Mpox vaccine compositions and methods of using same

Abstract

Disclosed are vaccine compositions, in particular, polyvalent icosahedral compositions for presentation of an mpox antigen. The disclosed compositions may contain an S particle comprising a norovirus (NoV) S domain protein and an mpox antigen, which may be linked via a linker protein domain operatively connected to the norovirus S domain protein and an mpox antigen. Fusion proteins for producing the vaccine compositions, and methods of using the disclosed vaccine composition are also provided.

Description

MPOX VACCINE COMPOSITIONS AND METHODS OF USING SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international application which claims priority to and benefit of U.S. provisional application Serial No. 63 / 535,611, filed August 31, 2024, the contents of which are incorporated in their entirety for all purposes.REFERENCE TO SEQUENCE LISTING

[0002] A Sequence Listing submitted as an ST.26 XML file via EFS-WEB is hereby incorporated by reference. The name of the XML file for the Sequence Listing is CHMC2023- 0702_ST26.xml, the date of the creation of the XML file is August 21, 2024, and the size of the XML file is 15,205 bytes.BACKGROUND

[0003] Mpox (formerly known as monkeypox) is a zoonotic infectious viral disease caused by the mpox virus that causes symptoms similar to those observed in smallpox patients, although these infections are less clinically severe. Typical signs of mpox include skin rash and mucosal lesions accompanied by fever, headache, and, in severe cases, even death. Both mpox and its closely related counterpart, smallpox, are listed as diseases with epidemic or pandemic potential by the World Health Organization (WHO). Smallpox and mpox are also considered potential bioterrorism agents. Historically, mpox was found to be endemic in central and western Africa, where several species of mammals are believed to be natural reservoirs of the virus. However, a recent outbreak starting in May 2022 documented approximately 91,788 confirmed cases in 116 countries, resulting in 167 deaths. This mpox outbreak was caused by clade lib mpox viruses. Notably, 85,525 cases occurred in locations that had not historically reported mpox disease, including 30,347 cases in the United States and 26,227 in Europe. Most recently, a surge of mpox in the Democratic Republic of the Congo, resulted from clade I mpox viruses, has caused nearly 13,000 suspected cases and 581 suspected deaths, raising serious concerns about another global mpox outbreak that may result in more deaths than the one starting in 2022. Therefore, mpox has emerged as a global public health threat.

[0004] Like smallpox virus and vaccinia virus, the mpox virus belongs to the Orthopoxvirus genus in the Poxviridae family, a group of double-stranded DNA viruses. Like in other poxviruses, an mpox virion is barrel shaped, with dimensions of approximately 360 x 270 x 250 nm. Each virion contains a linear, double-stranded DNA genome encoding approximately 250 genes (see, e.g., Lansiaux E, et al., The virology of human monkeypox virus (hMPXV): A brief overview. Virus re-search. 2022;322: 198932. Epub 2022 / 09 / 28. doi: 10.1016 / j.virusres.2022.198932. PubMed PMID: 36165924; PMCID: PMC9534104). The virions exist in two antigenically distinct forms: intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). For infection, the virus particle attaches to the host cell through recognition of cell surface glycosaminoglycans and then enters the host cells through various viral-host protein interactions. The mpox viruses reproduce in the cytoplasm using host ribosomes to generate the structural components of virions. Progeny poxviruses are enveloped by membranes before exiting infected cells via plasma membrane fusion or by cell rupture.

