RNA compositions for delivery of orthopox antigens and related methods

Abstract

The present disclosure provides pharmaceutical compositions for delivery of orthopoxvirus antigens (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) and related technologies (e.g., components thereof and / or methods relating thereto). For example, the present disclosure provides polyribonucleotides encoding one or more mpox antigens, fragments, or fusions thereof.

Description

Attorney Docket No.: 2013237-1080 RNA COMPOSITIONS FOR DELIVERY OF ORTHOPOX ANTIGENS AND RELATED METHODS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. US 63 / 575,618, filed April 5, 2024, the contents of which is incorporated herein in its entirety. BACKGROUND

[0002] Orthopoxvirus is a genus encompassing a number of viral species including mpox virus (previously known as “monkeypox”; also referred to herein as “MPXV”), vaccinia virus (also referred to herein as “VACV”), ectromelia virus (also referred to herein as “ECTV”), borealpox virus (previously known as “Alaskapox”; also referred to herein as “AKPV” or “BRPV”), cowpox virus (also referred to herein as “CPXV”), modified vaccinia virus Ankara (also referred to herein as “MVA”), volepox (also referred to herein as “VPXV”), and variola virus (also referred to herein as “VARV”). Some orthopoxviruses are restricted in the hosts they infect, while others have been identified in a broad range of host species. Orthopoxviruses share a number of biological phenotypes including: a lack of a specific receptor required for infection of mammalian cells, a relatively low mutation rate, environmental stability of virion, and the ability to infect hosts via a number of routes (e.g., mucosal, respiratory, parenteral, etc.).

[0003] Mpox was first discovered in 1958 when two outbreaks of a pox-like disease occurred in colonies of monkeys kept for research, hence the name “monkeypox.” The first human case of mpox was recorded in 1970 in the Democratic Republic of Congo during a period of intensified effort to eliminate smallpox. Since then, mpox has been reported in humans in other central and western African countries.

[0004] Beginning in May 2022, a multinational outbreak of mpox led to over 85,000 cases worldwide spanning 114 countries, many of which have not been previously considered endemic. Transmission of this zoonotic infection has sustained endemicity in West and Central Africa for many years, but the scale of the 2022 outbreak was unprecedented leading to its declaration as a public health emergency of international concern (PHEIC) by the World Health Organization (WHO) in July 2022. The PHEIC was discontinued in May 2023 due to a significant decline in the number of reported cases and no changes in the severity of the disease (WHO Emergency Committee Meeting 2023). However, the WHO declared another PHEIC in August 2024 due to increased transmission of a deadlier clade 1b. Cases continue to be documented in both historically endemic regions and newly affected regions, highlighting the limitations of the current vaccine supply. SUMMARY

[0005] Pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that deliver portions or components of a pathogen (e.g., virus) have advantages to live pathogen approaches by focusing the vaccine response only on targets with a high potential for eliciting protective responses. While specific antigens have been shown to be immunogenic following orthopoxvirus (e.g., MPXV, VACV, MPXV, ECTV, AKPV, CPXV, MVA, VPXV, or VARV) administration, the minimal set of antigens and the types of immunity to them that are essential for protection from disease are not fully understood. Therefore, despite the multivalency enabled by current RNA vaccine platforms, antigen selection for subunit vaccines against orthopoxviruses is still being explored. Page 1 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0006] Pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) of the present disclosure are designed to encode mpox antigens that have not previously been used or characterized for RNA vaccines. Orthopoxviruses have two distinct infectious forms, mature virions (MV) and enveloped virions (EV). Each viral form has a unique set of surface antigens. While immune responses raised to any single target can offer some protection from infection, both subunit vaccine and monoclonal antibody prophylaxis studies in animals illustrated that the value of combining antibodies to multiple EV and MV targets. Therefore, to effectively reduce viral replication and infectivity, exemplary vaccine compositions of the present disclosure elicit immune responses to mpox proteins from EVs and / or MVs.

[0007] The present disclosure provides a recognition of several challenges involved in development and administration of polypeptide-based therapies. First, development of polypeptide-based therapies can be time consuming and expensive. For example, polypeptide development is challenged by demanding and costly production, including purification and formulation methods. Second, polypeptide-based therapies present an increased number of regulatory challenges. In addition to demonstrating that a protein therapy will be safe and efficacious, there is tight regulation over manufacture of polypeptide-based therapies (e.g., assessing post- translational modifications, etc.) and quality control during storage and administration complicate the use of protein therapies. Third, administration of polypeptide-based therapies to a subject can be painful and time consuming. Generally, polypeptide-based therapies are administered intravenously over a longer period of time, increasing patient discomfort. Finally, polypeptide-based therapies can have a relatively short serum half-life. The present disclosure provides insights that address these challenges, making it possible to deliver one or more therapies, as described herein, to a subject safely, reliably, and with strong potency.

[0008] Among other things, the present disclosure describes one or more orthopoxvirus (e.g., MPXV, VACV, MPXV, ECTV, AKPV, CPXV, MVA, VPXV, or VARV) antigens, characteristic portions, fusions, or variants thereof that are delivered to a subject via one or more polyribonucleotides. Utilizing one or more polyribonucleotides as, e.g., a therapeutic agent (in contrast to administering a polypeptide itself) involves simpler and less expensive manufacturing processes. A less complex production of polyribonucleotide encoding antigens can streamline manufacture, mitigating regulatory and production challenges associated with developing and using an antigen themselves. Additionally, polyribonucleotides are effective at producing similar effects to recombinant polypeptides but tend to require much lower volumes be administered to a subject. This is because polyribonucleotides encoding an antigen, characteristic portion, or variant thereof, can be administered to a subject and the subject’s body produces the polypeptide itself. Without wishing to be bound by any particular theory, a polyribonucleotide encoding antigens, as a therapeutic agent, has higher therapeutic efficacy (in contrast to administering a polypeptide itself) due to its continuous translation into encoded antigen to trigger long-lasting expression compared to transient traditional antigen polypeptide delivery. The present disclosure also provides technologies that address certain limitations of recombinant polypeptide technologies, including for example, expression of antigens by utilizing RNA technologies as a modality to express antigens directly in a subject’s cells.

[0009] The inventors of the present disclosure have identified that certain MPXV antigens (specifically, A21, A36, A28, L5, H2, A30, C15, A14, B2, C2, D14, A17, G10, M5, and G2), variants thereof, and antigenic fragments thereof are useful in polyribonucleotide compositions (that encode orthopoxvirus polypeptide constructs) to immunize or vaccinate subjects against orthopoxvirus infection (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia infection). The inventors further identified that certain combinations, complexes, or fusions of two or more certain MPXV antigens are useful in Page 2 of 258 12621669v1Attorney Docket No.: 2013237-1080 pharmaceutical compositions for use in immunization or vaccination against orthopoxvirus infection (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia infection). For example, the inventors have recognized that a fusion protein comprising A17 and G10 antigenic fragments, or a pharmaceutical composition comprising a first polyribonucleotide encoding an A17 antigenic fragment and a second polyribonucleotide encoding a G10 antigenic fragment, is effective as a vaccine component for the prevention of orthopox (e.g., mpox) virus infections in subjects.

[0010] The present disclosure provides polyribonucleotides. In some embodiments, a polyribonucleotide described herein encodes a polypeptide. In some embodiments, a polypeptide described herein comprises one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof.

[0011] In some embodiments, one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof comprise one or more mpox virus (MPXV) antigens, variants thereof, or antigenic fragments thereof; one or more variola virus (VARV) antigens, variants thereof, or antigenic fragments thereof; one or more borealpox virus antigens (BPXV), variants thereof, or antigenic fragments thereof; one or more cowpox virus (CPXV) antigens, variants thereof, or antigenic fragments thereof; one or more modified vaccinia virus Ankara (MVA) antigens, variants thereof, or antigenic fragments thereof; one or more volepox antigens (VPXV), variants thereof, or antigenic fragments thereof; one or more variola virus (VARV) antigens, variants thereof, or antigenic fragments thereof; or any combination thereof.

[0012] In some embodiments, one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof comprise one or more MPXV antigens, variants thereof, or antigenic fragments thereof. In some embodiments, one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof comprise one or more VARV antigens, variants thereof, or antigenic fragments thereof. In some embodiments, one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof comprise one or more BPXV antigens, variants thereof, or antigenic fragments thereof. In some embodiments, one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof comprise one or more CPXV antigens, variants thereof, or antigenic fragments thereof. In some embodiments, one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof comprise one or more MVA antigens, variants thereof, or antigenic fragments thereof. In some embodiments, one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof comprise one or more VPXV, variants thereof, or antigenic fragments thereof. In some embodiments, one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof comprise one or more VARV antigens, variants thereof, or antigenic fragments thereof.

[0013] In some embodiments, one or more orthopoxvirus antigens, variants thereof, or antigenic fragments thereof comprise one or more MPXV antigens, variants thereof, or antigenic fragments thereof.

[0014] Some aspects of the disclosure provide a polyribonucleotide encoding a polypeptide that comprises one or more mpox virus (MPXV) antigens, variants thereof, or antigenic fragments thereof.

[0015] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises an A21L antigen, variant thereof, or antigenic fragment thereof.

[0016] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises an A36R antigen, variant thereof, or antigenic fragment thereof.

[0017] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises an A28L antigen, variant thereof, or antigenic fragment thereof.

[0018] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises an L5L antigen, variant thereof, or antigenic fragment thereof. Page 3 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0019] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises an H2R antigen, variant thereof, or antigenic fragment thereof.

[0020] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises an A30L antigen, variant thereof, or antigenic fragment thereof.

[0021] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises a C15L antigen, a variant thereof, or antigenic fragment.

[0022] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises an A14L antigen, variant thereof, or antigenic fragment thereof.

[0023] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises a B2R antigen, variant thereof, or antigenic fragment thereof.

[0024] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises a C2L antigen, variant thereof, or antigenic fragment thereof.

[0025] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises a D14L antigen, variant thereof, or antigenic fragment thereof.

[0026] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises an A17L antigen, variant thereof, or antigenic fragment thereof.

[0027] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises a G10R antigen, variant thereof, or antigenic fragment thereof.

[0028] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises an M5R antigen, variant thereof, or antigenic fragment thereof.

[0029] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises a G2L antigen, variant thereof, or antigenic fragment thereof.

[0030] In some embodiments, the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprises a combination of two or more of the following: an A21L antigen, variant thereof, or antigenic fragment thereof; an A36R antigen, variant thereof, or antigenic fragment thereof; an A28L antigen, variant thereof, or antigenic fragment thereof; an L5L antigen, variant thereof, or antigenic fragment thereof; an H2R antigen, variant thereof, or antigenic fragment thereof; an A30L antigen, variant thereof, or antigenic fragment thereof; a C15L antigen, a variant thereof, or antigenic fragment; an A14L antigen, variant thereof, or antigenic fragment thereof; a B2R antigen, variant thereof, or antigenic fragment thereof; a C2L antigen, variant thereof, or antigenic fragment thereof; a D14L antigen, variant thereof, or antigenic fragment thereof; an A17L antigen, variant thereof, or antigenic fragment thereof; a G10R antigen, variant thereof, or antigenic fragment thereof; an M5R antigen, variant thereof, or antigenic fragment thereof; and / or a G2L antigen, variant thereof, or antigenic fragment thereof

[0031] In some embodiments, an A21L antigen, variant thereof, or antigenic fragment thereof is a full-length A21L antigen, an extracellular domain of A21L, or an A21L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 23-115 of a reference A21L antigen having the amino acid sequence of SEQ ID NO: 305.

[0032] In some embodiments, an A36R antigen, variant thereof, or antigenic fragment thereof is a full-length A36R antigen, an extracellular domain of A36, or an A36 antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 39-168 of a reference A36 antigen having the amino acid sequence of SEQ ID NO: 273. Page 4 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0033] In some embodiments, an A28L antigen, variant thereof, or antigenic fragment thereof is a full-length A28L antigen, an extracellular domain of A28L, an A28L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-366 of a reference A28L antigen having the amino acid sequence of SEQ ID NO: 196, or an A28L antigen that comprises serines at amino acid residues corresponding to positions 450 and 451 of SEQ ID NO: 196.

[0034] In some embodiments, an L5L antigen, variant thereof, or antigenic fragment is a full-length L5L antigen, an extracellular domain of L5L, or an L5L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-109 of a reference L5L antigen having the amino acid sequence of SEQ ID NO: 348.

[0035] In some embodiments, an H2R antigen, variant thereof, or antigenic fragment thereof is a full- length H2R antigen, an extracellular domain of H2R, or an H2R antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 49-189 of a reference H2R antigen having the amino acid sequence of SEQ ID NO: 313.

[0036] In some embodiments, an A30L antigen, variant thereof, or antigenic fragment thereof is a full-length A30L antigen, an extracellular domain of A30L, or an A30L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 22-146 of a reference A30L antigen having the amino acid sequence of SEQ ID NO: 309.

[0037] In some embodiments, a C15L antigen, a variant thereof, or antigenic fragment thereof is a full-length C15L antigen.

[0038] In some embodiments, an A14L antigen, variant thereof, or antigenic fragment thereof is a full-length A14L antigen, an extracellular domain of A14L, or an A14L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 25-70 of a reference A14L antigen having the amino acid sequence of SEQ ID NO: 323.

[0039] In some embodiments, a B2R antigen, variant thereof, or antigenic fragment thereof is a full- length B2R antigen and / or a B2R antigen that comprises a serine at an amino acid residue corresponding to position 160 of SEQ ID NO: 275.

[0040] In some embodiments, a C2L antigen, variant thereof, or antigenic fragment thereof is a full- length C2L antigen, an extracellular domain of C2L, an C2L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 15-375 of a reference C2L antigen having the amino acid sequence of SEQ ID NO: 279, or an C2L antigen that comprises a serine at amino acid residue corresponding to position 115 of SEQ ID NO: 279.

[0041] In some embodiments, a D14L antigen, variant thereof, or antigenic fragment thereof is a full- length D14L antigen, an extracellular domain of D14L, a D14L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 20-216 of a reference D14L antigen having the amino acid sequence of SEQ ID NO: 283, or a D14L antigen that comprises a serine at an amino acid residue corresponding to position 204 of SEQ ID NO: 283.

[0042] In some embodiments, an A17L antigen, variant thereof, or antigenic fragment thereof is a full-length A17L antigen, an extracellular domain of A17L, or an A17L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-295 of a reference A17L antigen having the amino acid sequence of SEQ ID NO: 358.

[0043] In some embodiments, a G10R antigen, variant thereof, or antigenic fragment thereof is a full- length G10R antigen, an extracellular domain of G10R, or a G10R antigenic fragment that comprises or consists Page 5 of 258 12621669v1Attorney Docket No.: 2013237-1080 of an amino acid sequence corresponding to residues 1-271 of a reference G10R antigen having the amino acid sequence of SEQ ID NO: 359;

[0044] In some embodiments, a M5R antigen, variant thereof, or antigenic fragment thereof is a full- length M5R antigen, an extracellular domain of M5R, or an M5R antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 50-128 of a reference M5R antigen having the amino acid sequence of SEQ ID NO: 327;

[0045] In some embodiments, a G2L antigen, variant thereof, or antigenic fragment thereof is a full- length G2L antigen, an extracellular domain of G2L, or a G2L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 20-111 of a reference G2L antigen having the amino acid sequence of SEQ ID NO: 331.

[0046] In some embodiments, an A21L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 305, 307, or 342.

[0047] In some embodiments, an A36R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 273, 458, 459, or 461.

[0048] In some embodiments, an A28L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 196, 343, 344, 364, 291, or 293.

[0049] In some embodiments, a L5L antigen, variant thereof, or antigenic fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 348, 349, 317, or 319.

[0050] In some embodiments, a H2R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 313, 347, or 315.

[0051] In some embodiments, an A30L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 309, 339, or 311.

[0052] In some embodiments, a C15L antigen, a variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 345 or 321.

[0053] In some embodiments, an A14L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 323, 340, or 325. Page 6 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0054] In some embodiments, a B2R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 275 or 277.

[0055] In some embodiments, a C2L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 279, 281, 350, or 356.

[0056] In some embodiments, a D14L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 283, 285, 351, or 357.

[0057] In some embodiments, an A17L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 358, 360, 295, or 297.

[0058] In some embodiments, a G10R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 359, 361, 299, or 301.

[0059] In some embodiments, a M5R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 327, 352, or 329.

[0060] In some embodiments, a G2L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 331, 346, or 333.

[0061] In some embodiments, a polyribonucleotide comprises: a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 306, 369, 308, or 481; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to SEQ ID NO: 274, 226, 497, 498, or 464; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 197, 370 , 371, 388, 292, 294, or 474; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 375, 376, 318, or 320; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 314, 374, 316, 484, or 485; a nucleotide sequence having at Page 7 of 258 12621669v1Attorney Docket No.: 2013237-1080 least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 310, 367, or 312; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to SEQ ID NO: 372 or 322; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 324, 368, or 326; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 276, 278, 465, or 466; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 280, 282, 377, 380, 467, or 468; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 284, 286, 378, or 381; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 382, 384, 296, or 298; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 383, 385, 300, 302, 477, or 478; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 328, 379, 330, or 492; a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 332, 373, or 334; or any combination thereof.

[0062] In some embodiments, a polypeptide comprises, from N-terminus to C-terminus: (a) an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; (b) an A28 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 343; (c) an optional peptide linker, optionally wherein the peptide linker comprises an amino acid sequence having 100% identity to an amino acid sequence according to SEQ ID NO: 353; and (d) an HSV-1 or HSV-2 transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 254.

[0063] In some embodiments, a polypeptide comprises, from N-terminus to C-terminus: (a) an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; (b) an A36 antigen, variant thereof, or Page 8 of 258 12621669v1Attorney Docket No.: 2013237-1080 antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 458; (c) an optional peptide linker, optionally wherein the peptide linker comprises an amino acid sequence having 100% identity to an amino acid sequence according to SEQ ID NO: 353; and (d) an HSV-1 or HSV-2 transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 254.

[0064] In some embodiments, a polypeptide comprises, from N-terminus to C-terminus: (a) an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; and (b) an A17 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 358 or SEQ ID NO: 360.

[0065] In some embodiments, a polypeptide comprises, from N-terminus to C-terminus: (a) an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; and (b) a G10 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 359 or SEQ ID NO: 361.

[0066] In some embodiments, a polypeptide comprises (a) an A17 antigen, variant thereof, or antigenic fragment thereof; and a G10 antigen, variant thereof, or antigenic fragment thereof; or (b) a D14 antigen, variant thereof, or antigenic fragment thereof; and a C2 antigen, variant thereof, or antigenic fragment thereof; or (c) a D14 antigen, variant thereof, or antigenic fragment thereof; and a B2 antigen, variant thereof, or antigenic fragment thereof; or (d) a D14 antigen, variant thereof, or antigenic fragment thereof; a B2 antigen, variant thereof, or antigenic fragment thereof; and a C2 antigen, variant thereof, or antigenic fragment thereof.

[0067] Some aspects of the disclosure provide a polyribonucleotide encoding a polypeptide that comprises (i) an A17 antigen, variant thereof, or antigenic fragment thereof; and (ii) a G10 antigen, variant thereof, or antigenic fragment thereof.

[0068] In some embodiments, (i) the A17L antigen, variant thereof, or antigenic fragment thereof is a full-length A17L antigen, an extracellular domain of A17L, or an A17L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-295 of a reference A17L antigen having the amino acid sequence of SEQ ID NO: 358; and / or (ii) the G10R antigen, variant thereof, or antigenic fragment thereof is a full-length G10R antigen, an extracellular domain of G10R, or a G10R antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-271 of a reference G10R antigen having the amino acid sequence of SEQ ID NO: 359.

[0069] In some embodiments, a polypeptide comprises (i) the A17L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 358 or SEQ ID NO: 360; and / or (ii) the Page 9 of 258 12621669v1Attorney Docket No.: 2013237-1080 G10R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NOs: 359 or 361.

