wherein the amino acid sequence of the linker comprises the sequence of SEQ ID NO: 19 or a sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

28. A fusion protein comprising: a. a first Orthopoxvirus protein comprising an extracellular enveloped virus (EEV) envelope glycoprotein; b. a second Orthopoxvirus protein comprising an intracellular mature viral (IMV) surface membrane protein; and c. a third Orthopoxvirus protein comprising an extracellular enveloped virus (EEV) envelope glycoprotein.

29. The fusion protein of claim 28, wherein the fusion protein comprises one or more linkers.

30. The fusion protein of claim 28 or 29, wherein a. the first Orthopoxvirus protein comprises an extracellular enveloped virus (EEV) envelope glycoprotein selected from the group consisting of: B5, B6, and B7; b. the second Orthopoxvirus protein comprises an intracellular mature viral (IMV) surface membrane protein selected from the group consisting of: Ml and LI; and c. the third Orthopoxvirus protein comprises an extracellular enveloped virus (EEV) envelope glycoprotein selected from the group consisting of: A33, A35, and A36.

31. The fusion protein of claim 30, wherein the third Orthopoxvirus protein comprises a first protein selected from the group consisting of A33, A35, and A36, wherein the first protein is linked to a second protein selected from the group consisting of A33, A35, and A36; and wherein said third Orthopoxvirus protein forms a single chain dimer.

32. The fusion protein of claim 30, wherein: a. the first Orthopoxvirus protein comprises B5; b. the second Orthopoxvirus protein comprises LI; and c. the third Orthopoxvirus protein comprises A33.

33. The fusion protein of claim 32, wherein: a. the first Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 13, 20, or 22 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto; b. the second Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 14, 21, 48, or 49 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto; and c. the third Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 12, 51, or 52 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto..

34. The fusion protein of claim 30, wherein: a. the first Orthopoxvirus protein comprises B6; b. the second Orthopoxvirus protein comprises Ml; and c. the third Orthopoxvirus protein comprises A35.

35. The fusion protein of claim 34, wherein: a. the first Orthopoxvirus protein comprises the amino acid saequence of any one of SEQ ID NOs: 5 or 15, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto; b. the second Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 6, 7, or 50 or an amino acid sequence having at least 70%sequence identity thereto or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto; and c. the third Orthopoxvirus protein comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, 51, or 52 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

36. The fusion protein of any one claim of claims 28-35, wherein the fusion protein comprises, from N-terminal to C-terminal, an EEV envelope glycoprotein, an IMV surface membrane protein, and an EEV envelope glycoprotein.

37. The fusion protein of claim 36, wherein the fusion protein comprises, from N-terminal to C-terminal: a. A35, Ml, and B6; b. B6, Ml, and A35; or c. B5, LI, and A33.

38. The fusion protein of any one claim of claims 28-35, wherein the fusion protein comprises, from N-terminal to C-terminal, an IMV surface membrane protein, an EEV envelope glycoprotein, and an EEV envelope glycoprotein.

39. The fusion protein of claim 40, wherein the fusion protein comprises, from N-terminal to C-terminal: a. Ml, A35, and B6; or b. Ml, B6, and A35.

40. The fusion protein of any one claim of claims 28-35, wherein the fusion protein comprises, from N-terminal to C-terminal, an EEV envelope glycoprotein, an EEV envelope glycoprotein, and an IMV surface membrane protein.

41. The fusion protein of claim 40, wherein the fusion protein comprises, from N-terminal to C-terminal:a. A35, B6, and Ml; or b. B6, A35, and Ml.

42. The fusion protein of any one claim of claims 36-40, wherein the EEV envelope glycoprotein comprises a single chain dimer.

43. The fusion protein of any one claim of claims 28-42, wherein each of the Orthopoxvirus proteins comprises at least one modification relative to a wild type Orthopoxvirus protein.

44. The fusion protein of any one claim of claims 28-43, wherein the fusion protein comprises one or more ferritin subunits.