[0005] The peripheral proteins of the vaccinia virus have been extensively studied, revealing approximately 25 membrane proteins. Among them, 17 are suggested to play a functional role in viral penetration and / or virus-host cell membrane fusion. These proteins include A27, A17, H3, D8, LI, A28, H2, A21, L5, G3, G9, A16, J5, F9, 12, A26, and 03 (See, e.g., Moss B. Smallpox vaccines: targets of protective immunity. Immunol Rev. 2011 ;239(1): 8-26. Epub 2011 / 01 / 05. doi: 10.1 l l l / j, 1600-065X.2010.00975.x. PubMed PMID: 21198662; PMCID: PMC3074351). The entry-fusion complex comprises eight proteins: A16, A21, A28, G3, G9, H2, L5, and 03. The membrane proteins A27 and H3 facilitate intracellular IMV virion attachment to host cells via interactions with cell surface glycosaminoglycans. Immunization with the vaccinia vaccine in humans elicits antibodies against four major IMV surface proteins: A27, LI, D8, and H3 (See, e.g., Benhnia MR, et al., Redundancy and plasticity of neutralizing antibody responses are cornerstone attributes of the human immune response to the smallpox vaccine. J Virol. 2008;82(7):3751-68. Epub 2008 / 02 / 01. doi: 10.1128 / JVI.02244-07. PubMed PMID: 18234801; PMCID: PMC2268460 and Davies DH, et al., Proteome-wide analysis of the serological response to vaccinia and smallpox. Proteomics. 2007;7(10): 1678-86. Epub 2007 / 04 / 20. doi: 10.1002 / pmic.200600926. PubMed PMID: 17443847). To date, six membrane antigens of vaccinia viruses, namely, A27, A33, LI, D8, B5, and H3, are known to be important targets for protective immune responses in both mpox and vaccinia viruses. The crystal structures of the LI,A33, and D8 proteins have been elucidated. In particular, the structures of LI and D8 in complex with corresponding neutralizing antibodies were solved, with LI binding to the monoclonal antibody (mAb) 7D11 and D8 binding to the mAb LA5. Due to their close genetic and antigenic relationships with mpox and smallpox viruses, less virulent vaccinia viruses are employed as vaccines against these two pathogens. For instance, Jynneos™ (Bavarian Nordic) contains a live, modified, non-replicating vaccinia virus Ankara strain that provides -85% effectiveness against mpox. Due to the nature of these live vaccinia viruses, one of the currently licensed mpox / smallpox vaccines, ACAM2000, which comprises live replicating vaccinia viruses, is associated with a relatively high rate of adverse events in healthy persons. These include the risk of peri- and endocarditis, severe debilitation, and even death, posing a particular high risk to immunocompromised individuals and pregnant women. The Jynneos™ vaccine, which consists of non-replicating vaccinia virus, removes the observed pathologies associated with the ACAM2000 vaccine and is therefore much safer. However, Jynneos™ still has common side effects like injection site reactions and systemic symptoms such as headache and muscle pain. Additionally, future emerging variants of mpox virus may not be fully covered by existing vaccines. New vaccines may also be developed to target specific strains or clades of the mpox virus, providing more tailored protection. Finally, more effective vaccines with simpler administration processes will improve accessibility and distribution, particularly in resource-limited developing countries. Therefore, there is a high demand for a new generation of mpox vaccines with improved effectiveness and safety.BRIEF SUMMARY

[0006] Disclosed herein are vaccine compositions, in particular, polyvalent icosahedral compositions for presentation of an mpox antigen, and methods of using same. The disclosed compositions may contain an S particle comprising a modified norovirus (NoV) S domain protein and an mpox antigen. The modified S domain and mpox antigen may be linked via a linker protein domain operatively connected to the modified S domain protein and the mpox antigen. Methods of using the disclosed vaccine composition are also provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0008] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0009] FIG. 1. Generation and characterization of the S-Ll and S-Jl fusion proteins and their self-assembled PVNPs. (A) Schematic constructs of the S-Ll and S-Jl fusion proteins, as well as the free LI and JI proteins. Sig: secretory signal peptide; S: modified norovirus S domain; hinge: the hinge region of norovirus VP1; linker: a row of four glycines; Ll / Jl: the Ll / Jl protein; His: the Hisx6 tag. (B) An SDS-PAGE gel showing the quality of the resin-purified S-Jl and S-Ll fusion proteins, each revealing major bands at~45 kDa and ~52 kDa. Bovine serum albumin (BSA, ~68 kDa) served as a control. (C) A Western blot analysis of the purified S-Jl and S-Ll fusion proteins (each lane contained 0.25 pg of protein) was performed using hyperimmune serum against norovirus VLPs, which contain the S domain as a primary detection antibody. BSA was included as a negative control. (D) An SDS-PAGE gel showing the quality of the resin-purified JI and LI proteins, each revealing a major band at the expected -17 kDa and -20 kDa. In (B) and (C), the M lanes are protein standards with the indicated molecular weights in kDa. (E and F) TEM micrographs of the purified S-Ll (E) and S-Jl (F) proteins indicating PVNP formation. (G and H) The size distributions of the S-Ll (G) and S-Jl PVNPs were determined by dynamic light scattering (DLS).