[0070] In some embodiments, a polypeptide comprises, from N-terminus to C-terminus: (a) a first HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; (b) an A17 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 358 or SEQ ID NO: 360; (c) an optional peptide linker, optionally wherein the peptide linker comprises a P2A sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 363; (d) a second HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; and / or (e) a G10 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 359 or SEQ ID NO: 361.

[0071] Some aspects of the disclosure provide a polyribonucleotide encoding a polypeptide that comprises (i) a D14 antigen, variant thereof, or antigenic fragment thereof; and (ii) a C2 antigen, variant thereof, or antigenic fragment thereof; and / or a B2 antigen, variant thereof, or antigenic fragment thereof.

[0072] In some embodiments, a polypeptide comprises (a) a D14 antigen, variant thereof, or antigenic fragment thereof; and a C2 antigen, variant thereof, or antigenic fragment thereof; or (b) a D14 antigen, variant thereof, or antigenic fragment thereof; and a B2 antigen, variant thereof, or antigenic fragment thereof; or (c) a D14 antigen, variant thereof, or antigenic fragment thereof; a B2 antigen, variant thereof, or antigenic fragment thereof; and a C2 antigen, variant thereof, or antigenic fragment thereof.

[0073] In some embodiments, (i) the D14L antigen, variant thereof, or antigenic fragment thereof is a full-length D14L antigen, an extracellular domain of D14L, a D14L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 20-216 of a reference D14L antigen having the amino acid sequence of SEQ ID NO: 283, or a D14L antigen that comprises a serine at an amino acid residue corresponding to position 204 of SEQ ID NO: 283; and / or (ii) the C2L antigen, variant thereof, or antigenic fragment thereof is a full-length C2L antigen, an extracellular domain of C2L, an C2L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 15-375 of a reference C2L antigen having the amino acid sequence of SEQ ID NO: 279, or an C2L antigen that comprises a serine at amino acid residue corresponding to position 115 of SEQ ID NO: 279; and / or (iii) the B2R antigen, variant thereof, or antigenic fragment thereof is a full-length B2R antigen and / or a B2R antigen that comprises a serine at an amino acid residue corresponding to position 160 of SEQ ID NO: 275.

[0074] In some embodiments, (i) the D14L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, Page 10 of 258 12621669v1Attorney Docket No.: 2013237-1080 at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 283, 285, 351, or 357; (ii) the C2L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 279, 281, 350, or 356; and / or (iii) the B2R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 275 or 277.

[0075] In some embodiments, a polypeptide comprises a secretory signal, optionally wherein the secretory signal is a heterologous secretory signal.

[0076] In some embodiments, a secretory signal comprises or consists of an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160.

[0077] In some embodiments, a polyribonucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to SEQ ID NO: 161.

[0078] In some embodiments, a polypeptide comprises a transmembrane region that is N-terminal, C- terminal, or internal to the one or more MPXV antigens, variants thereof, or antigenic fragments thereof, optionally wherein the transmembrane region is a heterologous transmembrane region.

[0079] In some embodiments, a transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 254.

[0080] In some embodiments, a polyribonucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to SEQ ID NO: 255.

[0081] In some embodiments, one or more MPXV antigens, variants thereof, or antigenic fragments thereof is operably linked to the secretory signal and / or the transmembrane region via a peptide linker, optionally wherein the peptide linker is a GS linker.

[0082] Some aspects of the disclosure provide a pharmaceutical composition comprising (i) a first polyribonucleotide encoding an A17 antigen, variant thereof, or antigenic fragment thereof; and (ii) a second polyribonucleotide encoding an G10 antigen, variant thereof, or antigenic fragment thereof.

[0083] In some embodiments, (i) the A17L antigen, variant thereof, or antigenic fragment thereof is a full-length A17L antigen, an extracellular domain of A17L, or an A17L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-295 of a reference A17L antigen having the amino acid sequence of SEQ ID NO: 358; and / or (ii) the G10R antigen, variant thereof, or antigenic fragment thereof is a full-length G10R antigen, an extracellular domain of G10R, or a G10R antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-271 of a reference G10R antigen having the amino acid sequence of SEQ ID NO: 359. Page 11 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0084] In some embodiments, (i) the A17L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 358 or SEQ ID NO: 360; and / or (ii) the G10R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NOs: 359, or 361.

[0085] Some aspects of the disclosure provide a pharmaceutical composition comprising (i) a first polyribonucleotide encoding a D14 antigen, variant thereof, or antigenic fragment thereof; and (ii) a second polyribonucleotide encoding a C2 antigen, variant thereof, or antigenic fragment thereof.

[0086] Some aspects of the disclosure provide a pharmaceutical composition comprising (i) a first polyribonucleotide encoding a D14 antigen, variant thereof, or antigenic fragment thereof; (ii) a second polyribonucleotide encoding a B2 antigen, variant thereof, or antigenic fragment thereof; and optionally (iii) a third polyribonucleotide encoding a C2 antigen, variant thereof, or antigenic fragment thereof.

[0087] In some embodiments, (i) the D14L antigen, variant thereof, or antigenic fragment thereof is a full-length D14L antigen, an extracellular domain of D14L, a D14L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 20-216 of a reference D14L antigen having the amino acid sequence of SEQ ID NO: 283, or a D14L antigen that comprises a serine at an amino acid residue corresponding to position 204 of SEQ ID NO: 283; and / or (ii) the C2L antigen, variant thereof, or antigenic fragment thereof is a full-length C2L antigen, an extracellular domain of C2L, an C2L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 15-375 of a reference C2L antigen having the amino acid sequence of SEQ ID NO: 279, or an C2L antigen that comprises a serine at amino acid residue corresponding to position 115 of SEQ ID NO: 279; and / or (iii) the B2R antigen, variant thereof, or antigenic fragment thereof is a full-length B2R antigen and / or a B2R antigen that comprises a serine at an amino acid residue corresponding to position 160 of SEQ ID NO: 275.

[0088] In some embodiments, (i) the D14L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 283, 285, 351, or 357; (ii) the C2L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 279, 281, 350, or 356; and / or (iii) the B2R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 275, or 277.

[0089] In some embodiments, a first polyribonucleotide and / or the second polyribonucleotide further comprise a heterologous secretory signal, optionally wherein the heterologous secretory signal is a HSV gD secretory signal, optionally comprising an amino acid sequence having at least 85% identity to an amino acid sequence according to SEQ ID NO: 160.

[0090] Some aspects of the disclosure provide a pharmaceutical composition comprising one or more polyribonucleotides described herein. Page 12 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0091] In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises a lipid nanoparticle. In some embodiments, a pharmaceutical composition comprises a cationic lipid.

[0092] Some aspects of the disclosure provide a method of treating or preventing an orthopoxvirus infection in a subject, the method comprising administering one or more polyribonucleotides or a pharmaceutical composition described herein to the subject.

[0093] In some embodiments, an orthopoxvirus infection is a monkeypox infection, variola infection, borealpox infection, ectromelia infection, cowpox infection, volepox infection, modified vaccinia virus Ankara infection, or vaccinia infection.

[0094] In some embodiments, one or more polyribonucleotides or a pharmaceutical composition is administered to the subject prior to the orthopoxvirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG.1 depicts a schematic diagram of orthopox virus enveloped virion (EV) and mature virion (MV) membrane surface proteins (adapted from ViralZone©, Swiss Institute of Bioinformatics, see, https: / / viralzone.expasy.org).

[0096] FIGs.2A and 2B depict graphs showing orthopoxvirus-neutralizing activities of day 35 serum samples collected from Balb / C mice immunized with compositions comprising constructs encoding E8 or H3 antigens, in the presence or absence of complement. FIG.2A is a graph showing vaccinia virus (VACV) neutralizing activity of serum samples collected from Balb / C mice immunized with compositions comprising constructs encoding E8 or H3 antigens, in the presence or absence of complement. FIG.2B is a graph showing mpox virus (MPXV) neutralizing activity of serum samples collected from Balb / C mice immunized with compositions comprising constructs encoding E8 or H3 antigens, in the presence or absence of complement.

[0097] FIGs.3A to 3C depict Kaplan-Meier graphs from Paran et al. 2013., Virology Journal; 10:229. showing the survival of mice infected with various orthopoxviruses. FIG.3A is a Kaplan-Meier graph showing the survival of mice vaccinated with A33 antigen against VACV challenge. FIG.3B is a Kaplan-Meier graph showing the survival of mice vaccinated with A33 antigen against Ectromelia challenge. FIG.3C is a Kaplan-Meier graph showing the survival of mice vaccinated with A33 antigen against cowpox virus challenge.

[0098] FIG.4 depicts a sequence alignment of M1 antigen sequences of various old and new world orthopoxviruses.

[0099] FIG.5 depicts a sequence alignment of H3 antigen sequences of various old and new world orthopoxviruses.

[0100] FIG.6 depicts a sequence alignment of A35 antigen sequences of various old and new world orthopoxviruses.

[0101] FIG.7 depicts a sequence alignment of B6 antigen sequences of various old and new world orthopoxviruses.

[0102] FIGs.8 and 9 depict ribbon diagrams of orthopoxvirus antigens with key residues indicated that may be modified to facilitate plasma membrane display of EV antigens for an mRNA subunit vaccine. FIG.8 depicts a ribbon diagram of a VACV A34 homodimer with asparagine (N) residues indicated that may be modified to remove a mammalian glycosylation site. FIG.9 depicts a ribbon diagram of a VACV A56 antigen with asparagine (N) residues indicated that may be modified to remove a mammalian glycosylation site, and unpaired cysteine residues that may be modified to facilitate expression. Page 13 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0103] FIG.10 depicts a schematic diagram of modifications to MPXV A28 antigen to facilitate efficient plasma membrane display of the antigen.

[0104] FIGs.11 to 15 depict ribbon diagrams of orthopoxvirus antigens. FIG.11 depicts a ribbon diagram of an orthopoxvirus A28 antigen. FIG.12 depicts a ribbon diagram of an orthopoxvirus H2 antigen. FIG.13 depicts a ribbon diagram of an orthopoxvirus J5 antigen. FIG.14 depicts a ribbon diagram of an orthopoxvirus G3 antigen complexed with an L5 antigen. FIG.15 depicts a ribbon diagram of an orthopoxvirus A16 antigen complexed with a G9 antigen.

[0105] FIG.16 depicts a schematic diagram of a full-length A21L antigen and a corresponding amino acid sequence (SEQ ID NO: 305). FIG.17 depicts a schematic diagram of an A21L fragment corresponding to residues 23-115 with an N-terminal HSV-1 gD signal peptide / secretory signal and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 307).

[0106] FIG.18 depicts a schematic diagram of a full-length A36R antigen and a corresponding amino acid sequence (SEQ ID NO: 273).

[0107] FIG.19 depicts a schematic diagram of an A28L antigen fragment corresponding to residues 1-366 with an N-terminal HSV-1 gD signal peptide / secretory signal and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 291). FIG.20 depicts a schematic diagram of a full-length A28L antigen variant having C450S and C451S substitutions, an N-terminal HSV-1 gD signal peptide / secretory signal and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 293).

[0108] FIG.21 depicts a schematic diagram of a full-length L5L antigen with an N-terminal HSV-1 gD signal peptide / secretory signal, and a corresponding amino acid sequence (SEQ ID NO: 317). FIG.22 depicts a schematic diagram of a L5L antigen fragment corresponding to residues 1-109 with an N-terminal HSV-1 gD signal peptide / secretory signal and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 319).

[0109] FIG.23 depicts a schematic diagram of a full-length H2R antigen and a corresponding amino acid sequence (SEQ ID NO: 313). FIG.24 depicts a schematic diagram of an H2R antigen extracellular domain fragment corresponding to residues 49-189 with an N-terminal HSV-1 gD signal peptide / secretory signal and a C- terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 315).

[0110] FIG.25 depicts a schematic diagram of a full-length A30L antigen and a corresponding amino acid sequence (SEQ ID NO: 309). FIG.26 depicts a schematic diagram of an A30L antigen fragment corresponding to residues 22-146 with an N-terminal HSV-1 gD signal peptide / secretory signal and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 311).

[0111] FIG.27 depicts a schematic diagram of a full-length C15L antigen with an N-terminal HSV-1 gD signal peptide / secretory signal, and a corresponding amino acid sequence (SEQ ID NO: 321).

[0112] FIG.28 depicts a schematic diagram of a full-length A14L antigen and a corresponding amino acid sequence (SEQ ID NO: 323). FIG.29 depicts a schematic diagram of a A14L antigen extracellular domain fragment corresponding to residues 25-70 with an N-terminal HSV-1 gD signal peptide / secretory signal and a C- terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 325).

[0113] FIG.30 depicts a schematic diagram of a full-length B2R wild-type antigen and a corresponding amino acid sequence (SEQ ID NO: 275). FIG.31 depicts a schematic diagram of a full-length B2R antigen variant having a C160S substitution and a corresponding amino acid sequence (SEQ ID NO: 277). Page 14 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0114] FIG.32 depicts a schematic diagram of a full-length C2L wild-type antigen and a corresponding amino acid sequence (SEQ ID NO: 279). FIG.33 depicts a schematic diagram of a full-length C2L antigen variant having a C115S substitution and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 281).

[0115] FIG.34A depicts a schematic diagram of a full-length D14L wild-type antigen, and a corresponding amino acid sequence (SEQ ID NO: 283). FIG.34B depicts a schematic diagram of a full-length D14L antigen variant having a C204S substitution and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 285).

[0116] FIG.35A depicts a schematic diagram of an MPXV antigen fusion construct including a full- length D14L antigen variant having a C204S substitution operably linked via a GS linker to a C2L antigen variant fragment corresponding to residues 15-375, having a C115S substitution, and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 287). FIG.35B depicts a schematic diagram of an MPXV antigen fusion construct including a full-length C2L antigen variant having a C115S substitution operably linked via a GS linker to a D14L antigen variant fragment corresponding to residues 20-216 having a C204S substitution, and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 289).

[0117] FIG.36A depicts a schematic diagram of a full-length A17L antigen with an N-terminal HSV-1 gD signal peptide / secretory signal, and a corresponding amino acid sequence (SEQ ID NO: 295). FIG.36B depicts a schematic diagram of an A17L antigen fragment corresponding to residues 1-295 with an N-terminal HSV-1 gD signal peptide / secretory signal, and a corresponding amino acid sequence (SEQ ID NO: 297).

[0118] FIG.37A depicts a schematic diagram of a full-length G10R antigen with an N-terminal HSV-1 gD signal peptide / secretory signal, and a corresponding amino acid sequence (SEQ ID NO: 299). FIG.37B depicts a schematic diagram of an G10RL antigen fragment corresponding to residues 1-271 with an N-terminal HSV-1 gD signal peptide / secretory signal, and a corresponding amino acid sequence (SEQ ID NO: 301).

[0119] FIG.38 depicts a schematic diagram of an MPXV antigen construct including a full-length A17L antigen operably linked via a P2A cleavable sequence to a full-length G10R antigen, where both A17L and G10R antigens are operably linked at their respective N-termini to an HSV-1 gD signal peptide / secretory signal, and a corresponding amino acid sequence (SEQ ID NO: 303).

[0120] FIG.39A depicts a schematic diagram of a full-length M5R wild-type antigen, and a corresponding amino acid sequence (SEQ ID NO: 327). FIG.39B depicts a schematic diagram of an M5R antigen extracellular domain fragment corresponding to residues 50-128 with an N-terminal HSV-1 gD signal peptide / secretory signal and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 329).

[0121] FIG.40A depicts a schematic diagram of a full-length G2L wild-type antigen, and a corresponding amino acid sequence (SEQ ID NO: 331). FIG.40B depicts a schematic diagram of a G2L antigen extracellular domain fragment corresponding to residues 20-111 with an N-terminal HSV-1 gD signal peptide / secretory signal and a C-terminal HSV-1 gD transmembrane region, and a corresponding amino acid sequence (SEQ ID NO: 333).

[0122] FIG.41A depicts a schematic diagram of an MPXV antigen construct including an M5R antigen extracellular domain fragment corresponding to residues 50-128 operably linked via a 20GS linker to a G2L antigen extracellular domain fragment corresponding to residues 20-111. The construct includes an N-terminal HSV-1 gD signal peptide / secretory signal and a C-terminal HSV-1 gD transmembrane region. A corresponding Page 15 of 258 12621669v1Attorney Docket No.: 2013237-1080 amino acid sequence is also depicted (SEQ ID NO: 335). FIG.41B depicts a schematic diagram of an MPXV antigen construct including a full-length wild-type M5R antigen operably linked via a 20GS linker to a G2L antigen extracellular domain fragment corresponding to residues 20-111, and a corresponding amino acid sequence (SEQ ID NO: 337).

[0123] In the schematic diagrams of FIGs.21 to 41, sequences corresponding to: signal peptides / secretory signals are underlined, transmembrane regions are in bold, linkers / cleavage sequences are in italics, and substitutions are in bold, underlined, italics.

[0124] FIGs.42A to 42C are flow cytometry plots showing surface expression of A21 antigen (e.g., extracellular presentation of A21 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A21 Construct 1 (SEQ ID NO: 393).

[0125] FIGs.43A to 43C are flow cytometry plots showing surface expression of A21 antigen (e.g., extracellular presentation of A21 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A21 Construct 2 (SEQ ID NO: 394).

[0126] FIGs.44A to 44C are flow cytometry plots showing total protein expression of A21 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A21 Construct 1 (SEQ ID NO: 393).

[0127] FIGs.45A to 45C are flow cytometry plots showing total protein expression of A21 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A21 Construct 2 (SEQ ID NO: 394).

[0128] FIGs.46A to 46C are flow cytometry plots showing surface expression of A36 antigen (e.g., extracellular presentation of A36 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A36 Construct 1 (SEQ ID NO: 395).

[0129] FIGs.47A to 47C are flow cytometry plots showing surface expression of A36 antigen (e.g., extracellular presentation of A36 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A36 Construct 2 (SEQ ID NO: 460).

[0130] FIGs.47D to 47F are flow cytometry plots showing surface expression of A36 antigen (e.g., extracellular presentation of A36 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A36 Construct 3 (SEQ ID NO: 462).

[0131] FIGs.48A to 48C are flow cytometry plots showing total protein expression of A36 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A36 Construct 1 (SEQ ID NO: 395).

[0132] FIGs.49A to 49C are flow cytometry plots showing total protein expression of A36 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A36 Construct 2 (SEQ ID NO: 460).

[0133] FIGs.49D to 49F are flow cytometry plots showing total protein expression of A36 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A36 Construct 3 (SEQ ID NO: 462).

[0134] FIGs.50A to 50C are flow cytometry plots showing surface expression of A28 antigen (e.g., extracellular presentation of A28 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A28 Construct 1 (SEQ ID NO: 396). Page 16 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0135] FIGs.51A to 51C are flow cytometry plots showing surface expression of A28 antigen (e.g., extracellular presentation of A28 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A28 Construct 2 (SEQ ID NO: 397).

[0136] FIGs.52A to 52C are flow cytometry plots showing total protein expression of A28 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A28 Construct 1 (SEQ ID NO: 396).

[0137] FIGs.53A to 53C are flow cytometry plots showing total protein expression of A28 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A28 Construct 2 (SEQ ID NO: 397).

[0138] FIGs.54A to 54C are flow cytometry plots showing surface expression of L5 antigen (e.g., extracellular presentation of L5 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding L5 Construct 1 (SEQ ID NO: 398).

[0139] FIGs.55A to 55C are flow cytometry plots showing surface expression of L5 antigen (e.g., extracellular presentation of L5 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding L5 Construct 2 (SEQ ID NO: 399).

[0140] FIGs.56A to 56C are flow cytometry plots showing total protein expression of L5 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding L5 Construct 1 (SEQ ID NO: 398).