45. The fusion protein of claim 44, wherein the one or more ferritin subunits are selected from the group consisting of H. Pylori ferritin and H. Pylori hybrid bullfrog ferritin.

46. The fusion protein of any one claim of claims 28-45, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 46, 47, 66-70, 75, or 76 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

47. The fusion protein of any one claim of claims 28-43, wherein the fusion protein comprises an ESCRT-recruiting domain (ERD).

48. The fusion protein of claim 47, wherein the ERD comprises or is derived from an ESCRT and ALIX binding region (EABR).

49. The fusion protein of claim 48, wherein the ERD comprises the amino acid sequence of SEQ ID NO: 77 or 78, or a sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

50. The fusion protein of any one claim of claims 47-49, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 73 or 74 or an amino acid sequencehaving at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

51. The fusion protein of any one claim 28-50, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 23-26, 38, 43-44, 46, 47, or 66-76 or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

52. An engineered Orthopoxvirus protein comprising an engineered IMV surface membrane protein, wherein the engineered IMV surface membrane protein comprises at least one modification relative to a wild type IMV surface membrane protein.

53. The engineered Orthopoxvirus protein of claim 52, wherein the IMV surface membrane protein is selected from the group consisting of LI and Ml.

54. The engineered Orthopoxvirus protein of claim 52 or 53, wherein the engineered IMV surface membrane protein comprises the amino acid sequence of any one of SEQ ID NOs: 6-7, 14, 21, 31-32, 41-42, or 48-50, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

55. An engineered Orthopoxvirus protein comprising an engineered EEV envelope glycoprotein, wherein the engineered EEV envelope glycoprotein comprises at least one modification relative to a wild type EEV envelope glycoprotein.

56. The engineered Orthopoxvirus protein of claim 55, wherein the EEV envelope glycoprotein is selected from the group consisting of A33, A35, A36, B5, B6, and B7.

57. The engineered Orthopoxvirus protein of claim 56, wherein the EEV envelope glycoprotein is selected from the group consisting of B5, B6, and B7; and wherein the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 5, 11, 13, 15, 16, 20, 22, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

58. The engineered Orthopoxvirus protein of claim 56, wherein the EEV envelope glycoprotein is selected from the group consisting of A33, A35, and A36; and wherein the EEV envelope glycoprotein comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, 8-10, 12, 45, or 51-52, or an amino acid sequence having at least about 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

59. The engineered Orthopoxvirus protein of any one of claims 55-58, wherein the at least one modification comprises a substitution or deletion, wherein the substitution or deletion removes one or more N-linked glycosylation sites.

60. The engineered Orthopoxvirus protein of claim 59, wherein the engineered Orthopoxvirus protein comprises a B6 glycoprotein, wherein the at least one modification is a substitution.

61. The engineered Orthopoxvirus protein of claim 60, wherein the substitution is in the B6 glycoprotein at a position corresponding to position 139 relative to SEQ ID NO: 39.

62. The engineered Orthopoxvirus protein of claim 61, wherein the substitution comprises substitution of cysteine (C) for isoleucine (I) at position 139 relative to SEQ ID NO: 39.

63. The engineered Orthopoxvirus protein of claim any one of claims 58-62, wherein the engineered Orthopoxvirus protein comprises two EEV envelope glycoproteins, wherein the two EEV envelope glycoproteins form a dimer.

64. The engineered Orthopoxvirus protein of claim 63, wherein the two EEV envelope glycoproteins are the same.

65. The engineered Orthopoxvirus protein of claim 64, wherein the two EEV envelope glycoproteins are A33.

66. The engineered Orthopoxvirus protein of claim 63, wherein the two EEV envelope glycoproteins are not the same.

67. The engineered Orthopoxvirus protein of claim 66, wherein the two EEV envelope glycoproteins comprise: a. A33 and A35; b. A33 and A36; or c. A35 and A36.

68. The engineered Orthopoxvirus protein of any one of claims 63-67, wherein the dimer is a single chain dimer.

69. The engineered Orthopoxvirus protein of any one of claims 52-68, wherein the engineered Orthopoxvirus protein is fused to a binding partner.