[0010] FIG. 2. Three-dimensional (3D) structural modeling of the S60-L1 PVNP. With the help of UCSF ChimeraX software, a 3D model was generated using the previously elucidated cryoEM map of S60-VP8* PVNP (40), in which the N-termini of 60 LI crystal structures (PBD code: lypy) (22) were docked to the C-termini of the S domains of the S60 nanoparticle to replace the VP8*s. (A) Surface representations of the S60 nanoparticle at the five-fold axis, showing 60 exposed S domain termini in green. (B and C) Surface representations of the S60-L1 PVNPs in the frontal (B) and section (C) views, respectively. (D to F) Cartoon representations of the abovethree S-Ll PVNP structures, including the S60 nanoparticle (D) and the front (E) and section (F) views of the PVNP. (G to I) Transparent surface representations showing the cartoon representations of the S-Ll PVNP, including its S60 nanoparticle (G), as well as the front (H) and section (H) views of the PVNP. (J) The crystal structure of the LI antigen in cartoon representation showing its N-terminus at the base and residue D35 in the red sphere representation representing the major neutralizing epitope on the distal top of the antigen. (K and L) Surface representations of S60-L1 PVNPs viewed at two-fold (K) and three-fold (L) axes. In all the images, the S60 nanoparticles are shown in orange, while the LI proteins are shown in cyan. All the images except J to L are viewed at the five-fold axes.

[0011] FIG. 3. Ll / Jl -specific serum antibody responses elicited by the S-L1 / S-J1 PVNPs in mice. (A, B) LI- and Ji-specific serum IgG titers elicited by the S-Ll and S-Jl PVNPs, as well as by free LI and JI proteins after two (A) and three (B) immunizations. (C) LI- and Jl-specific serum IgA titers elicited by the S-Ll and S-Jl PVNPs, as well as by free LI and JI proteins after three immunizations. The S60 nanoparticle (S) was used as a negative control. Y-axes indicate IgG (A and B) or IgA (C) titers, while X-axes indicate various immunogens. Purified LI and JI proteins were used as capture antigens for determination of the LI- (left panels) and Jl-specific (right panels) IgG (A and B) and IgA (C) titers, respectively. Significant differences between the data sets are shown as for significance with p values <0.05, “**” for high significance with p values < 0.01, or “****” for extreme significance with p values < 0.0001 . The dashed lines indicate the limits of detection (LODs).

[0012] FIG. 4. Neutralizing antibody titers of the mouse sera after immunization with the S- L1 PVNPs, the S-Jl PVNPs, the free LI protein, or the free JI protein for two (A) or three (B) times against vaccinia virus (Western Reserve strain) replication in cell culture. Y-axes show 50% neutralizing antibody titers of mouse sera, whereas X-axes indicate the mouse serum samples after immunization with various immunogens. The sera after immunization with the S60 nanoparticles (S) served as negative controls. Significant differences between the data groups are shown as for significance with p values < 0.05 or “**” for high significance with p values < 0.01. The dashed lines indicate the limits of detection (LODs).

[0013] FIG. 5. The S-Ll PVNPs protect mice against mortality and body weight loss caused by vaccinia virus challenge, (a) LI (left two columns) and JI (right two columns) specific IgG responses after three immunizations with the S-Ll PVNP (S-Ll) and S-Jl PVNP (S-Jl), respectively, using the S60 nanoparti cl es(s) as a negative control, the Y-axis indicates Ll / Jl- specific IgG titers, while the X-axis shows different immunogens. The dashed lines indicate the limits of detection (LODs). (b) survival curves of three groups of mice after immunization with S- L1 PVNPs (S-Ll), S-Jl PVNPs (S-Jl), or S60 nanoparticles (S), followed by vaccinia virus challenge. The Y-axis indicates survival rates as a percentage, while the X-axis indicates Day Post Challenge (DPCs). (c) body weight change curves of the same three groups of mice after immunization with S-Ll PVNPs (S-Ll), S-Jl PVNPs (S-Jl), or S60 nanoparticles (S), followed by vaccinia virus challenge. The Y-axis indicates the body weight change as a percentage, while the X-axis indicates the days post challenge (DPCs). Significant differences between the data sets are shown as for significance with p values <0.05, “**” for high significance with p values < 0.01, or “***” for extreme significance with p values < 0.0001. In (c), the symbol ‘+’ indicates significant differences between the S-Ll PVNP (S-Ll)-immunized group and the S60 nanoparticle (S)-immunized group, while the symbol indicates significant differences between the S-Ll PVNP (S-Ll)-immunized group and the S-Jl PVNP (S-Jl)-immunized group.DETAILED DESCRIPTIONDEFINITIONS