[0141] FIGs.57A to 57C are flow cytometry plots showing total protein expression of L5 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding L5 Construct 2 (SEQ ID NO: 399).

[0142] FIGs.58A to 58C are flow cytometry plots showing surface expression of H2 antigen (e.g., extracellular presentation of H2 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding H2 Construct 1 (SEQ ID NO: 400).

[0143] FIGs.59A to 59C are flow cytometry plots showing surface expression of H2 antigen (e.g., extracellular presentation of H2 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding H2 Construct 2 (SEQ ID NO: 401).

[0144] FIGs.60A to 60C are flow cytometry plots showing total protein expression of H2 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding H2 Construct 1 (SEQ ID NO: 400).

[0145] FIGs.61A to 61C are flow cytometry plots showing total protein expression of H2 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding H2 Construct 2 (SEQ ID NO: 401).

[0146] FIGs.62A to 62C are flow cytometry plots showing surface expression of A30 antigen (e.g., extracellular presentation of A30 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A30 Construct 1 (SEQ ID NO: 402).

[0147] FIGs.63A to 63C are flow cytometry plots showing surface expression of A30 antigen (e.g., extracellular presentation of A30 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A30 Construct 2 (SEQ ID NO: 403).

[0148] FIGs.64A to 64C are flow cytometry plots showing total protein expression of A30 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A30 Construct 1 (SEQ ID NO: 402). Page 17 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0149] FIGs.65A to 65C are flow cytometry plots showing total protein expression of A30 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A30 Construct 2 (SEQ ID NO: 403).

[0150] FIGs.66A to 66C are flow cytometry plots showing surface expression of C15 antigen (e.g., extracellular presentation of C15 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding C15 Construct 1 (SEQ ID NO: 404).

[0151] FIGs.67A to 67C are flow cytometry plots showing total protein expression of C15 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding C15 Construct 1 (SEQ ID NO: 404).

[0152] FIGs.68A to 68C are flow cytometry plots showing surface expression of A14 antigen (e.g., extracellular presentation of A14 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A14 Construct 1 (SEQ ID NO: 407).

[0153] FIGs.69A to 69C are flow cytometry plots showing surface expression of A14 antigen (e.g., extracellular presentation of A14 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A14 Construct 2 (SEQ ID NO: 408).

[0154] FIGs.70A to 70C are flow cytometry plots showing total protein expression of A14 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A14 Construct 1 (SEQ ID NO: 407).

[0155] FIGs.71A to 71C are flow cytometry plots showing total protein expression of A14 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A14 Construct 2 (SEQ ID NO: 408).

[0156] FIGs.72A to 72C are flow cytometry plots showing surface expression of B2 antigen (e.g., extracellular presentation of B2 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 1 (SEQ ID NO: 409).

[0157] FIGs.73A to 73C are flow cytometry plots showing surface expression of B2 antigen (e.g., extracellular presentation of B2 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 2 (SEQ ID NO: 410).

[0158] FIGs.74A to 74C are flow cytometry plots showing total protein expression of B2 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 1 (SEQ ID NO: 409).

[0159] FIG.75A to 75C are flow cytometry plots showing total protein expression of B2 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 2 (SEQ ID NO: 410).

[0160] FIG.76A to 76C are flow cytometry plots showing surface expression of C2L antigen (e.g., extracellular presentation of C2L antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding C2L Construct 1 (SEQ ID NO: 411).

[0161] FIG.77A to 77C are flow cytometry plots showing surface expression of C2L antigen (e.g., extracellular presentation of C2L antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding C2L Construct 2 (SEQ ID NO: 412).

[0162] FIG.78A to 78C are flow cytometry plots showing total protein expression of C2L antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding C2L Construct 1 (SEQ ID NO: 411). Page 18 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0163] FIG.79A to 79C are flow cytometry plots showing total protein expression of C2L antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding C2L Construct 2 (SEQ ID NO: 412).

[0164] FIG.80A to 80C are flow cytometry plots showing surface expression of D14L antigen (e.g., extracellular presentation of D14L antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding D14L Construct 1 (SEQ ID NO: 413).

[0165] FIG.81A to 81C are flow cytometry plots showing surface expression of D14L antigen (e.g., extracellular presentation of D14L antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding D14L Construct 2 (SEQ ID NO: 414).

[0166] FIG.82A to 82C are flow cytometry plots showing total protein expression of D14L antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding D14L Construct 1 (SEQ ID NO: 413).

[0167] FIG.83A to 83C are flow cytometry plots showing total protein expression of D14L antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding D14L Construct 2 (SEQ ID NO: 414).

[0168] FIG.84A to 84C are flow cytometry plots showing surface expression of a fusion protein comprising D14L and C2L (e.g., extracellular presentation of D14L and C2L antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding D14_C2 Construct 1 (SEQ ID NO: 415).

[0169] FIG.85A to 85C are flow cytometry plots showing surface expression of a fusion protein comprising D14L and C2L (e.g., extracellular presentation of D14L and C2L antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding C2_D14 Construct 1 (SEQ ID NO: 416).

[0170] FIG.86A to 86C are flow cytometry plots showing total protein expression of a fusion protein comprising D14L and C2L in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding D14_C2 Construct 1 (SEQ ID NO: 415).

[0171] FIG.87A to 87C are flow cytometry plots showing total protein expression of a fusion protein comprising D14L and C2L in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding C2_D14 Construct 1 (SEQ ID NO: 416).

[0172] FIG.88A to 88C are flow cytometry plots showing surface expression of B2 and C2 antigens (e.g., extracellular presentation of B2 and C2 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 1 (SEQ ID NO: 409) and an exemplary plasmid encoding C2 Construct 1 (SEQ ID NO: 411).

[0173] FIG.89A to 89C are flow cytometry plots showing total protein expression of B2 and C2 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 1(SEQ ID NO: 409) and an exemplary plasmid encoding C2 Construct 1 (SEQ ID NO: 411).

[0174] FIG.90A to 90C are flow cytometry plots showing surface expression of B2 and C2 antigens (e.g., extracellular presentation of B2 and C2 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 2 (SEQ ID NO: 410) and an exemplary plasmid encoding C2 Construct 1 (SEQ ID NO: 411).

[0175] FIG.91A to 91C are flow cytometry plots showing total protein expression of B2 and C2 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary Page 19 of 258 12621669v1Attorney Docket No.: 2013237-1080 plasmid encoding B2 Construct 2 (SEQ ID NO: 410) and an exemplary plasmid encoding C2 Construct 1 (SEQ ID NO: 411).

[0176] FIG.92A to 92C are flow cytometry plots showing surface expression of B2 and D14 antigens (e.g., extracellular presentation of B2 and D14 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 1 (SEQ ID NO: 409) and an exemplary plasmid encoding D14 Construct 1 (SEQ ID NO: 413).

[0177] FIG.93A to 93C are flow cytometry plots showing total protein expression of B2 and D14 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 1 (SEQ ID NO: 409) and an exemplary plasmid encoding D14 Construct 1 (SEQ ID NO: 413).

[0178] FIG.94A to 94C are flow cytometry plots showing surface expression of B2 and D14 antigens (e.g., extracellular presentation of B2 and D14 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 2 (SEQ ID NO: 410) and an exemplary plasmid encoding D14 Construct 1 (SEQ ID NO: 413).

[0179] FIG.95A to 95C are flow cytometry plots showing total protein expression of B2 and D14 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding B2 Construct 2 (SEQ ID NO: 410) and an exemplary plasmid encoding D14 Construct 1 (SEQ ID NO: 413).

[0180] FIG.96A to 96C are flow cytometry plots showing surface expression of A17 antigen (e.g., extracellular presentation of A17 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 1 (SEQ ID NO: 417).

[0181] FIG.97A to 97C are flow cytometry plots showing surface expression of A17 antigen (e.g., extracellular presentation of A17 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 2 (SEQ ID NO: 419).

[0182] FIG.98A to 98C are flow cytometry plots showing total protein expression of A17 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 1 (SEQ ID NO: 417).

[0183] FIG.99A to 99C are flow cytometry plots showing total protein expression of A17 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 2 (SEQ ID NO: 419).

[0184] FIG.100A to 100C are flow cytometry plots showing surface expression of G10 antigen (e.g., extracellular presentation of G10 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding G10 Construct 1 (SEQ ID NO: 418).

[0185] FIG.101A to 101C are flow cytometry plots showing surface expression of G10 antigen (e.g., extracellular presentation of G10 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding G10 Construct 2 (SEQ ID NO: 420).

[0186] FIG.102A to 102C are flow cytometry plots showing total protein expression of G10 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding G10 Construct 1 (SEQ ID NO: 418).

[0187] FIG.103A to 103C are flow cytometry plots showing total protein expression of G10 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding G10 Construct 2 (SEQ ID NO: 420). Page 20 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0188] FIG.104A to 104C are flow cytometry plots showing surface expression of A17 and G10 antigens (e.g., extracellular presentation of A17 and G10 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 1 (SEQ ID NO: 417) and an exemplary plasmid encoding G10 Construct 1 (SEQ ID NO: 418).

[0189] FIG.105A to 105C are flow cytometry plots showing total protein expression of A17 and G10 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 1 (SEQ ID NO: 417) and an exemplary plasmid encoding G10 Construct 1 (SEQ ID NO: 418).

[0190] FIG.106A to 106C are flow cytometry plots showing surface expression of A17 and G10 antigens (e.g., extracellular presentation of A17 and G10 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 1 (SEQ ID NO: 417) and an exemplary plasmid encoding G10 Construct 2 (SEQ ID NO: 420).

[0191] FIG.107A to 107C are flow cytometry plots showing total protein expression of A17 and G10 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 1 (SEQ ID NO: 417) and an exemplary plasmid encoding G10 Construct 2 (SEQ ID NO: 420).

[0192] FIG.108A to 108C are flow cytometry plots showing surface expression of A17 and G10 antigens (e.g., extracellular presentation of A17 and G10 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 2 (SEQ ID NO: 419) and an exemplary plasmid encoding G10 Construct 1 (SEQ ID NO: 418).

[0193] FIG.109A to 109C are flow cytometry plots showing total protein expression of A17 and G10 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 2 (SEQ ID NO: 419) and an exemplary plasmid encoding G10 Construct 1 (SEQ ID NO: 418).

[0194] FIG.110A to 110C are flow cytometry plots showing surface expression of A17 and G10 antigens (e.g., extracellular presentation of A17 and G10 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 2 (SEQ ID NO: 419) and an exemplary plasmid encoding G10 Construct 2 (SEQ ID NO: 420).

[0195] FIG.111A to 111C are flow cytometry plots showing total protein expression of A17 and G10 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17 Construct 2 (SEQ ID NO: 419) and an exemplary plasmid encoding G10 Construct 2 (SEQ ID NO: 420).

[0196] FIG.112A to 112C are flow cytometry plots showing surface expression of a fusion protein comprising A17 and G10 antigens (e.g., extracellular presentation of A17 and / or G10 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17_G10 Construct 1 (SEQ ID NO: 421).

[0197] FIG.113A to 113C are flow cytometry plots showing total protein expression of a fusion protein comprising A17 and G10 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding A17_G10 Construct 1 (SEQ ID NO: 421).

[0198] FIG.114A to 114C are flow cytometry plots showing surface expression of G2 antigen (e.g., extracellular presentation of G2 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding G2 Construct 1 (SEQ ID NO: 423). Page 21 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0199] FIG.115A to 115C are flow cytometry plots showing surface expression of G2 antigen (e.g., extracellular presentation of G2 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding G2 Construct 2 (SEQ ID NO: 424).

[0200] FIG.116A to 116C are flow cytometry plots showing total protein expression of G2 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding G2 Construct 1 (SEQ ID NO: 423). FIG.116A is a dot plot showing fixed, permeabilized cells intracellularly stained with anti-FLAG -Brilliant Violet™ 421 and anti-c-Myc-phycoerythrin (PE). FIG.116B is a histogram showing anti-FLAG staining (negative control). FIG.116C is a histogram showing intracellular staining for G2 (c-Myc-tagged).

[0201] FIG.117A to 117C are flow cytometry plots showing total protein expression of G2 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding G2 Construct 2 (SEQ ID NO: 424).

[0202] FIG.118A to 118C are flow cytometry plots showing surface expression of M5 antigen (e.g., extracellular presentation of M5 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 1 (SEQ ID NO: 422).

[0203] FIG.119A to 119C are flow cytometry plots showing surface expression of M5 antigen (e.g., extracellular presentation of M5 antigen) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 2 (SEQ ID NO: 425).

[0204] FIG.120A to 120C are flow cytometry plots showing total protein expression of M5 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 1 (SEQ ID NO: 422).

[0205] FIG.121A to 121C are flow cytometry plots showing total protein expression of M5 antigen in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 2 (SEQ ID NO: 425).

[0206] FIG.122A to 122C are flow cytometry plots showing surface expression of M5 and G2 antigens (e.g., extracellular presentation of M5 and G2 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 1 (SEQ ID NO: 422) and an exemplary plasmid encoding G2 Construct 1 (SEQ ID NO: 423).

[0207] FIG.123A to 123C are flow cytometry plots showing total protein expression of M5 and G2 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 1 (SEQ ID NO: 422) and an exemplary plasmid encoding G2 Construct 1 (SEQ ID NO: 423).

[0208] FIG.124A to 124C are flow cytometry plots showing surface expression of M5 and G2 antigens (e.g., extracellular presentation of M5 and G2 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 1 (SEQ ID NO: 422) and an exemplary plasmid encoding G2 Construct 2 (SEQ ID NO: 424).

[0209] FIG.125A to 125C are flow cytometry plots showing total protein expression of M5 and G2 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 1 (SEQ ID NO: 422) and an exemplary plasmid encoding G2 Construct 2 (SEQ ID NO: 424).

[0210] FIG.126A to 126C are flow cytometry plots showing surface expression of M5 and G2 antigens (e.g., extracellular presentation of M5 and G2 antigens) in HEK293T cells following transient Page 22 of 258 12621669v1Attorney Docket No.: 2013237-1080 transfection of the cells with an exemplary plasmid encoding M5 Construct 2 (SEQ ID NO: 425) and an exemplary plasmid encoding G2 Construct 1 (SEQ ID NO: 423).

[0211] FIG.127A to 127C are flow cytometry plots showing total protein expression of M5 and G2 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 2 (SEQ ID NO: 425) and an exemplary plasmid encoding G2 Construct 1 (SEQ ID NO: 423).

[0212] FIG.128A to 128C are flow cytometry plots showing surface expression of M5 and G2 antigens (e.g., extracellular presentation of M5 and G2 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 2 (SEQ ID NO: 425) and an exemplary plasmid encoding G2 Construct 2 (SEQ ID NO: 424).

[0213] FIG.129A to 129C are flow cytometry plots showing total protein expression of M5 and G2 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5 Construct 2 (SEQ DI NO: 425) and an exemplary plasmid encoding G2 Construct 2 (SEQ ID NO: 424).

[0214] FIG.130A to 130C are flow cytometry plots showing surface expression of a fusion protein comprising M5 and G2 antigens (e.g., extracellular presentation of M5 and / or G2 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5_G2 Construct 1 (SEQ ID NO: 426).

[0215] FIG.131A to 131C are flow cytometry plots showing surface expression of a fusion protein comprising M5 and G2 antigens (e.g., extracellular presentation of M5 and / or G2 antigens) in HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5_G2 Construct 2 (SEQ ID NO: 427).

[0216] FIG.132A to 132C are flow cytometry plots showing total protein expression of M5 and G2 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5_G2 Construct 1 (SEQ ID NO: 426).

[0217] FIG.133A to 133C are flow cytometry plots showing total protein expression of M5 and G2 antigens in fixed, permeabilized HEK293T cells following transient transfection of the cells with an exemplary plasmid encoding M5_G2 Construct 2 (SEQ ID NO: 427).

[0218] FIGs.134A to 159B provide graphs showing average body weight change and percent survival of populations of BALB / c mice immunized with saline (FIGs.134A to 134B) or an exemplary polyribonucleotide composition prior to intranasal challenge with vaccinia virus (VACV).

[0219] FIGs.135A to 135B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A28 Construct 1 (SEQ ID NO: 291).

[0220] FIG.136A to 136B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A21 Construct 2 (SEQ ID NO: 307).

[0221] FIG.137A to 137B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding L5 Construct 2 (SEQ ID NO: 319).

[0222] FIG.138A to 138B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding C15 Construct 1 (SEQ ID NO: 321).

[0223] FIG.139A to 139B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A17 Construct 1 (SEQ ID NO: 295). Page 23 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0224] FIG.140A to 140B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding G10 Construct 1 (SEQ ID NO: 299).

[0225] FIG.141A to 141B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A17 Construct 1 (SEQ ID NO: 295) and an exemplary polyribonucleotide encoding G10 Construct 1 (SEQ ID NO: 299).

[0226] FIG.142A to 142B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A17 Construct 1 (SEQ ID NO: 295) and an exemplary polyribonucleotide encoding G10 Construct 2 (SEQ ID NO: 301).

[0227] FIG.143A to 143B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A30 Construct 2 (SEQ ID NO: 311).

[0228] FIG.144A to 144B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A14 Construct 1 (SEQ ID NO: 323).

[0229] FIG.145A to 145B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A17_G10 Construct 1 (SEQ ID NO: 303).

[0230] FIG.146A to 146B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding H2 Construct 2 (SEQ ID NO: 315).

[0231] FIG.147A to 147B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A14 Construct 2 (SEQ ID NO: 325).

[0232] FIG.148A to 148B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding B2 Construct 1 (SEQ ID NO: 275).

[0233] FIG.149A to 149B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding C2 Construct 1 (SEQ ID NO: 279).

[0234] FIG.150A to 150B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding D14 Construct 1 (SEQ ID NO: 283).

[0235] FIG.151A to 151B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding B2 Construct 1 (SEQ ID NO: 275) and an exemplary polyribonucleotide encoding C2 Construct 1 (SEQ ID NO: 279).

[0236] FIG.152A to 152B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding B2 Construct 1 (SEQ ID NO: 275) and an exemplary polyribonucleotide encoding D14 Construct 1 (SEQ ID NO: 283).

[0237] FIG.153A to 153B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding B2 Construct 1 (SEQ ID NO: 275) and D14 Construct 1 (SEQ ID NO: 283) and an exemplary polyribonucleotide encoding C2 Construct 1 (SEQ ID NO: 279).

[0238] FIG.154A to 154B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding D14_C2 Construct 1 (SEQ ID NO: 287).

[0239] FIG.155A to 155B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding M5 Construct 2 (SEQ ID NO: 329).

[0240] FIG.156A to 156B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding G2 Construct 2 (SEQ ID NO: 333).

[0241] FIG.157A to 157B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding M5 Construct 2 (SEQ ID NO: 329) and an exemplary polyribonucleotide encoding G2 Construct 2 (SEQ ID NO: 333). Page 24 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0242] FIG.158A to 158B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding M5_G2 Construct 1 (SEQ ID NO: 335).

[0243] FIG.159A to 159B: BALB / c mice (n=10) immunized with an exemplary polyribonucleotide encoding A36 Construct 2 (SEQ ID NO: 459).

[0244] FIGs.160A to 160F provide graphs showing the ability of serum from mice vaccinated with exemplary polyribonucleotides of the disclosure to neutralize vaccinia virus (in presence or absence of complement).

[0245] FIGs.161A to 161B provide graphs showing the ability of serum from mice vaccinated with exemplary polyribonucleotides of the disclosure to neutralize monkeypox virus (in presence or absence of complement). DEFINITIONS

[0246] Compositions and compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0247] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, provided compounds show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and / or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.

[0248] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this disclosure.