70. The engineered Orthopoxvirus protein of claim 69, wherein the binding partner is a ferritin subunit.

71. The engineered Orthopoxvirus protein of claim 70, wherein the ferritin subunit comprises a H. Pylori ferritin or a H. Pylori hybrid bullfrog ferritin.

72. The engineered Orthopoxvirus protein of claim 71, wherein the ferritin subunit comprises the amino acid sequence of SEQ ID NO: 1, 18, or 82, or an amino acid sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

73. The engineered Orthopoxvirus protein of any one of claims 70-72, wherein the ferritin subunit is fused to the engineered Orthopoxvirus protein by a linker.

74. The engineered Orthopoxvirus protein of claim 73, wherein the linker is a flexible linker.

75. The engineered Orthopoxvirus protein of claim 73, wherein the linker is a rigid linker.

76. The engineered Orthopoxvirus protein of claim 73, wherein the amino acid sequence of the linker comprises the sequence of SEQ ID NO: 19 or a sequence having at least 70% sequence identity thereto, or a sequence having at least 1, 2, 3, 4, or 5 amino acid modifications thereto.

77. A ferritin nanoparticle comprising the composition of any one of claims 23-27, the fusion protein of any one of claims 44-46, or the engineered Orthopoxvirus protein of any one of claims 70-76.

78. An eVLP comprising the fusion protein of any one of claims 28-43 or 47-50 or the engineered Orthopoxvirus protein of any one of claims 52-69.

79. A polynucleotide encoding the amino acid sequence of: a. the one or more of the engineered Orthopoxvirus proteins of the compositions of any one of claims 1-27, b. the fusion protein of any one of claims 28-51; or c. the engineered Orthopoxvirus protein of any one of claims 52-76.

80. The polynucleotide of claim 79, wherein the polynucleotide comprises DNA.

81. The polynucleotide of claim 79, wherein the polynucleotide comprises RNA.

82. The polynucleotide of claim 81, wherein the RNA is mRNA.

83. A vaccine composition comprising one or more of: a. the compositions of any one of claims 1-27, b. the fusion protein of any one of claims 28-51; c. the engineered Orthopoxvirus protein of any one of claims 52-76; d. the ferritin nanoparticle of claim 77; e. the eVLP of claim 78; or f. the polynucleotide of any one of claims 79-82; and g. one or more adjuvants.

84. The vaccine composition of claim 83, wherein the one or more adjuvants comprises alum and / or CpG.

85. A pharmaceutical composition comprising:a. the compositions of any one of claims 1-27, b. the fusion protein of any one of claims 28-51; c. the engineered Orthopoxvirus protein of any one of claims 52-76; d. the ferritin nanoparticle of claim 77; e. the eVLP of claim 78; f. the polynucleotide of any one of claims 79-82; or g. the vaccine compositions of claim 83 or 84.

86. A method of preventing an Orthopoxvirus infection or a disease or symptom associated with an Orthopoxvirus infection in a subject, comprising administering to the subject an effective amount of: a. the compositions of any one of claims 1-27, b. the fusion protein of any one of claims 28-51; c. the engineered Orthopoxvirus protein of any one of claims 52-76; d. the ferritin nanoparticle of claim 77; e. the eVLP of claim 78; f. the polynucleotide of any one of claims 79-82; g. the vaccine compositions of claim 83 or 84; or h. the pharmaceutical composition of claim 85.

87. A method of vaccinating a subject against an Orthopoxvirus infection or a disease associated with an Orthopoxvirus infection, comprising administering to the subject an effective amount of: a. the compositions of any one of claims 1-27, b. the fusion protein of any one of claims 28-51; c. the engineered Orthopoxvirus protein of any one of claims 52-76; d. the ferritin nanoparticle of claim 77; e. the eVLP of claim 78; f. the polynucleotide of any one of claims 79-82; g. the vaccine compositions of claim 83 or 84; or h. the pharmaceutical composition of claim 85.

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