[0249] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0250] Agent: As used herein, the term “agent,” may refer to a physical entity. In some embodiments, an agent may be characterized by a particular feature and / or effect. For example, as used herein, the term “therapeutic agent” refers to a physical entity has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, an agent may be a compound, molecule, or entity of any Page 25 of 258 12621669v1Attorney Docket No.: 2013237-1080 chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.

[0251] Aliphatic: The term “aliphatic” refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “cycloaliphatic”), that has a single point or more than one points of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., C1-6). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6alkyl.

[0252] Alkyl: The term “alkyl,” used alone or as part of a larger moiety, refers to a saturated, optionally substituted straight or branched chain hydrocarbon group having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C1-12,C1-3, or C1-2). Exemplary alkyl groupsinclude methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0253] Alkylene: The term “alkylene” is refers to a bivalent alkyl group. In some embodiments, “alkylene” is a bivalent straight or branched alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, e.g., from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is optionally replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group and also include those described in the specification herein. It will be appreciated that two substituents of the alkylene group may be taken together to form a ring system. In some embodiments, two substituents can be taken together to form a 3- to 7-membered ring. The substituents can be on the same or different atoms. The suffix “-ene” or “-enyl” when appended to certain groups herein are intended to refer to a bifunctional moiety of said group. For example, “-ene” or “-enyl”, when appended to “cyclopropyl” becomes “cyclopropylene” or “cyclopropylenyl” and is intended to refer to a bifunctional cyclopropyl group.

[0254] Alkenyl: The term “alkenyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain or cyclic hydrocarbon group having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term “cycloalkenyl” refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0255] Alkynyl: The term “alkynyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl. Page 26 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0256] Amino acid: In its broadest sense, as used herein, the term “amino acid” refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N– C(H)(R)–COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0257] Antigen: The term “antigen”, as used herein, refers to an agent that (i) elicits an immune response (e.g., adaptive humoral immune response, cell-mediated immunity, or both); and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody.

[0258] Anti-orthopoxvirus immune response: The term “anti-orthopoxvirus immune response”, as used herein, refers to an immune response produced through pre-exposure to one or more orthopoxvirus antigens, e.g., through administration (e.g., vaccination) of the polyribonucleotide compositions or constructs as described herein directed to orthopoxvirus.

[0259] Aryl: The term “aryl” refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, an “aryl” group contains between six and twelve total ring members (e.g., C6-C12). The term “aryl” may be used interchangeably with the term “aryl ring”. In some embodiments, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons. In some embodiments, an “aryl” ring system is an aromatic ring (e.g., phenyl) that is fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aryl rings include that are fused include

[0260] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). Page 27 of 258 12621669v1Attorney Docket No.: 2013237-1080 In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0261] Co-administration: As used herein, the term “co-administration” refers to use of a composition (e.g., a pharmaceutical composition) described herein and one or more additional therapeutic agents. In some embodiments, one or more additional therapeutic agents comprises at least one polyribonucleotide. In some embodiments, two or more of the polyribonucleotides described herein can be co- administered to a subject. For example, in some embodiments, a first polyribonucleotide encoding an A17 antigen and a second polyribonucleotide encoding a G10 antigen can be co-administered to a subject. The combined use of a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent may be performed concurrently or separately (e.g., sequentially in any order). In some embodiments, a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent may be combined in one pharmaceutically acceptable excipient, or they may be placed in separate excipient and delivered to a target cell or administered to a subject at different times. Each of these situations is contemplated as falling within the meaning of “co-administration” or “combination,” provided that a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent are delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.

[0262] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.

[0263] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences Page 28 of 258 12621669v1Attorney Docket No.: 2013237-1080 in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0264] Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0265] Cycloaliphatic: As used herein, the term “cycloaliphatic” refers to a monocyclic C3-8hydrocarbon or a bicyclic C6-10hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point or more than one points of attachment to the rest of the molecule.

[0266] Cycloalkyl: As used herein, the term “cycloalkyl” refers to an optionally substituted saturated ring monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0267] Derived: In the context of an amino acid sequence (peptide or polypeptide), one amino acid sequence can be “derived from” a designated or reference amino acid sequence (peptide or polypeptide) if it is a structural analogue of the designated or reference amino acid sequence. In some embodiments, an amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a designated or reference amino acid sequence may be variants of that designated or reference sequence or a fragment thereof.

[0268] Detecting: The term “detecting” is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest (e.g., an antigen or antigenic fragment) or any form of measurement of an entity of interest in a sample. Thus, “detecting” may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and / or location of an entity of interest. Page 29 of 258 12621669v1Attorney Docket No.: 2013237-1080 Quantitative and qualitative determinations, measurements or assessments are included, including semi- quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest is being detected relative to a control reference, or absolute. As such, the term “quantifying” when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification. Absolute quantification may be accomplished by correlating a detected level of an entity of interest to known control standards (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different entities of interest to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.

[0269] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” (or “therapeutic regimen”) may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic (or prophylactic or immunizing) agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises two doses of the same composition (e.g., the same polyribonucleotide composition). In some embodiments, a second dose is administered to a subject 2-4 weeks after a first dose.

[0270] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, a DNA molecule, or an RNA molecule encodes a polypeptide if transcription and / or translation of said gene, cDNA, DNA molecule, or RNA molecule produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen or antigenic fragment refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0271] Endogenous: As used herein, the term “endogenous”, with respect to a secretory signal or a transmembrane region operably linked to a polypeptide (e.g., antigen, variant thereof, or fragment thereof), refers to a secretory signal or transmembrane region from an orthopoxvirus that is the same as the orthopoxvirus from which the polypeptide (e.g., antigen, variant thereof, or fragment thereof) was derived.

[0272] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.

[0273] Epitope: As used herein, the term “epitope” refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope may be recognized by a T cell, a B cell, or an antibody. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface- exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such Page 30 of 258 12621669v1Attorney Docket No.: 2013237-1080 chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Accordingly, in some embodiments, an epitope of an antigen may include a continuous or discontinuous portion of the antigen. In some embodiments, an epitope is or comprises a T cell epitope. In some embodiments, an epitope may have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.

[0274] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In addition, the term “expression” in the context of an amino acid sequence refers to the generation of polypeptide from the amino acid sequence (or a nucleic acid sequence encoding the amino acid sequence). In some embodiments, a gene product can be a transcript, e.g., a polyribonucleotide as provided herein. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0275] Fragment: As used herein, “fragment” refers a structure that is or includes a discrete portion of a reference agent (sometimes referred to as the “parent” agent). In some embodiments, a fragment lacks one or more moieties found in the reference agent. In some embodiments, a fragment is or includes one or more moieties found in the reference agent. In some embodiments, the reference agent is a polymer such as a polynucleotide or polypeptide. In some embodiments, a fragment of a polymer is or includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., amino acid residues) of the reference polymer. In some embodiments, a fragment of a polymer is or includes at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the reference polymer. A fragment of a reference polymer is not necessarily identical to a corresponding portion of the reference polymer. For example, a fragment of a reference polymer can be a polymer having a sequence of residues having at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the reference polymer. A fragment may, or may not, be generated by physical fragmentation of a reference agent. In some instances, a fragment is generated by physical fragmentation of a reference agent. In some instances, a fragment is not generated by physical fragmentation of a reference agent and can be instead, for example, produced by de novo synthesis or other means.

[0276] Fusion: The term “fusion” or “fusion protein” or “fusion polypeptide” as used herein, refers to two or more polypeptides connected via one or more linkers to form a contiguous polypeptide chain. In some embodiments, two polypeptides may be connected via a linker peptide. In some embodiments, a linker peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. In some embodiments, a linker peptide comprises or consists of a series of glycine and serine residues. In some embodiments, a fusion protein comprises two or more antigens or antigenic fragments connected via one or more linker peptides. Page 31 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0277] Heteroaliphatic: The term “heteroaliphatic” or “heteroaliphatic group,” as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight–chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. The term “nitrogen” also includes a substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1–10 carbon atoms wherein 1–3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1–4 carbon atoms, wherein 1–2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1–3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2-CH2-O-CH2-CH2-O- CH3, and the like.

[0278] terms “heteroaryl” and “heteroar–”, used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 π- electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridyl, imidazo[4,5- b]pyridyl, imidazo[4,5-c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar–”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H–quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3–b]–1,4–oxazin–3(4H)–one, 4H-thieno[3,2- b]pyrrole, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted.

[0279] Heteroatom : The term “heteroatom” as used herein refers to nitrogen, oxygen, or sulfur, andincludes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.

[0280] Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, a 6- to 10- membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0–3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+(as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or Page 32 of 258 12621669v1Attorney Docket No.: 2013237-1080 carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms.

[0281] Heterologous: As used herein, the term “heterologous”, with respect to a secretory signal or a transmembrane region operably linked to a polypeptide (e.g., antigen, variant thereof, or fragment thereof), refers to a secretory signal or transmembrane region from a virus or an organism other than the orthopoxvirus from which the polypeptide (e.g., antigen, variant thereof, or fragment thereof) was derived. For example, a glycoprotein D (gD) secretory signal from herpes simplex virus (HSV) is a heterologous secretory signal when operably linked to a MPXV antigen (e.g., a MPXV antigen as described herein)

[0282] Homology: As used herein, the term “homology” or “homolog” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0283] Identity: As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of a sequence aligned for comparison purposes is at Page 33 of 258 12621669v1Attorney Docket No.: 2013237-1080 least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0284] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be “increased” relative to that obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0285] In order: As used herein with reference to a polynucleotide or polyribonucleotide, “in order” refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with reference to a polypeptide, “in order” refers to the order of features moving from the N-terminal-most of the features to the C-terminal-most of the features along the polypeptide. “In order” does not mean that no additional features can be present among the listed features. For example, if Features A, B, and C of a polynucleotide are described herein as being “in order, Feature A, Feature B, and Feature C,” this description does not exclude, e.g., Feature D being located between Features A and B. Page 34 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0286] Ionizable: The term “ionizable” refers to a compound or group or atom that is charged at a certain pH. In the context of an ionizable amino lipid, such a lipid or a function group or atom thereof bears a positive charge at a certain pH. In some embodiments, an ionizable amino lipid is positively charged at an acidic pH. In some embodiments, an ionizable amino lipid is predominately neutral at physiological pH values, e.g., in some embodiments about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, an ionizable amino lipid may have a pKa within a range of about 5 to about 7.

[0287] Isolated: The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0288] Linker: As used herein, the term “linker” refers to a portion of a polypeptide that connects different regions, portions, or antigens to one another.

[0289] Lipid: As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0290] RNA lipid nanoparticle: As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as provided herein. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0291] Neutralization: As used herein, the term “neutralization” refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term “neutralization” refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells.

[0292] Nucleic acid / Polynucleotide: As used herein, the terms “nucleic acid” or “polynucleotide”refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid Page 35 of 258 12621669v1Attorney Docket No.: 2013237-1080 comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-natural residues. In some embodiments, a non- natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro), reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0293] Operably linked: As used herein, “operably linked” refers to the association of at least a first element and a second element such that the component elements are in a relationship permitting them to function in their intended manner. For example, a nucleic acid sequence or amino acid sequence is operably linked with another sequence if it modifies the expression, structure, or activity of the linked sequence, e.g., in an intended manner. In many cases, two nucleic acid sequences are operably linked if they contribute to the expression, structure, or activity of a gene or encoded polypeptide. For example, a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence if the regulatory sequence and coding sequence are associated in a manner that permits control of expression of the coding sequence by the regulatory sequence. In some embodiments, an "operably linked" regulatory sequence is directly or indirectly covalently associated with a coding sequence (e.g., in a single nucleic acid). In some embodiments, a regulatory sequence controls expression of a coding sequence in trans and inclusion of the regulatory sequence in the same nucleic acid as the coding sequence is not a requirement of operable linkage. In many cases, two amino acid sequences are operably linked if they are expressed as a single polypeptide.

[0294] Pharmaceutically effective amount: The term “pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease (e.g., orthopoxvirus infection), a desired reaction in some embodiments relates to inhibition of the course of the disease (e.g., orthopoxvirus infection). In some embodiments, such inhibition may comprise slowing down the progress of a disease (e.g., orthopoxvirus infection) and / or interrupting or reversing the progress of the disease (e.g., Page 36 of 258 12621669v1Attorney Docket No.: 2013237-1080 orthopoxvirus infection). In some embodiments, a desired reaction in a treatment of a disease (e.g., orthopoxvirus infection) may be or comprise delay or prevention of the onset of a disease (e.g., orthopoxvirus infection) or a condition (e.g., an orthopoxvirus infection associated condition). An effective amount of a composition (e.g., a pharmaceutical composition) described herein will depend, for example, on disease (e.g., orthopoxvirus infection) or a condition (e.g., a orthopoxvirus infection associated condition) to be treated, the severity of such a disease (e.g., orthopoxvirus infection) or a condition, individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of a composition (e.g., a pharmaceutical composition) described herein may depend on various such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0295] Polypeptide: As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non- natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at Page 37 of 258 12621669v1Attorney Docket No.: 2013237-1080 least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0296] Prevent: As used herein, the terms “prevent” or “prevention” when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0297] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0298] Ribonucleic acid (RNA) or Polyribonucleotide: As used herein, the terms “ribonucleic acid,” “RNA,” or “polyribonucleotide” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA). In some embodiments, an RNA is an mRNA. In some embodiments in which an RNA is a mRNA, said mRNA typically comprises at its 3' end a poly(A) region. In some embodiments in which an RNA is a mRNA, said mRNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0299] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0300] Risk: As will be understood from context, “risk” of a disease, disorder, and / or condition refers to a likelihood that a particular individual will develop the disease, disorder, and / or condition. In some Page 38 of 258 12621669v1Attorney Docket No.: 2013237-1080 embodiments, risk is expressed as a percentage. In some embodiments, risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and / or event. In some embodiments, a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and / or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes.

[0301] Secretory signal: As used herein, the terms “secretory signal” or “signal peptide” or “signal sequence” refer to an amino acid sequence motif that targets associated polypeptides for translocation to a secretory pathway. In some embodiments, a secretory signal or signal peptide or signal sequence is abbreviated and referred to as a “SP.”

[0302] Selective or specific: The terms “selective” or “specific,” when used herein in reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a target- binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.

[0303] Substituted or optionally substituted: As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structuremay have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in some embodiments, their recovery, purification, and use for one or more of the purposes provided herein. Groups described as being “substituted” preferably have Page 39 of 258 12621669v1Attorney Docket No.: 2013237-1080 between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.

[0304] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; –(CH2)0–4R°; –(CH2)0–4OR°; -O(CH2)0-4Ro, –O–(CH2)0–4C(O)OR°; –(CH2)0–4CH(OR°)2; –(CH2)0–4SR°; –(CH2)0–4Ph, which may be substituted with R°; –(CH2)0–4O(CH2)0–1Ph which may be substituted with R°; –CH=CHPh, which may be substituted with R°; –(CH2)0–4O(CH2)0–1-pyridyl which may be substituted with R°; –NO2; –CN; –N3; -(CH2)0–4N(R°)2; –(CH2)0–4N(R°)C(O)R°; –N(R°)C(S)R°; –(CH2)0–4N(R°)C(O)NR°2; -N(R°)C(S)NR°2; –(CH2)0–4N(R°)C(O)OR°; - N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; –(CH2)0–4C(O)R°; C(S)R°; –(CH2)0–4C(O)OR°; – (CH2)0–4C(O)SR°; -(CH2)0–4C(O)OSiR°3; –(CH2)0–4OC(O)R°; –OC(O)(CH2)0–4SR°; –(CH2)0–4SC(O)R°; –(CH2)0–4C(O)NR°2; –C(S)NR°2; –C(S)SR°; –SC(S)SR°, -(CH2)0–4OC(O)NR°2; -C(O)N(OR°)R°; –C(O)C(O)R°; – C(O)CH2C(O)R°; –C(NOR°)R°; -(CH2)0–4SSR°; –(CH2)0–4S(O)2R°; –(CH2)0–4S(O)2OR°; –(CH2)0–4OS(O)2R°; – S(O)2NR°2; -(CH2)0–4S(O)R°; -N(R°)S(O)2NR°2; –N(R°)S(O)2R°; –N(OR°)R°; –C(NH)NR°2; – P(O)2R°; -P(O)R°2; -OP(O)R°2; –OP(O)(OR°)2; SiR°3; –(C1–4straight or branched alkylene)O–N(R°)2; or –(C1–4straight or branched alkylene)C(O)O–N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C1–6aliphatic, –CH2Ph, –O(CH2)0–1Ph, -CH2-(5- to 6-membered heteroaryl ring), or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3- to 12-membered saturated, partially unsaturated, or aryl mono– or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0305] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, –(CH2)0–2R^, –(haloR^), –OSiR^3, -C(O)SR^, –(C1–4 straight or branched alkylene)C(O)OR^, or –SSR^wherein each R^is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1–4aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =O and =S.

[0306] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =O (“oxo”), =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*, =NOR*, – O(C(R*2))2–3O–, or –S(C(R*2))2–3S–, wherein each independent occurrence of R*is selected from hydrogen, C1–6aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: –O(CR*2)2–3O–, wherein each independent occurrence of R*is selected from hydrogen, C1–6aliphatic which may be substituted as defined below, or an unsubstituted 5–6–membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0307] Suitable substituents on the aliphatic group of R*include halogen, –R^, -(haloR^), -OH, –OR^, –O(haloR^), –CN, –C(O)OH, –C(O)OR^, –NH2, –NHR^, –NR^2, or –NO2, wherein each R^is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1–4aliphatic, – Page 40 of 258 12621669v1Attorney Docket No.: 2013237-1080 CH2Ph, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0308] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include – R†, –NR†2, –C(O)R†, –C(O)OR†, –C(O)C(O)R†, –C(O)CH2C(O)R†, -S(O)2R†, -S(O)2NR†2, –C(S)NR†2, –C(NH)NR†2, or –N(R†)S(O)2R†; wherein each R†is independently hydrogen, C1–6aliphatic which may be substituted as defined below, unsubstituted –OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0309] Suitable substituents on the aliphatic group of R†are independently halogen, –R^, -(haloR^), – OH, –OR^, –O(haloR^), –CN, –C(O)OH, –C(O)OR^, –NH2, –NHR^, –NR^2, or -NO2, wherein each R^is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1–4aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0310] Subject: As used herein, the term “subject” refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered. In some embodiments, a subject is a vaccinia-naïve subject. In some embodiments, a vaccinia-naïve subject has no prior history of known or suspected orthopox (e.g., smallpox) vaccination. In some embodiments, a subject is a vaccinia-experienced subject. In some embodiments, a vaccinia-experienced subject has prior history of an orthopox (e.g., smallpox) vaccination. In some embodiments, a subject has not been previously infected with an orthopox virus (e.g., MPXV, VACV, MPXV, ECTV, AKPV, CPXV, MVA, VPXV, or VARV). In some embodiments, a subject has been previously infected with an orthopox virus (e.g., MPXV, VACV, MPXV, ECTV, AKPV, CPXV, MVA, VPXV, or VARV). In some embodiments, a subject has 1, 2, 3, 4, 5, or more previous orthopox virus (e.g., MPXV, VACV, MPXV, ECTV, AKPV, CPXV, MVA, VPXV, or VARV) infections. In some embodiments, a subject has not experienced an Page 41 of 258 12621669v1Attorney Docket No.: 2013237-1080 orthopox virus infection in at least 6, 12, 18, or 24 months prior to immunization with a polyribonucleotide of the present disclosure.

[0311] Suffering from : An individual who is “suffering from” a disease, disorder, and / or condition(e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, or vaccinia) has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0312] Susceptible to: An individual who is “susceptible to” a disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) is one who has a higher risk of developing the disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) may not have been diagnosed with the disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) may exhibit symptoms of the disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) may not exhibit symptoms of the disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) will develop the disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) will not develop the disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia).

[0313] Therapy: The term “therapy” refers to an administration or delivery of an agent or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a polyribonucleotide of the present disclosure that can be used to prevent, delay onset of, reduce severity of, and / or reduce incidence of an orthopox infection (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia infection) Page 42 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0314] Transmembrane region: As used herein, the terms “transmembrane region” or “transmembrane domain” refer to a region of a polypeptide that spans a biological membrane, such as the plasma membrane of a cell. In some embodiments, a transmembrane region or transmembrane domain is abbreviated and referred to as a “TM region” or “TM domain.”

[0315] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). Treatments of the present disclosure include prophylactic administration of one or more polyribonucleotides of the present disclosure to a subject or population of subjects (e.g., to immunize or vaccinate the subject or population of subjects against orthopox infections such as mpox infections). Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia), for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition (e.g., orthopox, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia).

[0316] Variant: As used herein, the term “variant” refers to a molecule that shows significant structural (e.g., primary or secondary) identity with a reference molecule but differs structurally from the reference molecule. For example, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0317] The present disclosure provides compositions (e.g., pharmaceutical compositions, e.g., immunogenic compositions, e.g., vaccines) for delivering particular orthopoxvirus antigen constructs to a subject (e.g., a patient) and related technologies (e.g., methods). In particular, the present disclosure provides orthopoxvirus vaccine compositions and related technologies (e.g., methods of immunization or vaccination). I. Mpox and Other Orthopoxviruses

[0318] Orthopoxviruses (OPVs) are characterized by structural and lifecycle complexity. Orthopoxviruses include, for example, mpox virus (previously known as “monkeypox”; also referred to herein as “MPXV”), vaccinia virus (also referred to herein as “VACV”), ectromelia virus (also referred to herein as “ECTV”), borealpox virus (previously known as “Alaskapox”; also referred to herein as “AKPV” or “BRPV”), cowpox virus (also referred to herein as “CPXV”), modified vaccinia virus Ankara (also referred to herein as “MVA”), volepox (also referred to herein as “VPXV”), and variola virus (also referred to herein as “VARV”). Mpox is closely related to variola virus, and the present disclosure encompasses a recognition that the relatedness of orthopoxviruses is high in general. Page 43 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0319] Resulting from successful campaigns to promote vaccination against variola virus, smallpox (a disease that is caused by variola virus infection(s)) was declared eradicated in 1980. However, since then, the world population has largely become unvaccinated against orthopoxviruses. Low levels of transmission of mpox have supported endemicity in West and Central Africa for years, but recently there has been a community spread of mpox in non-endemic regions including Europe and North America. The waning global population-level immunity against orthopoxviruses is at least partly responsible for this recent spread of mpox.

[0320] Mpox and other orthopoxviruses are a continuing global threat. Variola virus is classified as a category A bioterrorism agent that is especially of concern given the current limitations in supply of canonically produced vaccines. The recent spread of mpox and emergence of borealpox virus also highlights a continuing risk of emergence of a novel orthopoxvirus.

[0321] The present disclosure provides orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) polyribonucleotides, antigen constructs, and / or pharmaceutical compositions that are effective for vaccination against orthopox (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia). The present disclosure also provides the insight that polyribonucleotides, antigen constructs, and pharmaceutical compositions targeting one orthopox species may cross-protect against other orthopoxviruses. For example, in some embodiments, provided MPXV polyribonucleotides, antigen constructs, and / or pharmaceutical compositions are effective for vaccination against mpox and one or more other orthopox viruses. In some embodiments, provided MPXV polyribonucleotides, antigen constructs, and / or pharmaceutical compositions are effective for vaccination against mpox and one or more of variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia. In some embodiments, provided MPXV polyribonucleotides, antigen constructs, and / or pharmaceutical compositions are effective for vaccination against mpox and a novel orthopox virus.

[0322] In some embodiments, provided orthopoxvirus polyribonucleotides encode one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) antigens, variants thereof, or antigenic fragments thereof. In some embodiments, provided orthopoxvirus antigen constructs include one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) antigens, variants thereof, or antigenic fragments thereof. In some embodiments, provided pharmaceutical compositions include (i) a pharmaceutically acceptable carrier and (ii) one or more polyribonucleotides encoding one or more orthopoxvirus (e.g. mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) antigens, variants thereof, or antigenic fragments thereof, or one or more antigen constructs including one or more orthopoxvirus (e.g. mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) antigens, variants thereof or antigenic fragments thereof. Mpox Structure

[0323] Mpox virions are ovoid or brick-shaped particles which are enclosed by geometrically corrugated lipoprotein outer membrane. Mature mpox virions have a densely packed core containing enzymes, a double-stranded DNA genome, and transcription factors that are protected by a protein core.

[0324] The mpox genome consists of a linear double-stranded DNA (about 197 kb) covalently joined at its ends by palindromic hairpins, and the inverted terminal repeats (ITRs) are made up of a hairpin loop, tandem repeats, and some open reading frames (ORF). Although MPXV is a DNA virus, its entire life cycle occurs in the cytoplasm of infected cells. All the proteins required for viral DNA replication, transcription, virion assembly, and egress are encoded by the mpox genome. The genes encoding for housekeeping functions are Page 44 of 258 12621669v1Attorney Docket No.: 2013237-1080 highly conserved among OPVs and are present in the central region of the genome while those that encode for the genes mediating virus–host interactions are less conserved and located in the terminal regions of the genome.

[0325] In the context of vaccinia virus (and most likely in mpox virus), intracellular mature virus (referred to as the “MV” form throughout the present disclosure) and extracellular-enveloped virus (referred to as the “EV” form throughout the present disclosure) are two forms of infectious virions produced in poxvirus- infected cells. MV is released upon cell lysis, while EV is released from cells via interaction with actin tails, and this is said to be the cause of rapid long-distance spread of the virus within the infected host. Although the aforementioned features are for VACV, it is likely that these features are common to all orthopoxviruses, including MPXV. Cell-associated virions (CEVs) are formed following the microtubule-mediated transport of intracellular enveloped virus (IEV) to the cell periphery, in which the outer membrane of IEV fuses with the plasma membrane and remains attached to the cell surface. CEVs are mostly responsible for cell-to-cell spread. IEV is formed when IMV is wrapped by a double membrane derived from early endosomal component or the trans-Golgi network (TGN). However, apart from IEV exocytosis, an alternative route for the formation of EV is by the budding of MV through the plasma membrane. In the prototype vaccinia virus, virion morphogenesis can be defective resulting in non-infectious dense particles (DPs), but this has not yet been reported for mpox. In addition, unlike some strains of CPXV in which IMVs are occluded within A-type inclusions (ATI), mpox does not form ATIs or sequester MVs into ATIs because of truncation in the ATIP gene. Mpox Transmission

[0326] The two possible means of mpox transmission are animal-to-human transmission and human- to-human transmission. Respiratory droplets and contact with body fluids, contaminated patient’s environment or items, and skin lesion of an infected person have been found to be associated with inter-human transmission. Contact between broken skin or mucous membranes and an infected patient’s body fluids, respiratory droplets, or scabs is considered a “high risk” exposure that warrants post-exposure vaccination as soon as possible. Congo Basin (CB) clade (Central Africa clade) is reported to be more virulent than West Africa (WA) clade and thereby contributes more to inter-human transmission. Animal-to-human transmission, which is also known as zoonotic transmission, occurs via direct contact with any of the aforementioned natural viral hosts or consumption of these hosts. In addition, zoonotic transmission could occur by direct contact with the blood, body fluids, and inoculation from mucocutaneous lesions of an infected animal. Nosocomial transmission has been reported for CB and WA clades of mpox while sexual transmission has been speculated for infected individuals with groin and genital lesions. At present human-to-animal transmission has not been reported. Human-to-human transmission, secondary attack rates (SARS), and serial transmission events is much higher with the CB clade compared to the WA clade. The reproduction number R0 for the CB clade is estimated to be in the range of 0.6–1.0. The R0 has not be estimated for the WA clade of mpoxes, but it is presumed to be lower than that of the CB clade. The upper limit R0 of 1.0 in the CB clade indicates that the viruses will not only sustain human-to-human transmission but may persist in the human population. Presumably, if as expected the R0 of the WA clade is much lower than what was estimated for the CB clade, then sustained human-to-human transmission and persistence in human population are highly unlikely and outbreaks will be largely due to spillover events from zoonotic hosts. Mpox Treatment

[0327] Currently, there are no specific clinically proven treatments for mpox infection. As with most viral illnesses, the treatment is supportive symptom management. There are, however, prevention measures that Page 45 of 258 12621669v1Attorney Docket No.: 2013237-1080 can help prevent and / or reduce severity of an outbreak. Infected individuals should remain in isolation, wear a surgical mask, and keep lesions covered as much as reasonably possible until all lesion crusts have naturally fallen off and a new skin layer has formed. For severe cases, investigational use can be considered for compounds with demonstrated benefit against orthopoxviruses in animal studies and severe vaccinia vaccine complications. The oral DNA polymerase inhibitor brincidofovir, oral intracellular viral release inhibitor tecovirimat, and intravenous vaccinia immune globulin have unknown efficacy against the mpox virus. For individuals exposed to the virus, temperature and symptoms should be monitored twice per day for 21 days because that is the accepted upper limit of the mpox incubation period. Infectiousness aligns with symptom onset; therefore, close contacts need not isolate while asymptomatic. According to the Centers for Disease Control and Prevention (CDC), vaccination within four days of exposure may prevent disease onset, and vaccination within 14 days may reduce disease severity. II. Orthopoxvirus Polypeptide Constructs

[0328] The present disclosure, among other things, utilizes RNA technologies as a modality to express one or more orthopoxvirus polypeptide constructs that include one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) antigens, variants thereof, or antigenic fragments thereof, as described herein. For example, in some embodiments, an orthopoxvirus polypeptide construct includes one or more MPXV antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct additionally includes one or more additional amino acid sequences, such as a secretory signal (e.g., a heterologous secretory signal), a transmembrane region (e.g., a heterologous transmembrane region), and / or a linker, as described herein.

[0329] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes a fusion polypeptide incorporating two or more operably linked orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, modified vaccinia virus Ankara, or vaccinia) proteins, variants thereof, or antigenic fragments thereof, as described herein. In some embodiments, a fusion polypeptide additionally includes one or more additional amino acid sequences, such as a secretory signal (e.g., a heterologous secretory signal), a transmembrane region (e.g., a heterologous transmembrane region), a linker, and / or a cleavable linker, as described herein. MPXV Antigens

[0330] The inventors of the present disclosure have identified that certain MPXV antigens (specifically, A21, A36, A28, L5, H2, A30, C15, A14, B2, C2, D14, A17, G10, M5, and G2), variants thereof, antigenic fragments thereof, and fusions thereof, are useful in polyribonucleotide compositions (that encode orthopoxvirus polypeptide constructs) to immunize or vaccinate subjects against orthopox virus infection (e.g., mpox virus infection).

[0331] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more MPXV antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct includes a full-length MPXV antigen. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of a full-length MPXV antigen. In some embodiments, an orthopoxvirus polypeptide construct includes an MPXV antigen variant or an antigenic fragment thereof. In some embodiments, an MPXV antigen variant, or antigenic fragment thereof, incorporates one or more amino acid substitutions. For example, in some embodiments, an MPXV antigen variant, or antigenic fragment thereof, incorporates one or more (e.g., one, two, three, four, five, six, or more) cysteine substitutions. Page 46 of 258 12621669v1Attorney Docket No.: 2013237-1080 A21L

[0332] In its native context in the mature virion (MV) membrane, A21L (also referred to as “A21” herein) is a type II transmembrane protein with an extracellular domain (ECD) positioned at its C-terminus. Mpox A21L sequences include, e.g., UniProt accession number Q8V4V7, the content of which is incorporated herein by reference in its entirety. Wild-type MPXV A21L antigen includes an N-terminal transmembrane region corresponding to residues 1-22. MPXV A21L antigen is an ortholog of VACV A12L antigen.

[0333] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more A21L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length A21L antigen. In some embodiments, an A21L antigenic fragment does not include the N-terminal transmembrane region of full-length A21L. In some embodiments, an A21L antigenic fragment comprises or consists of an extracellular domain of A21L. Thus, in some embodiments, an A21L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 23-115 of a full-length A21L antigen (e.g., having the amino acid sequence of SEQ ID NO: 305). In some embodiments, a full-length A21L antigen comprises or consists of 100-125 amino acid residues or 110-120 amino acid residues, optionally 115 amino acid residues.

[0334] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length A21L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 305. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an A21L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 342. A36R

[0335] A36R (also referred to as “A36” herein) is a type II integral membrane protein embedded in the enveloped virus (EV) membrane. Although A36 may traffic normally to the cell surface when ectopically expressed in mammalian cells, it has several proposed viral protein binding partners and independent expression of A36 in the absence of other viral co-factors may not result in cell surface display of the A36 C-terminal extracellular domain. Mpox A36R sequences include, e.g., UniProt accession numbers Q8V4U3, Q911X0, and Q911W9, the contents of which are incorporated herein by reference in their entireties. Wild-type MPXV A36 antigen is 168 amino acids in length. MPXV A36R antigen is an ortholog of VACV A34R antigen.

[0336] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more A36R antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length A36R antigen. In some embodiments, an A36 antigenic fragment does not include the transmembrane region of full-length A36. In some embodiments, an A36 antigenic fragment comprises or consists of an extracellular domain of A36. Thus, in some embodiments, an A36 antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 39-168 of a full-length A36 antigen (e.g., having the amino acid sequence of SEQ ID NO: 273). In some embodiments, a full-length A36R antigen comprises or consists of 150-190 amino acid residues or 160-180 amino acid residues, optionally 168 amino acid residues. Page 47 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0337] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length A36R antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 273. In some embodiments, an orthopoxvirus polypeptide construct includes a full-length A36R antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 458. A28L

[0338] A28L (also referred to as “A28” herein) is a secreted antigen that is tethered to the plasma membrane via interaction with other viral proteins (including A29L). Mpox A28L sequences include, e.g., UniProt accession numbers VpNSD8, V9NKU4, V9NWM3, V9NNF4, Q8V4V0, V9NR63, A0A0F7G921, A0A2L1F535, A0A0F7GAP0, and A0A0F7GB12, the contents of which are incorporated herein by reference in their entireties. Wild-type MPXV A28L includes two cysteine residues at positions 450 and 451 that form disulfide bonds with another viral protein. The C-terminal region of A28L includes a disordered region. MPXV A28L antigen is an ortholog of VACV A26L antigen. In some embodiments, A28L is an MV-associated protein.

[0339] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more A28L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length A28L antigen. In some embodiments, an A28L antigenic fragment does not include the disordered region of full-length A28L. In some embodiments, an A28L antigenic fragment comprises or consists of an extracellular domain of A28L. Thus, in some embodiments, an A28L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 1-366 of a full- length A28L antigen (e.g., having the amino acid sequence of SEQ ID NO: 196). In some embodiments, an orthopoxvirus polypeptide construct encoding an A28L antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface) and / or a heterologous transmembrane region (e.g., to assist with insertion of the polypeptide into the cell membrane for presentation of an antigenic fragment of the polypeptide). In some embodiments, an orthopoxvirus polypeptide construct encoding an A28L antigen is engineered to comprise an HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct encoding an A28L antigen is engineered to comprise an HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an A28L antigen is engineered to comprise an HSV gD secretory signal and an HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an A28L antigen is engineered to remove surface-exposed cysteine residues (e.g., at amino acid residues corresponding to positions 450 and 451 of SEQ ID NO: 196). In some embodiments, an orthopoxvirus polypeptide construct encoding an A28L antigen is engineered to comprise serines at amino acid residues corresponding to positions 450 and 451 of SEQ ID NO: 196. In some embodiments, a full-length A28L antigen comprises or consists of 470-550 amino acid residues or 500-530 amino acid residues, optionally 509 or 520 amino acid residues.

[0340] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length A28L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 196 or SEQ ID NO: 364. In some Page 48 of 258 12621669v1Attorney Docket No.: 2013237-1080 embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an A28L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 343. In some embodiments, an orthopoxvirus polypeptide construct includes an A28L variant incorporating one or more amino acid substitutions compared to a wild-type A28L sequence. In some embodiments, an orthopoxvirus polypeptide construct includes an A28L variant incorporating one or more amino acid substitutions to remove cysteine residues compared to a wild-type A28L sequence. In some embodiments, an orthopoxvirus polypeptide construct includes an A28L variant incorporating one or more amino acid substitutions to remove cysteine residues at positions 450 and 451 compared to a wild-type A28L sequence. In some embodiments, an orthopoxvirus polypeptide construct includes an A28L variant incorporating C450S and C451S substitutions. In some embodiments, an orthopoxvirus polypeptide construct includes an A28L variant comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 344. L5L

[0341] L5L (also referred to as “L5” herein) is a type I transmembrane protein in the MV membrane with an extracellular domain positioned at its N-terminus. WT L5 lacks cytosolic residues C-terminal of the TM domain. Without wishing to be bound by any particular theory, the absence of cytosolic residues following a TM domain is known to cause expression problems for mammalian proteins. However, this may not be a problem in the context of the viral MV membrane. MV type I integral membrane proteins lack a SP. Therefore, addition of an N-terminal SP may be a minimal modification for correct trafficking of L5 protein to the cell surface. Mpox L5L sequences include, e.g., UniProt accession numbers Q77HN1 and V9NSL7, the contents of which are incorporated herein by reference in their entireties. MPXV L5L antigen includes a C-terminal transmembrane region corresponding to residues 110-133. MPXV L5L antigen is an ortholog of VACV J5L antigen.

[0342] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more L5L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length L5L antigen. In some embodiments, an L5L antigenic fragment does not include the C-terminal transmembrane region of full-length L5L. In some embodiments, an L5L antigenic fragment comprises or consists of an extracellular domain of L5L. Thus, in some embodiments, an L5L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 1-109 of a full-length L5L antigen (e.g., having the amino acid sequence of SEQ ID NO: 348). In some embodiments, an orthopoxvirus polypeptide construct encoding an L5L antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface) and / or a heterologous transmembrane region (e.g., to assist with insertion of the polypeptide into the cell membrane for presentation of an antigenic fragment of the polypeptide). In some embodiments, an orthopoxvirus polypeptide construct encoding an L5L antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct encoding an L5L antigen is engineered to comprise a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an L5L antigen is engineered to comprise a HSV gD secretory signal and a HSV gD transmembrane region. In some embodiments, a full-length L5L antigen comprises or consists of 110-155 amino acid residues or 120-140 amino acid residues, optionally 133 amino acid residues. Page 49 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0343] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length L5L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 348. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an L5L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 349. H2R

[0344] In its native context in the MV membrane, H2R (also referred to as “H2” herein) is a type II transmembrane protein with an extracellular domain positioned at its C-terminus (corresponding to residues 49- 189). Mpox H2R sequences include, e.g., UniProt accession numbers Q77HN0, Q5IXT3, and Q3I7M5, the contents of which are incorporated herein by reference in their entireties. MPXV H2R antigen is an ortholog of VACV H2R antigen.

[0345] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more H2R antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length H2R antigen. In some embodiments, an H2R antigenic fragment does not include the putative N-terminal transmembrane region of full-length H2R. In some embodiments, an H2R antigenic fragment comprises or consists of an extracellular domain of H2R. Thus, in some embodiments, an H2R antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 49-189 of a full-length H2R antigen (e.g., having the amino acid sequence of SEQ ID NO: 313). In some embodiments, an orthopoxvirus polypeptide construct encoding an H2R antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface) and / or a heterologous transmembrane region (e.g., to assist with insertion of the polypeptide into the cell membrane for presentation of an antigenic fragment of the polypeptide). In some embodiments, an orthopoxvirus polypeptide construct encoding an H2R antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct encoding an H2R antigen is engineered to comprise a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an H2R antigen is engineered to comprise a HSV gD secretory signal and a HSV gD transmembrane region. In some embodiments, a full-length H2R antigen comprises or consists of 170-210 amino acid residues or 180-200 amino acid residues, optionally 189 amino acid residues.

[0346] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length H2R antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 313. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an H2R antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 347. Page 50 of 258 12621669v1Attorney Docket No.: 2013237-1080 A30L

[0347] In its native context in the MV membrane, A30L (also referred to as “A30” herein) is a type II transmembrane protein with its extracellular domain on the C-terminus (corresponding to residues 22-146). Mpox A30L sequences include, e.g., UniProt accession number Q8V4U9, the contents of which is incorporated herein by reference in its entirety. MPXV A30L antigen is an ortholog of VACV A28L antigen.

[0348] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more A30L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length A30L antigen. In some embodiments, an A30L antigenic fragment does not include the N-terminal transmembrane region of full-length A30L. In some embodiments, an A30L antigenic fragment comprises or consists of an extracellular domain of A30L. Thus, in some embodiments, an A30L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 22-146 of a full-length A30L antigen (e.g., having the amino acid sequence of SEQ ID NO: 309) In some embodiments, an orthopoxvirus polypeptide construct encoding an A30L antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface) and / or a heterologous transmembrane region (e.g., to assist with insertion of the polypeptide into the cell membrane for presentation of an antigenic fragment of the polypeptide). In some embodiments, an orthopoxvirus polypeptide construct encoding an A30L antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct encoding an A30L antigen is engineered to comprise a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an A30L antigen is engineered to comprise a HSV gD secretory signal and a HSV gD transmembrane region. In some embodiments, a full-length A30L antigen comprises or consists of 125-165 amino acid residues or 140-155 amino acid residues, optionally 146 amino acid residues.

[0349] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length A30L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 309. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an A30L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 339. C15L

[0350] C15L (also referred to as “C15” herein) is a type I transmembrane protein in the MV membrane with its extracellular domain positioned at its N-terminus. MV type I integral membrane proteins lack a SP. Therefore, addition of an N-terminal SP may be a modification for trafficking of C15 protein to the cell surface. Mpox C15L sequences include, e.g., UniProt accession numbers Q8V539 and Q5IXY4, the contents of which are incorporated herein by reference in their entireties. MPXV C15L antigen is an ortholog of VACV F9L antigen.

[0351] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more C15L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length C15L antigen. In some embodiments, an orthopoxvirus polypeptide construct encoding an C15L antigen is engineered to comprise a heterologous secretory signal (e.g., Page 51 of 258 12621669v1Attorney Docket No.: 2013237-1080 to assist with trafficking the antigen to the cell surface). In some embodiments, an orthopoxvirus polypeptide construct encoding an C15L antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, a full-length C15L antigen comprises or consists of 190-230 amino acid residues or 200-220 amino acid residues, optionally 212 amino acid residues.

[0352] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length C15L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 345. A14L

[0353] In its native context in the MV membrane, A14L (also referred to as “A14” herein) is a type II transmembrane protein with an extracellular domain positioned at its C-terminus (corresponding to residues 25- 70). Mpox A14L sequences include, e.g., UniProt accession numbers Q77HM8 and Q3T624, the content of which are incorporated herein by reference in their entireties. MPXV A14L antigen is an ortholog of VACV A13L antigen.

[0354] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more A14L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length A14L antigen. In some embodiments, an A14L antigenic fragment does not include the N-terminal transmembrane region of full-length A14L. In some embodiments, an A14L antigenic fragment comprises or consists of an extracellular domain of A14L. Thus, in some embodiments, an A14L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 25-70 of a full-length A14L antigen (e.g., having the amino acid sequence of SEQ ID NO: 323). In some embodiments, an orthopoxvirus polypeptide construct encoding an A14L antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface) and / or a heterologous transmembrane region (e.g., to assist with insertion of the polypeptide into the cell membrane for presentation of an antigenic fragment of the polypeptide). In some embodiments, an orthopoxvirus polypeptide construct encoding an A14L antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct encoding an A14L antigen is engineered to comprise a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an A14L antigen is engineered to comprise a HSV gD secretory signal and a HSV gD transmembrane region. In some embodiments, a full-length A14L antigen comprises or consists of 55-85 amino acid residues or 60-80 amino acid residues, optionally 70 amino acid residues.

[0355] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length A14L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 323. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an A14L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 340. Page 52 of 258 12621669v1Attorney Docket No.: 2013237-1080 B2R

[0356] B2R (also referred to as poxvirus hemagglutinin and “B2” herein) is a type I integral membrane protein that contains a native secretory signal and is considered to be a hemagglutinin (HA) protein. B2 interacts with C2 and D14 to attach them to the EV membrane. Current models suggest that a B2 molecule interacts with either C2 or D14, not both simultaneously. B2 bonds to D14 via a disulfide bond mediated by a cysteine residue at position 160. Mpox B2R sequences include, e.g., UniProt accession numbers Q8VS6, Q8AZ47 and Q3T6V2, the contents of which are incorporated herein by reference in their entireties. MPXV B2R antigen has a cysteine residue at position 160 that may form disulfide bonds with other viral proteins. MPXV B2R antigen is an ortholog of VACV A56L antigen.

[0357] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more B2R antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length B2R antigen. In some embodiments, an orthopoxvirus polypeptide construct encoding a B2R antigen is engineered to remove surface-exposed cysteine residues (e.g., at amino acid residues corresponding to position 160 of SEQ ID NO: 275). In some embodiments, an orthopoxvirus polypeptide construct encoding a B2R antigen is engineered to comprise a serine at an amino acid residue corresponding to position 160 of SEQ ID NO: 275. In some embodiments, a full-length B2R antigen comprises or consists of 290-335 amino acid residues or 305-325 amino acid residues, optionally 313 amino acid residues.

[0358] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length B2R antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 275. In some embodiments, an orthopoxvirus polypeptide construct includes a B2R variant incorporating one or more amino acid substitutions compared to a wild-type B2R sequence. In some embodiments, an orthopoxvirus polypeptide construct includes an B2R variant incorporating one or more cysteine substitutions compared to a wild-type B2R sequence. In some embodiments, an orthopoxvirus polypeptide construct includes a B2R variant incorporating a cysteine substitutions at position 160 compared to a wild-type B2R sequence. In some embodiments, an orthopoxvirus polypeptide construct includes a B2R variant incorporating a C160S substitution. In some embodiments, an orthopoxvirus polypeptide construct includes a B2R variant comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 277. C2L

[0359] Mpox C2L (also referred to as serine protease inhibitor 3 [SPI-3] and “”C2” herein) is a type I integral membrane protein that contains a native secretory signal. C2L sequences include, e.g., UniProt accession numbers Q8V552, Q3T784, Q3I8Y7, and Q5QC70, the contents of which are incorporated herein by reference in their entireties. MPXV C2L antigen has a cysteine residue at position 115 that may form disulfide bonds with other viral proteins. MPXV C2L antigen is an ortholog of VACV K2 antigen.

[0360] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more C2L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length C2L antigen. In some embodiments, an C2L antigenic fragment does not include an N-terminal transmembrane region of full-length C2L. In some embodiments, an Page 53 of 258 12621669v1Attorney Docket No.: 2013237-1080 C2L antigenic fragment comprises or consists of an extracellular domain of C2L. Thus, in some embodiments, an C2L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 15-375 of a full-length C2L antigen (e.g., having the amino acid sequence of SEQ ID NO: 279). In some embodiments, an orthopoxvirus polypeptide construct encoding an C2L antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface) and / or a heterologous transmembrane region (e.g., to assist with insertion of the polypeptide into the cell membrane for presentation of an antigenic fragment of the polypeptide). In some embodiments, an orthopoxvirus polypeptide construct encoding an C2L antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct encoding an C2L antigen is engineered to comprise a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an C2L antigen is engineered to comprise a HSV gD secretory signal and a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding a C2L antigen is engineered to remove surface-exposed cysteine residues (e.g., at amino acid residues corresponding to position 115 of SEQ ID NO: 279). In some embodiments, an orthopoxvirus polypeptide construct encoding an C2L antigen is engineered to comprise a serine at amino acid residue corresponding to position 115 of SEQ ID NO: 279. In some embodiments, a full-length C2L antigen comprises or consists of 350-400 amino acid residues or 365-385 amino acid residues, optionally 375 amino acid residues.

[0361] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length C2L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 279. In some embodiments, an orthopoxvirus polypeptide construct includes a C2L variant incorporating one or more amino acid substitutions compared to a wild-type C2L sequence. In some embodiments, an orthopoxvirus polypeptide construct includes a C2L variant incorporating one or more cysteine substitutions compared to a wild-type C2L sequence. In some embodiments, an orthopoxvirus polypeptide construct includes a C2L variant incorporating a cysteine substitutions at position 115 compared to a wild-type C2L sequence. In some embodiments, an orthopoxvirus polypeptide construct includes a C2L variant incorporating a C115S substitution. In some embodiments, an orthopoxvirus polypeptide construct includes a C2L variant comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 350. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of a C2L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 356. D14L

[0362] Mpox D14L (also referred to as virus complement-control protein [VCP], and “D14” herein) is a type I integral membrane protein that contains a native secretory signal. D14L sequences include, e.g., UniProt accession numbers Q98VL5 and Q77HN7, the contents of which are incorporated herein by reference in their entireties. MPXV D14L antigen has a cysteine residue at position 204 that may form disulfide bonds with other viral proteins. MPXV D14L antigen is an ortholog of VACV C3L antigen. Page 54 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0363] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more D14L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length D14L antigen. In some embodiments, an D14L antigenic fragment does not include the N-terminal transmembrane region of full-length D14L. In some embodiments, an D14L antigenic fragment comprises or consists of an extracellular domain of D14L. Thus, in some embodiments, an D14L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 20-216 of a full-length D14L antigen (e.g., having the amino acid sequence of SEQ ID NO: 283). In some embodiments, an orthopoxvirus polypeptide construct encoding an D14L antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface) and / or a heterologous transmembrane region (e.g., to assist with insertion of the polypeptide into the cell membrane for presentation of an antigenic fragment of the polypeptide). In some embodiments, an orthopoxvirus polypeptide construct encoding an D14L antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct encoding an D14L antigen is engineered to comprise a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an D14L antigen is engineered to comprise a HSV gD secretory signal and a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding a D14L antigen is engineered to remove surface- exposed cysteine residues (e.g., at amino acid residues corresponding to position 204 of SEQ ID NO: 283). In some embodiments, an orthopoxvirus polypeptide construct encoding an D14L antigen is engineered to comprise a serine at amino acid residue corresponding to position 204 of SEQ ID NO: 283. In some embodiments, a full- length D14L antigen comprises or consists of 200-230 amino acid residues or 210-225 amino acid residues, optionally 216 amino acid residues.

[0364] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length D14L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 283. In some embodiments, an orthopoxvirus polypeptide construct includes a D14L variant incorporating one or more amino acid substitutions compared to a wild-type D14L sequence. In some embodiments, an orthopoxvirus polypeptide construct includes a D14L variant incorporating one or more cysteine substitution compared to a wild-type D14L sequence. In some embodiments, an orthopoxvirus polypeptide construct includes a D14L variant incorporating a cysteine substitution at position 204 compared to a wild-type D14L sequence. In some embodiments, an orthopoxvirus polypeptide construct includes a D14L variant incorporating a C204S substitution. In some embodiments, an orthopoxvirus polypeptide construct includes a D14L variant comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 351. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of a D14L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 357. A17L

[0365] A17L (also referred to as “A17” herein) is a type I integral membrane protein embedded in the MV envelope that forms a heterodimeric complex with G10. Without wishing to be bound by any particular Page 55 of 258 12621669v1Attorney Docket No.: 2013237-1080 theory, the interface between G10 and A17 is sufficiently large to suggest that co-expression is necessary for these proteins to adopt the correct conformation. As an MV type I integral membrane protein, the native protein sequence of A17 lacks a secretory signal. Mpox A17L sequences include, e.g., UniProt accession numbers V9NMP4, Q8V4W1, V9NT62, and Q5IXP7, the contents of which are incorporated herein by reference in their entireties. MPXV A17L antigen is an ortholog of VACV A16L antigen.

[0366] In some embodiments, an orthopoxvirus polypeptide construct is a full-length A17L antigen. In some embodiments, an A17L antigenic fragment does not include a C-terminal transmembrane region of full- length A17L. In some embodiments, an A17L antigenic fragment comprises or consists of an extracellular domain of A17L. In some embodiments, an A17L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 1-295 of a full-length A17L antigen (e.g., having the amino acid sequence of SEQ ID NO: 358). In some embodiments, an orthopoxvirus polypeptide construct encoding an A17L antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface). In some embodiments, an orthopoxvirus polypeptide construct encoding an A17L antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, a full-length A17L antigen comprises or consists of 350-400 amino acid residues or 370-385 amino acid residues, optionally 377 amino acid residues.

[0367] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more A17L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct includes a full-length A17L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 358. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an A17L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 360. G10R

[0368] G10R (also referred to as “G10” herein) is a type I integral membrane protein embedded in the MV envelope that forms a heterodimeric complex with A17. As an MV type I integral membrane protein, the native protein sequence of G10 lacks a secretory signal. Mpox G10R sequences include, e.g., UniProt accession numbers Q8V503, V9NPW6, and Q3I7N8, the contents of which are incorporated herein by reference in their entireties. G10R forms a heterodimer with a large interface. MPXV G10R antigen is an ortholog of VACV G9R antigen.

[0369] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more G10R antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length G10R antigen. In some embodiments, a G10R antigenic fragment does not include a C-terminal transmembrane region of full-length G10R. In some embodiments, an G10R antigenic fragment comprises or consists of an extracellular domain of G10R. Thus, in some embodiments, an G10R antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 1-271 of a full-length G10R antigen (e.g., having the amino acid sequence of SEQ ID NO: 359). In some embodiments, an orthopoxvirus polypeptide construct encoding an G10R antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface). In some embodiments, an orthopoxvirus polypeptide construct encoding an G10R antigen is engineered to comprise a HSV gD secretory Page 56 of 258 12621669v1Attorney Docket No.: 2013237-1080 signal. In some embodiments, a full-length G10R antigen comprises or consists of 320-360 amino acid residues or 330-350 amino acid residues, optionally 340 amino acid residues.

[0370] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length G10R antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 359. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an G10R antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 361. M5R

[0371] In the native viral MV membrane, M5R (also referred to as “M5” herein) is a type II transmembrane proteins with an extracellular domain positioned at its C-terminus. The M5 extracellular domain forms a heterodimer with G2 on the virion surface. Mpox M5R sequences include, e.g., UniProt accession numbers Q8V4Z8 and Q5IXU1, the contents of which are incorporated herein by reference in their entireties. MPXV M5R antigen is an ortholog of VACV L5R antigen.

[0372] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more M5R antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length M5R antigen. In some embodiments, an M5R antigenic fragment does not include the N-terminal transmembrane region of full-length M5R. In some embodiments, an M5R antigenic fragment comprises or consists of an extracellular domain of M5R. Thus, in some embodiments, an M5R antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 50-128 of a full-length M5R antigen (e.g., having the amino acid sequence of SEQ ID NO: 327). In some embodiments, an orthopoxvirus polypeptide construct encoding an M5R antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface) and / or a heterologous transmembrane region (e.g., to assist with insertion of the polypeptide into the cell membrane for presentation of an antigenic fragment of the polypeptide). In some embodiments, an orthopoxvirus polypeptide construct encoding an M5R antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct encoding an M5R antigen is engineered to comprise a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an M5R antigen is engineered to comprise a HSV gD secretory signal and a HSV gD transmembrane region. In some embodiments, a full-length M5R antigen comprises or consists of 110-140 amino acid residues or 120-135 amino acid residues, optionally 128 amino acid residues.

[0373] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length M5R antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 327. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an M5R antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 352. Page 57 of 258 12621669v1Attorney Docket No.: 2013237-1080 G2L

[0374] In the native viral MV membrane, G2L (also referred to as “G2” herein) is a type II transmembrane proteins with an extracellular domain positioned at its C-terminus. The G2 extracellular domain forms a heterodimer with M5 on the virion surface. Mpox G2L sequences include, e.g., UniProt accession numbers Q8V509 and A0A0F6N9M0, the contents of which are incorporated herein by reference in their entireties. MPXV G2L antigen is an ortholog of VACV G3L antigen.

[0375] In some embodiments, an orthopoxvirus polypeptide construct as described herein includes one or more G2L antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct is a full-length G2L antigen. In some embodiments, an G2L antigenic fragment does not include the N-terminal transmembrane region of full-length G2L. In some embodiments, an G2L antigenic fragment comprises or consists of an extracellular domain of G2L. Thus, in some embodiments, an G2L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 20-111 of a full-length G2L antigen (e.g., having the amino acid sequence of SEQ ID NO: 331). In some embodiments, an orthopoxvirus polypeptide construct encoding an G2L antigen is engineered to comprise a heterologous secretory signal (e.g., to assist with trafficking the antigen to the cell surface) and / or a heterologous transmembrane region (e.g., to assist with insertion of the polypeptide into the cell membrane for presentation of an antigenic fragment of the polypeptide). In some embodiments, an orthopoxvirus polypeptide construct encoding an G2L antigen is engineered to comprise a HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct encoding an G2L antigen is engineered to comprise a HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct encoding an G2L antigen is engineered to comprise a HSV gD secretory signal and a HSV gD transmembrane region. In some embodiments, a full-length G2L antigen comprises or consists of 100-125 amino acid residues or 105-120 amino acid residues, optionally 111 amino acid residues.

[0376] In some embodiments, an orthopoxvirus polypeptide construct includes a full-length G2L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 331. In some embodiments, an orthopoxvirus polypeptide construct includes an antigenic fragment of an G2L antigen, or variant thereof, comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 346. Table 1 and Table 2 list exemplary MPXV antigen amino acid and nucleotide sequences, respectively. In some embodiments, an orthopoxvirus polypeptide construct as described herein comprises or consists of any one of the amino acid sequences provided in Table 1. In some embodiments, an orthopoxvirus polypeptide construct as described herein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 97% identity to any one of the amino acid sequences provided in Table 1. In some embodiments, an orthopoxvirus polypeptide construct as described herein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 97% identity to any one of the amino acid sequences provided in Table 10, Table 11, or Table 12. In some embodiments, an orthopoxvirus construct as described herein comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, or at least 97% identity to any one of the nucleic acid sequences provided in Table 2 or Table 12. Page 58 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0377] In some embodiments, an antigenic fragment described herein comprises or consists of 50-500 amino acid residues. In some embodiments, an antigenic fragment described herein comprises or consists of 30- 40, 30-50, 40-60, 50-70, 60-80, 50-100, 50-150, 75-125, 100-150, 125-175, 150-200, 175-225, 200-250, 225- 275, or 250-500 amino acid residues. Table 1: Exemplary MPXV Antigen Amino Acid SequencesPage 59 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 60 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 61 of 258 12621669v1Attorney Docket No.: 2013237-1080 Table 2: Exemplary MPXV Antigen Nucleotide SequencesPage 62 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 63 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 64 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 65 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 66 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 67 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 68 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 69 of 258 12621669v1Attorney Docket No.: 2013237-1080Page 70 of 258 12621669v1Attorney Docket No.: 2013237-1080 the mpox viral envelope. H3L polypeptide sequences include, e.g., UniProt accession numbers, Q8V4Z2, Q3I8S1, Q5IXT2, A0A0F6N9X0, each of which is incorporated herein by reference in its entirety. Mpox H3L was found to bear high sequence similarity to vaccinia H3L.

[0386] A29L (also referred to as 14 kDa protein, 14K membrane protein, IMV surface protein fusion protein, MPXV-COP-132, MPXV-SL-132, and “A29” herein) is a ~ 14 kD polypeptide. A29L localizes to the viral envelope and is involved in fusion of the viral membrane with the host plasma membrane. Mpox A29L sequences include, e.g., UniProt accession number Q77HM6, Q9YN60, Q3I824, each of which is incorporated herein by reference in its entirety.

[0387] Mpox A29L is homologous to vaccinia A27L. Vaccinia A27L (see, e.g., accession number AAN78218.2, incorporated herein by reference in its entirety) is implicated in viral attachment, virus-host cell fusion, cell-cell fusion, plaque size and the formation of enveloped virions. Secretory Signals

[0388] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a secretory signal, e.g., that is functional in mammalian cells. In some embodiments, a utilized secretory signal is a heterologous secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal). In some embodiments, an HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, an HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal according to SEQ ID NO: 160 (MGGAAARLGAVILFVVIVGLHGVRG). In some embodiments, an HSV gD secretory signal is encoded by the nucleic acid sequence of atggggggggctgccgccaggttgggggccgtgattttgtttgtcgtcatagtgggcctccatggggtccgcggc (SEQ ID NO: 161).

[0389] In some embodiments, a secretory signal is characterized by a length of about 15 to 30 amino acids. In some embodiments, a secretory signal is characterized by a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.

[0390] In many embodiments, a secretory signal is positioned at the N-terminus of an orthopoxvirus polypeptide construct described herein. In some embodiments, a secretory signal preferably allows transport of a orthopoxvirus polypeptide construct with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER) or endosomal-lysosomal compartment.

[0391] In some embodiments, a secretory signal is one listed in Table 3 or a secretory signal having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a signal sequence is selected from those included in the Table 3 below and / or those encoded by the sequences in Table 4 below. Table 3: Exemplary secretory signalsPage 71 of 258 12621669v1Attorney Docket No.: 2013237-1080Table 4: Exemplary polynucleotide sequences encoding secretory signalsTransmembrane Regions

[0392] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a transmembrane region (also referred to herein as a “transmembrane domain”). In some embodiments, a utilized transmembrane region is at the N-terminus, C-terminus or internal to an orthopoxvirus antigen construct described herein. In some embodiments, a utilized transmembrane region is at the C-terminus of an orthopoxvirus antigen construct described herein. In some embodiments, a utilized transmembrane region is a heterologous transmembrane region. In some embodiments, a utilized transmembrane region is a non-human transmembrane region. In some embodiments, a utilized transmembrane region is a viral transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of an HSV transmembrane region, e.g., an HSV-1 or HSV-2 transmembrane region. In some embodiments, an HSV transmembrane region comprises or consists of an HSV gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR (SEQ ID NO: 254).

[0393] In some embodiments, a heterologous transmembrane region comprises or consists of a human transmembrane region. In some embodiments, a human transmembrane region comprises or consists of a human decay accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region. In some embodiments, an hDAF-GPI anchor region comprises or consists of an amino acid sequence of PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 222). In some embodiments, an HSV transmembrane region comprises or consists of an HSV gB transmembrane region, e.g., comprising or consisting of an amino acid sequence of MSNPFGALAVGLLVLAGLAAAFFAFRYVMRL (SEQ ID NO: 223) or MSNPFGALAVGLLVLAGLVAAFFAFRYVLQL (SEQ ID NO: 224).

[0394] In some embodiments, a heterologous transmembrane region comprises or consists of a vesicular stomatitis virus G (VSV-G) transmembrane region. In some embodiments, a VSV-G transmembrane region comprises or consists of an amino acid sequence of IASFFFIIGLIIGLFLVLRVGIYLCIKLKHTKKRQIYTDIEMN (SEQ ID NO: 225).

[0395] In some embodiments, an orthopoxvirus polypeptide construct described herein utilizes a transmembrane region that is internal to the orthopoxvirus polypeptide construct. For example, in some Page 72 of 258 12621669v1Attorney Docket No.: 2013237-1080 embodiments, an orthopoxvirus polypeptide construct described herein utilizes an endogenous transmembrane region.

[0396] In some embodiments, an orthopoxvirus polypeptide construct described herein does not comprise a transmembrane region. Linkers

[0397] In some embodiments, an orthopoxvirus polypeptide construct described herein includes one or more linkers. In some embodiments, a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments, a linker is or comprises no more than about 30, 25, 20, 15, 10 or fewer amino acids. A linker can include any amino acid sequence and is not limited to any particular amino acids. In some embodiments, a linker comprises one or more glycine (G) amino acids. In some embodiments, a linker comprises one or more serine (S) amino acids. In some embodiments, a linker comprises a glycine-serine linker. A “glycine- serine linker” as used herein refers to a linker that comprises predominantly (e.g., 80% or more) glycine and serine amino acids. In some embodiments, a linker includes amino acids selected based on a cleavage predictor to generate highly-cleavable linkers.

[0398] In some embodiments, a linker is or comprises GGSGGS (SEQ ID NO: 353). In some embodiments, a linker is or comprises GSG. In some embodiments, a linker is or comprises a 20GS linker (SEQ ID NO: 355). In some embodiments, a linker is one presented in Table 5. In some embodiments, a linker is or comprises a sequence as set forth in WO2017 / 081082, which is incorporated herein by reference in its entirety (see SEQ ID NOs: 1509-1565, or a fragment or variant thereof).

[0399] In some embodiments, a linker comprises a self-cleaving peptide. In some embodiments, a linker comprises a viral 2A peptide. In some embodiments, a viral 2A peptide allows the expression of multiple polypeptides (e.g., multiple orthopoxvirus antigens, variants thereof, or fragments thereof) from a single open reading frame. In some embodiments, a viral 2A peptide comprises or consists of a foot-and-mouth disease virus 2A peptide, an equine rhinitis A virus 2A peptide, a Thosea asigna virus 2A peptide, or a porcine teschovirus-12A peptide.

[0400] In some embodiments, a linker comprises or consists of a P2A linker. In some embodiments, a P2A linker comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 363). In some embodiments, a P2A linker is encoded by a polyribonucleotide comprising a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to AGCTACTAACTTCAGC CTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTA (SEQ ID NO: 387).

[0401] In some embodiments, a linker comprises or consists of a F2A linker. In some embodiments, a F2A linker comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 227.

[0402] In some embodiments, a linker comprises or consists of a T2A linker. In some embodiments, a T2A linker comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 228. Page 73 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0403] In some embodiments, a linker comprises or consists of a E2A linker. In some embodiments, a E2A linker comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 229.

[0404] In some embodiments, an orthopoxvirus polypeptide construct described herein comprises a linker between a C-terminal region or portion thereof and a transmembrane region. In some embodiments, an orthopoxvirus construct described herein comprises a linker between two orthopoxvirus antigens, variants thereof, or antigenic fragments thereof. Table 5: Exemplary linkersEmbodiments of orthopoxvirus polypeptide constructs

[0405] In some embodiments, an orthopoxvirus polypeptide construct described herein includes one or more MPXV polypeptide antigens, variants thereof, or fragments thereof as described above. Exemplary combinations of regions are described below.

[0406] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length A21 antigen that includes an N-terminal TM domain. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A21 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 305.

[0407] In some embodiments, an orthopoxvirus polypeptide construct comprises an A21 antigenic fragment, an N-terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A21 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 307. Page 74 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0408] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length A36 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A36 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 273.

[0409] In some embodiments, an orthopoxvirus polypeptide construct comprises an A36 antigenic fragment, an N-terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A36 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 459.

[0410] In some embodiments, an orthopoxvirus polypeptide construct comprises an extracellular fragment of A36 (e.g., that can be secreted). In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A36 Construct 3. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 461.

[0411] In some embodiments, an orthopoxvirus polypeptide construct comprises an A28 antigenic fragment (e.g., an N-terminal globular domain), an N-terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A28 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 291.

[0412] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length A28 antigen having amino acid substitutions corresponding to C450S and C451S, an N-terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A28 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 293.

[0413] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length L5 antigen and an N-terminal HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of L5 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 317.

[0414] In some embodiments, an orthopoxvirus polypeptide construct comprises a L5 antigenic fragment lacking the endogenous L5 TM domain, an N-terminal HSV gD secretory signal, and an HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of L5 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct Page 75 of 258 12621669v1Attorney Docket No.: 2013237-1080 comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 319.

[0415] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length H2 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of H2 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 313.

[0416] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length H2 antigen, an N-terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of H2 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 315.

[0417] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length A30 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A30 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 309.

[0418] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length A30 antigen, an N-terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A30 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 311.

[0419] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length C15 antigen and an N-terminal HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of C15 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 321.

[0420] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length A14 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A14 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 323.

[0421] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length A14 antigen, an N-terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A14 Page 76 of 258 12621669v1Attorney Docket No.: 2013237-1080 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 325.

[0422] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length B2 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of B2 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 275.

[0423] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length B2 antigen having an amino acid substitution corresponding to C160S. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of B2 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 277.

[0424] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length C2 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of C2 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 279.

[0425] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length C2 antigen having an amino acid substitution corresponding to C115S. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of C2 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 281.

[0426] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length D14 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of D14 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 283.

[0427] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length D14 antigen having an amino acid substitution corresponding to C204S. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of D14 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 285.

[0428] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length D14 antigen having an amino acid substitution corresponding to C204S, a C2 antigenic fragment having an amino acid substitution corresponding to C115S, and a C-terminal HSV gD transmembrane region. In some Page 77 of 258 12621669v1Attorney Docket No.: 2013237-1080 embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of D14_C2 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 287.

[0429] In some embodiments, an orthopoxvirus polypeptide construct comprises a D14 antigenic fragment having an amino acid substitution corresponding to C204S, a full-length C2 antigen having an amino acid substitution corresponding to C115S, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of C2_D14 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 289.

[0430] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length A17 antigen and an N-terminal HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A17 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 295.

[0431] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length G10 antigen and an HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of G10 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 299.

[0432] In some embodiments, an orthopoxvirus polypeptide construct comprises an A17 antigenic fragment (e.g., a C-terminally truncated A17 antigenic fragment) and an N-terminal HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of A17 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 297.

[0433] In some embodiments, an orthopoxvirus polypeptide construct comprises a G10 antigenic fragment (e.g., a C-terminally truncated G10 antigenic fragment) and an N-terminal HSV gD secretory signal. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of G10 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 301.

[0434] In some embodiments, an orthopoxvirus polypeptide construct comprises an N-terminal HSV gD secretory signal, a full-length A17 antigen, a cleavable linker (e.g., P2A linker), an internal HSV gD secretory signal, and a full-length G10 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises Page 78 of 258 12621669v1Attorney Docket No.: 2013237-1080 or consists of the amino acid sequence of A17_G10 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 303.

[0435] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length M5 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of M5 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 327.

[0436] In some embodiments, an orthopoxvirus polypeptide construct comprises a full-length G2 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of G2 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 331.

[0437] In some embodiments, an orthopoxvirus polypeptide construct comprises a G2 antigenic fragment (e.g., a G2 extracellular domain), an N-terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of G2 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 333.

[0438] In some embodiments, an orthopoxvirus polypeptide construct comprises a M5 antigenic fragment (e.g., a M5 extracellular domain), an N-terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of M5 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 329.

[0439] In some embodiments, an orthopoxvirus polypeptide construct comprises a G2 antigenic fragment (e.g., a G2 extracellular domain), a M5 antigenic fragment (e.g., a M5 extracellular domain), an N- terminal HSV gD secretory signal, and a C-terminal HSV gD transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of M5_G2 Construct 1. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 335.

[0440] In some embodiments, an orthopoxvirus polypeptide construct comprises a G2 antigenic fragment (e.g., a G2 extracellular domain) and a full-length M5 antigen. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of M5_G2 Construct 2. In some embodiments, an orthopoxvirus polypeptide construct comprises or consists of the amino acid sequence of an Page 79 of 258 12621669v1Attorney Docket No.: 2013237-1080 amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 337.

[0441] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV A21L antigen sequence according to SEQ ID NO: 305.

[0442] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; (ii) an MPXV A21L antigen, variant thereof, or antigenic fragment thereof; and (iii) a linker; and (iv) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; (ii) an MPXV A21L antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 342; (iii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iv) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 307.

[0443] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV A36R antigen sequence according to SEQ ID NO: 273.

[0444] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; (ii) an MPXV A28L antigen, variant thereof, or antigenic fragment thereof; (iii) a linker; and (iv) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; (ii) an MPXV A28L antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 343; (iii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iv) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 291.

[0445] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; (ii) an MPXV A28L variant Page 80 of 258 12621669v1Attorney Docket No.: 2013237-1080 including C450s and C451S substitutions and comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 344; (iii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iv) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 293.

[0446] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; and (ii) an MPXV L5L antigen, variant thereof, or antigenic fragment thereof. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; and (ii) an MPXV L5L antigen comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 348. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 317.

[0447] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; (ii) an MPXV L5L antigen, variant thereof, or antigenic fragment thereof; (iii) a linker; and (iv) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; (ii) an MPXV L5L antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 349; (iii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iv) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 319.

[0448] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV H2R antigen sequence according to SEQ ID NO: 313.

[0449] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; (ii) an MPXV H2 antigen, variant thereof, or antigenic fragment thereof; (iii) a Page 81 of 258 12621669v1Attorney Docket No.: 2013237-1080 linker; and (iv) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; (ii) an MPXV H2R antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 347; (iii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iv) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 315.

[0450] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV A30L antigen sequence according to SEQ ID NO: 309.

[0451] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; (ii) an MPXV A30L antigen, variant thereof, or antigenic fragment thereof; (iii) a linker; and (iv) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; (ii) an MPXV A30L antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 339; (iii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iv) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 311.

[0452] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; and (ii) an MPXV C15L antigen, variant thereof, or antigenic fragment thereof. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; and (ii) an MPXV C15L antigen comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 345. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least Page 82 of 258 12621669v1Attorney Docket No.: 2013237-1080 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 321.

[0453] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV A14L antigen sequence according to SEQ ID NO: 323.

[0454] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; (ii) an MPXV A14L antigen, variant thereof, or antigenic fragment thereof; (iii) a linker; and (iv) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; (ii) an MPXV A14L antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 340; (iii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iv) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 325.

[0455] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV B2R antigen sequence according to SEQ ID NO: 275.

[0456] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV B2R antigen variant incorporating a C160S substitution and having an amino acid sequence according to SEQ ID NO: 277.

[0457] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV C2L antigen sequence according to SEQ ID NO: 279.

[0458] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an MPXV C2L antigen, variant thereof, or antigenic fragment thereof; (ii) a linker; and (iii) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an MPXV C2L antigen comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 350; (ii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iii) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 281.

[0459] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV D14L antigen sequence according to SEQ ID NO: 283. Page 83 of 258 12621669v1Attorney Docket No.: 2013237-1080

[0460] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an MPXV D14L antigen, variant thereof, or antigenic fragment thereof; (ii) a linker; and (iii) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an MPXV D14L variant incorporating a C204S substitution and comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 351; (ii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iii) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 285.

[0461] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; and (ii) an MPXV A17L antigen, variant thereof, or antigenic fragment thereof. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; and (ii) an MPXV A17L antigen comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 358. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 295.

[0462] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; and (ii) an MPXV A17L antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 360. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 297.

[0463] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; and (ii) an MPXV G10R antigen, variant thereof, or antigenic fragment thereof. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; and (ii) an MPXV G10R antigen comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, Page 84 of 258 12621669v1Attorney Docket No.: 2013237-1080 at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 359. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 299.

[0464] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; and (ii) an MPXV G10R antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 361. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 301.

[0465] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV M5R antigen sequence according to SEQ ID NO: 327.

[0466] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; (ii) an MPXV M5R antigen, variant thereof, or antigenic fragment thereof; (iii) a linker; and (iv) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; (ii) an MPXV M5R antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 352; (iii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iv) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 329.

[0467] In some embodiments, an orthopoxvirus polypeptide construct described herein includes a full- length MPXV G2L antigen sequence according to SEQ ID NO: 331.

[0468] In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an N-terminal secretory signal; (ii) an MPXV G2L antigen, variant thereof, or antigenic fragment thereof; (iii) a linker; and (iv) a transmembrane region. In some embodiments, an orthopoxvirus polypeptide construct described herein includes: (i) an HSV-1 gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 160; (ii) an MPXV G2L antigenic fragment comprising an amino acid sequence having at least 85%, at least 90%, at Page 85 of 258 12621669v1Attorney Docket No.: 2013237-1080 least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 346; (iii) a linker having an amino acid sequence according to SEQ ID NO: 353; and (iv) an HSV-1 gD transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 254. In some embodiments, an orthopoxvirus polypeptide construct described herein includes a polypeptide having a sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 333. Embodiments of fusion proteins comprising two or more orthopoxvirus antigens

[0469] In some embodiments, an orthopoxvirus polypeptide construct described herein includes two or more MPXV polypeptide antigens, variants thereof, or fragments thereof, operably linked to form a contiguous fusion polypeptide. In some embodiments, two or more MPXV antigens, variants thereof, or fragments thereof are operably linked via linker sequences to form a contiguous fusion polypeptide. In some embodiments, a fusion polypeptide further includes one or more secretory signal and / or a transmembrane region. In some embodiments, a transmembrane region is heterologous and at the C-terminus of the fusion polypeptide. In some embodiments, a transmembrane region is endogenous and internal to the fusion polypeptide. Exemplary orthopoxvirus polypeptide construct fusions are described below. Fusion proteins comprising a D14 antigen

[0470] An orthopoxvirus polypeptide construct can, in some embodiments, comprise a MPXV D14 antigen, a MPXV B2 antigen, and / or a MPXV C2 antigen. In some embodiments, an orthopoxvirus polypeptide construct is a fusion protein comprising a B2 antigen and a D14 antigen. In some embodiments, an orthopoxvirus polypeptide construct is a fusion protein comprising a C2 antigen and a D14 antigen.

[0471] A fusion protein comprising a B2 antigen and a D14 antigen can comprise (i) one or more B2R antigens, variants thereof, or antigenic fragments thereof; and (ii) one or more D14 antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct comprises (i) one or more B2R antigens, variants thereof, or antigenic fragments thereof; and (ii) one or more D14 antigens, variants thereof, or antigenic fragments thereof. In some embodiments, a B2 antigen is a full-length B2R antigen. In some embodiments, a B2R antigen comprises or consists of an amino acid sequence according to SEQ ID NO: 275. In some embodiments, a B2 antigen is engineered to remove surface-exposed cysteine residues (e.g., at amino acid residues corresponding to position 160 of SEQ ID NO: 275). In some embodiments, a B2 antigen is engineered to comprise a serine at an amino acid residue corresponding to position 160 of SEQ ID NO: 275. In some embodiments, a B2R antigen comprises or consists of an amino acid sequence according to SEQ ID NO: 277. In some embodiments, a D14 antigen is a full-length D14L antigen or a D14L antigenic fragment that comprises or consists of an extracellular domain of D14L. In some embodiments, a B2R antigen comprises or consists of an amino acid sequence according to SEQ ID NO: 283. In some embodiments, a D14L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 20-216 of a full-length D14L antigen (e.g., having the amino acid sequence of SEQ ID NO: 283). In some embodiments, a D14L antigen is engineered to remove surface-exposed cysteine residues (e.g., at amino acid residues corresponding to position 204 of SEQ ID NO: 283). In some embodiments, a D14L antigen is engineered to comprise a serine at Page 86 of 258 12621669v1Attorney Docket No.: 2013237-1080 amino acid residue corresponding to position 204 of SEQ ID NO: 283. In some embodiments, a D14L antigen comprises or consists of an amino acid sequence according to SEQ ID NO: 351. In some embodiments, a D14L antigenic fragment comprises or consists of an amino acid sequence corresponding to residues 20-216 of a full- length D14L antigen and is engineered to comprise a serine at amino acid residue corresponding to position 204 of SEQ ID NO: 283. In some embodiments, a D14L antigen comprises or consists of an amino acid sequence according to SEQ ID NO: 357.

[0472] In some embodiments, a fusion protein comprising a B2 antigen and a D14 antigen comprises a full-length B2 antigen (e.g., having the amino acid sequence of SEQ ID NO: 275) and a full-length D14 antigen (e.g., having the amino acid sequence of SEQ ID NO: 283).

[0473] In some embodiments, a fusion protein comprising a B2 antigen and a D14 antigen comprises a B2 antigen that is engineered to remove surface-exposed cysteine residues (e.g., having the amino acid sequence of SEQ ID NO: 277) and a D14 antigen that is engineered to remove surface-exposed cysteine residues (e.g., having the amino acid sequence of SEQ ID NO: 351 or 357).

[0474] In some embodiments, a fusion protein comprising a B2 antigen and a D14 antigen is arranged such that the B2 antigen is positioned to be closer to the N-terminus than the D14 antigen (e.g., order of the protein: N-term—[B2 antigen]—[D14 antigen]—C-term). In some embodiments, a fusion protein comprising a B2 antigen and a D14 antigen is arranged such that the D14 antigen is positioned to be closer to the N-terminus than the B2 antigen (e.g., order of the protein: N-term—[D14 antigen]—[B2 antigen]—C-term).

[0475] In some embodiments, a fusion protein comprising a B2 antigen and a D14 antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 275 or 277. In some embodiments, a fusion protein comprising a B2 antigen and a D14 antigen comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 283, 285, 351, or 357.

[0476] A fusion protein comprising a C2 antigen and a D14 antigen can comprise (i) one or more C2 antigens, variants thereof, or antigenic fragments thereof; and (ii) one or more D14 antigens, variants thereof, or antigenic fragments thereof. In some embodiments, an orthopoxvirus polypeptide construct comprises (i) one or more C2 antigens, variants thereof, or antigenic fragments thereof; and (ii) one or more D14 antigens, variants thereof, or antigenic fragments thereof. In some embodiments, a C2 antigen is a full-length C2 antigen. In some embodiments, a C2 antigenic...

Claims

Attorney Docket No.: 2013237-1080 CLAIMS 1. A polyribonucleotide encoding a polypeptide that comprises one or more mpox virus (MPXV) antigens, variants thereof, or antigenic fragments thereof, wherein the one or more MPXV antigens, variants thereof, or antigenic fragments thereof comprise: a) an A21L antigen, variant thereof, or antigenic fragment thereof; b) an A36R antigen, variant thereof, or antigenic fragment thereof; c) an A28L antigen, variant thereof, or antigenic fragment thereof; d) an L5L antigen, variant thereof, or antigenic fragment thereof; e) an H2R antigen, variant thereof, or antigenic fragment thereof; f) an A30L antigen, variant thereof, or antigenic fragment thereof; g) a C15L antigen, a variant thereof, or antigenic fragment; h) an A14L antigen, variant thereof, or antigenic fragment thereof; i) a B2R antigen, variant thereof, or antigenic fragment thereof; j) a C2L antigen, variant thereof, or antigenic fragment thereof; k) a D14L antigen, variant thereof, or antigenic fragment thereof; l) an A17L antigen, variant thereof, or antigenic fragment thereof; m) a G10R antigen, variant thereof, or antigenic fragment thereof; n) an M5R antigen, variant thereof, or antigenic fragment thereof; o) a G2L antigen, variant thereof, or antigenic fragment thereof; or p) any combination thereof.

2. The polyribonucleotide of claim 1, wherein: a) the A21L antigen, variant thereof, or antigenic fragment thereof is a full-length A21L antigen, an extracellular domain of A21L, or an A21L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 23-115 of a reference A21L antigen having the amino acid sequence of SEQ ID NO: 305; b) the A36R antigen, variant thereof, or antigenic fragment thereof is a full-length A36R antigen, an extracellular domain of A36, or an A36 antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 39-168 of a reference A36 antigen having the amino acid sequence of SEQ ID NO: 273; c) the A28L antigen, variant thereof, or antigenic fragment thereof is a full-length A28L antigen, an extracellular domain of A28L, an A28L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-366 of a reference A28L antigen having the amino acid sequence of SEQ ID NO: 196, or an A28L antigen that comprises serines at amino acid residues corresponding to positions 450 and 451 of SEQ ID NO: 196; d) the L5L antigen, variant thereof, or antigenic fragment is a full-length L5L antigen, an extracellular domain of L5L, or an L5L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-109 of a reference L5L antigen having the amino acid sequence of SEQ ID NO: 348; e) the H2R antigen, variant thereof, or antigenic fragment thereof is a full-length H2R antigen, an extracellular domain of H2R, or an H2R antigenic fragment that comprises or consists of an amino Page 247 of 258 12621669v1Attorney Docket No.: 2013237-1080 acid sequence corresponding to residues 49-189 of a reference H2R antigen having the amino acid sequence of SEQ ID NO: 313; f) the A30L antigen, variant thereof, or antigenic fragment thereof is a full-length A30L antigen, an extracellular domain of A30L, or an A30L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 22-146 of a reference A30L antigen having the amino acid sequence of SEQ ID NO: 309; g) the C15L antigen, a variant thereof, or antigenic fragment thereof is a full-length C15L antigen; h) the A14L antigen, variant thereof, or antigenic fragment thereof is a full-length A14L antigen, an extracellular domain of A14L, or an A14L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 25-70 of a reference A14L antigen having the amino acid sequence of SEQ ID NO: 323; i) the B2R antigen, variant thereof, or antigenic fragment thereof is a full-length B2R antigen and / or a B2R antigen that comprises a serine at an amino acid residue corresponding to position 160 of SEQ ID NO: 275; j) the C2L antigen, variant thereof, or antigenic fragment thereof is a full-length C2L antigen, an extracellular domain of C2L, an C2L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 15-375 of a reference C2L antigen having the amino acid sequence of SEQ ID NO: 279, or an C2L antigen that comprises a serine at amino acid residue corresponding to position 115 of SEQ ID NO: 279; k) the D14L antigen, variant thereof, or antigenic fragment thereof is a full-length D14L antigen, an extracellular domain of D14L, a D14L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 20-216 of a reference D14L antigen having the amino acid sequence of SEQ ID NO: 283, or a D14L antigen that comprises a serine at an amino acid residue corresponding to position 204 of SEQ ID NO: 283; l) the A17L antigen, variant thereof, or antigenic fragment thereof is a full-length A17L antigen, an extracellular domain of A17L, or an A17L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-295 of a reference A17L antigen having the amino acid sequence of SEQ ID NO: 358; m) the G10R antigen, variant thereof, or antigenic fragment thereof is a full-length G10R antigen, an extracellular domain of G10R, or a G10R antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-271 of a reference G10R antigen having the amino acid sequence of SEQ ID NO: 359; n) the M5R antigen, variant thereof, or antigenic fragment thereof is a full-length M5R antigen, an extracellular domain of M5R, or an M5R antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 50-128 of a reference M5R antigen having the amino acid sequence of SEQ ID NO: 327; o) the G2L antigen, variant thereof, or antigenic fragment thereof is a full-length G2L antigen, an extracellular domain of G2L, or a G2L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 20-111 of a reference G2L antigen having the amino acid sequence of SEQ ID NO: 331; or p) any combination thereof. Page 248 of 258 12621669v1Attorney Docket No.: 2013237-1080 3. The polyribonucleotide of claim 1 or 2, wherein: a) the A21L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 305, 307, or 342; b) the A36R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 273, 458, 459, or 461; c) the A28L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 196, 343, 344, 364, 291, or 293; d) the L5L antigen, variant thereof, or antigenic fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 348, 349, 317, or 319; e) the H2R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 313, 347, or 315; f) the A30L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 309, 339, or 311; g) the C15L antigen, a variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 345 or 321; h) the A14L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 323, 340, or 325; i) the B2R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 275 or 277; j) the C2L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 279, 281, 350, or 356; k) the D14L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence Page 249 of 258 12621669v1Attorney Docket No.: 2013237-1080 having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 283, 285, 351, or 357; l) the A17L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 358, 360, 295, or 297; m) the G10R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 359, 361, 299, or 301; n) the M5R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 327, 352, or 329; o) the G2L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 331, 346, or 333; or p) any combination thereof.

4. The polyribonucleotide of any one of claims 1 to 3, wherein the polyribonucleotide comprises: a) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 306, 369, 308, or 481; b) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to SEQ ID NO: 274, 226, 497, or 464; c) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 197, 370 , 371, 388, 292, 294, or 474; d) an nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 375, 376, 318, or 320; e) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 314, 374, 316, 484, or 485; f) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 310, 367, or 312; g) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, Page 250 of 258 12621669v1Attorney Docket No.: 2013237-1080 at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to SEQ ID NO: 372 or 322; h) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 324, 368, or 326; i) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 276, 278, 465, or 466; j) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 280, 282, 377, 380, 467, or 468; k) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 284, 286, 378, or 381; l) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 382, 384, 296, or 298; m) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 383, 385, 300, 302, 477, or 478; n) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 328, 379, 330, or 492; o) a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to any one of SEQ ID NOs: 332, 373, , or 334; or p) any combination thereof.

5. The polyribonucleotide of any one of claims 1 to 4, wherein the polypeptide comprises, from N-terminus to C-terminus: a) an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; b) an A28 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 343; c) an optional peptide linker, optionally wherein the peptide linker comprises an amino acid sequence having 100% identity to an amino acid sequence according to SEQ ID NO: 353; and d) an HSV-1 or HSV-2 transmembrane region comprising an amino acid sequence having at least 85%, Page 251 of 258 12621669v1Attorney Docket No.: 2013237-1080 at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO:

254.

6. The polyribonucleotide of any one of claims 1 to 4, wherein the polypeptide comprises, from N-terminus to C-terminus: a) an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; b) an A36 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 458; c) an optional peptide linker, optionally wherein the peptide linker comprises an amino acid sequence having 100% identity to an amino acid sequence according to SEQ ID NO: 353; and d) an HSV-1 or HSV-2 transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO:

254.

7. The polyribonucleotide of any one of claims 1 to 4, wherein the polypeptide comprises, from N-terminus to C-terminus: a) an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; and b) an A17 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 358 or SEQ ID NO:

360.

8. The polyribonucleotide of any one of claims 1, 2, 3, 4, or 7, wherein the polypeptide comprises, from N- terminus to C-terminus: a) an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; and b) a G10 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 359 or SEQ ID NO:

361. Page 252 of 258 12621669v1Attorney Docket No.: 2013237-1080 9. The polyribonucleotide of any one of claims 1 to 4 comprising a) an A17 antigen, variant thereof, or antigenic fragment thereof; and a G10 antigen, variant thereof, or antigenic fragment thereof; or b) a D14 antigen, variant thereof, or antigenic fragment thereof; and a C2 antigen, variant thereof, or antigenic fragment thereof; or c) a D14 antigen, variant thereof, or antigenic fragment thereof; and a B2 antigen, variant thereof, or antigenic fragment thereof; or d) a D14 antigen, variant thereof, or antigenic fragment thereof; a B2 antigen, variant thereof, or antigenic fragment thereof; and a C2 antigen, variant thereof, or antigenic fragment thereof.

10. A polyribonucleotide encoding a polypeptide that comprises (i) an A17 antigen, variant thereof, or antigenic fragment thereof; and (ii) a G10 antigen, variant thereof, or antigenic fragment thereof.

11. The polyribonucleotide of claim 10, wherein: i. the A17L antigen, variant thereof, or antigenic fragment thereof is a full-length A17L antigen, an extracellular domain of A17L, or an A17L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-295 of a reference A17L antigen having the amino acid sequence of SEQ ID NO: 358; and / or ii. the G10R antigen, variant thereof, or antigenic fragment thereof is a full-length G10R antigen, an extracellular domain of G10R, or a G10R antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-271 of a reference G10R antigen having the amino acid sequence of SEQ ID NO:

359.

12. The polyribonucleotide of claim 10 or 11, wherein: i. the A17L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 358 or SEQ ID NO: 360; and / or ii. the G10R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NOs: 359, or 361.

13. The polyribonucleotide of any one of claims 1 to 12 wherein the polypeptide comprises, from N-terminus to C-terminus: a) a first HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; b) an A17 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid Page 253 of 258 12621669v1Attorney Docket No.: 2013237-1080 sequence according to SEQ ID NO: 358 or SEQ ID NO: 360; c) an optional peptide linker, optionally wherein the peptide linker comprises a P2A sequence comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 363; d) a second HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 160; and / or e) a G10 antigen, variant thereof, or antigenic fragment thereof comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 359 or SEQ ID NO:

361.

14. A polyribonucleotide encoding a polypeptide that comprises i. a D14 antigen, variant thereof, or antigenic fragment thereof; and ii. a C2 antigen, variant thereof, or antigenic fragment thereof; and / or a B2 antigen, variant thereof, or antigenic fragment thereof.

15. The polyribonucleotide of claim 14, wherein the polypeptide comprises: a) a D14 antigen, variant thereof, or antigenic fragment thereof; and a C2 antigen, variant thereof, or antigenic fragment thereof; or b) a D14 antigen, variant thereof, or antigenic fragment thereof; and a B2 antigen, variant thereof, or antigenic fragment thereof; or c) a D14 antigen, variant thereof, or antigenic fragment thereof; a B2 antigen, variant thereof, or antigenic fragment thereof; and a C2 antigen, variant thereof, or antigenic fragment thereof.

16. The polyribonucleotide of claim 14 or 15, wherein: i. the D14L antigen, variant thereof, or antigenic fragment thereof is a full-length D14L antigen, an extracellular domain of D14L, a D14L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 20-216 of a reference D14L antigen having the amino acid sequence of SEQ ID NO: 283, or a D14L antigen that comprises a serine at an amino acid residue corresponding to position 204 of SEQ ID NO: 283; and / or ii. the C2L antigen, variant thereof, or antigenic fragment thereof is a full-length C2L antigen, an extracellular domain of C2L, an C2L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 15-375 of a reference C2L antigen having the amino acid sequence of SEQ ID NO: 279, or an C2L antigen that comprises a serine at amino acid residue corresponding to position 115 of SEQ ID NO: 279; and / or iii. the B2R antigen, variant thereof, or antigenic fragment thereof is a full-length B2R antigen and / or a B2R antigen that comprises a serine at an amino acid residue corresponding to position 160 of SEQ ID NO:

275. Page 254 of 258 12621669v1Attorney Docket No.: 2013237-1080 17. The polyribonucleotide of any one of claims 14 to 16, wherein: i. the D14L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 283, 285, 351, or 357; ii. the C2L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 279, 281, 350, or 356; and / or iii. the B2R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 275 or 277.

18. The polyribonucleotide of any one of claims 1 to 17, wherein the polypeptide comprises a secretory signal, optionally wherein the secretory signal is a heterologous secretory signal.

19. The polyribonucleotide of claim 18, wherein the secretory signal comprises or consists of an HSV gD secretory signal comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO:

160.

20. The polyribonucleotide of claim 18 or 19, wherein the polyribonucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to SEQ ID NO:

161.

21. The polyribonucleotide of any one of claims 1 to 20, wherein the polypeptide comprises a transmembrane region that is N-terminal, C-terminal, or internal to the one or more MPXV antigens, variants thereof, or antigenic fragments thereof, optionally wherein the transmembrane region is a heterologous transmembrane region.

22. The polyribonucleotide of claim 21, wherein the transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO:

254.

23. The polyribonucleotide of claim 21 or 22, wherein the polyribonucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleotide sequence according to SEQ ID NO:

255. Page 255 of 258 12621669v1Attorney Docket No.: 2013237-1080 24. The polyribonucleotide of any one of claims 18 to 23, wherein the one or more MPXV antigens, variants thereof, or antigenic fragments thereof is operably linked to the secretory signal and / or the transmembrane region via a peptide linker, optionally wherein the peptide linker is a GS linker.

25. A pharmaceutical composition comprising (i) a first polyribonucleotide encoding an A17 antigen, variant thereof, or antigenic fragment thereof; and (ii) a second polyribonucleotide encoding an G10 antigen, variant thereof, or antigenic fragment thereof.

26. The pharmaceutical composition of claim 25, wherein: i. the A17L antigen, variant thereof, or antigenic fragment thereof is a full-length A17L antigen, an extracellular domain of A17L, or an A17L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-295 of a reference A17L antigen having the amino acid sequence of SEQ ID NO: 358; and / or ii. the G10R antigen, variant thereof, or antigenic fragment thereof is a full-length G10R antigen, an extracellular domain of G10R, or a G10R antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 1-271 of a reference G10R antigen having the amino acid sequence of SEQ ID NO:

359.

27. The pharmaceutical composition of claim 25 or 26, wherein: i. the A17L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NO: 358 or SEQ ID NO: 360; and / or ii. the G10R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to SEQ ID NOs: 359 or 361.

28. A pharmaceutical composition comprising (i) a first polyribonucleotide encoding a D14 antigen, variant thereof, or antigenic fragment thereof; and (ii) a second polyribonucleotide encoding a C2 antigen, variant thereof, or antigenic fragment thereof.

29. A pharmaceutical composition comprising (i) a first polyribonucleotide encoding a D14 antigen, variant thereof, or antigenic fragment thereof; (ii) a second polyribonucleotide encoding a B2 antigen, variant thereof, or antigenic fragment thereof; and optionally (iii) a third polyribonucleotide encoding a C2 antigen, variant thereof, or antigenic fragment thereof.

30. The pharmaceutical composition of claim 28 or 29, wherein: i. the D14L antigen, variant thereof, or antigenic fragment thereof is a full-length D14L antigen, an extracellular domain of D14L, a D14L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 20-216 of a reference D14L antigen having the amino acid sequence of SEQ ID NO: 283, or a D14L antigen that comprises a serine at an amino acid residue corresponding to position 204 of SEQ ID NO: 283; and / or Page 256 of 258 12621669v1Attorney Docket No.: 2013237-1080 ii. the C2L antigen, variant thereof, or antigenic fragment thereof is a full-length C2L antigen, an extracellular domain of C2L, an C2L antigenic fragment that comprises or consists of an amino acid sequence corresponding to residues 15-375 of a reference C2L antigen having the amino acid sequence of SEQ ID NO: 279, or an C2L antigen that comprises a serine at amino acid residue corresponding to position 115 of SEQ ID NO: 279; and / or iii. the B2R antigen, variant thereof, or antigenic fragment thereof is a full-length B2R antigen and / or a B2R antigen that comprises a serine at an amino acid residue corresponding to position 160 of SEQ ID NO:

275.

31. The polyribonucleotide of any one of claims 28 to 30, wherein: i. the D14L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 283, 285, 351, or 357; ii. the C2L antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 279, 281, 350, or 356; and / or iii. the B2R antigen, variant thereof, or antigenic fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence according to any one of SEQ ID NOs: 275, or 277.

32. The pharmaceutical composition of any one of claims 25 to 31, wherein the first polyribonucleotide and / or the second polyribonucleotide further comprise a heterologous secretory signal, optionally wherein the heterologous secretory signal is a HSV gD secretory signal, optionally comprising an amino acid sequence having at least 85% identity to an amino acid sequence according to SEQ ID NO:

160.

33. A pharmaceutical composition comprising one or more polyribonucleotides of any one of claims 1 to 24 and a pharmaceutically acceptable excipient.

34. A method of treating or preventing an orthopoxvirus infection in a subject, the method comprising administering one or more polyribonucleotides of any one of claims 1 to 24 or a pharmaceutical composition of any one of claims 25 to 33 to the subject.

35. The method of claim 34, wherein the orthopoxvirus infection is a monkeypox infection, variola infection, borealpox infection, ectromelia infection, cowpox infection, volepox infection, modified vaccinia virus Ankara infection, or vaccinia infection.

36. The method of claim 34 or 35, wherein the one or more polyribonucleotides or the pharmaceutical composition is administered to the subject prior to the orthopoxvirus infection. Page 257 of 258 12621669v1

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