[0014] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The methods may comprise, consist of, or consist essentially of the elements of the compositions and / or methods as described herein, as well as any additional or optional element described herein or otherwise useful in an mpox vaccine composition and / or methods of using same.

[0015] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0016] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0017] As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0018] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and / or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.

[0019] As used herein, the term “antigen” may be used interchangeably with the terms “immunogen” and “immunogenic antigen”, as defined below. Technically speaking, an antigen is a substance that is able to combine with the products of an immune response once they are made, but is not necessarily able to induce an immune response (i.e. while all immunogens are antigens,the reverse is not true); however, the antigens that are discussed herein as the subject of the present invention are assumed to be immunogenic antigens, even when referred to as antigens.

[0020] The term “fusion protein” means a protein created through translation of a fusion gene, resulting in a single polypeptide with functional properties derived from each of the original proteins.

[0021] The term “immunity” means the state of having sufficient biological defenses to avoid infection, disease, or other biological invasion by a disease-causing organism.

[0022] The term “immunogenicity” means the ability of an immunogen to elicit a humoral and / or cell-mediated immune response.

[0023] The terms “immunogen” and “immunogenic antigen” mean a specific type of antigen that is able to induce or provoke an adaptive immune response in the form of the production of one or more antibodies.

[0024] The terms “immunogenic response” and “immune response” mean an alteration in the reactivity of an organisms' immune system in response to an immunogen. This can involve antibody production, induction of cell-mediated immunity, complement activation or development of acquired immunity or immunological tolerance to a certain disease or pathogen.

[0025] The terms “immunization” and “vaccination” mean the deliberate induction of an immune response and involve effective manipulation of the immune system's natural specificity, as well as its inducibility. The principle behind immunization is to introduce an antigen, derived from a disease-causing organism, which stimulates the immune system to develop protective immunity against that organism, but wherein the antigen itself does not cause the pathogenic effects of that organism. The desired outcome of a prophylactic or therapeutic immune response resulting from an immunization may vary according to the disease. For example, an immune response against a pathogen may inhibit or prevent colonization and replication of the pathogen, effecting protective immunity and the absence or reduction of any disease symptoms. A vaccine against pathogens may also be considered effective if it reduces the number, severity, or duration of symptoms; if it reduces the number of individuals in a population with symptoms; or even if it merely reduces the transmission of an infectious pathogen.

[0026] The term “infection” means the invasion of an animal or plant host's body tissues by a pathogen, as well as the multiplication of the pathogen within the body and the body's reaction to the pathogen and any toxins that it may produce.

[0027] The term “vaccine” means a biological preparation or composition that improves immunity to a particular disease. Vaccines are examples of immunogenic antigens intentionally administered to induce an immune response in the recipient.Mpox Vaccine Methods and Compositions

[0028] Disclosed are methods and compositions for immunization of an individual against an mpox infection, in particular, mpox vaccines employing a P particle platform comprising a modified P domain. In aspects, a P particle platform displaying an mpox antigen is disclosed. In aspects, a fusion protein comprising a P domain and an mpox antigen is disclosed, such fusion protein being useful for the manufacture of a P particle platform displaying the mpox antigen, the fusion proteins aggregating to form the antigen-presenting composition (PVNP as described herein) which may then be used as the basis of a vaccine composition for eliciting an immune response to the selected antigen in an individual administered the composition. The disclosed vaccine compositions may be administered to an individual for immunization against an mpox infection. In aspects, the vaccine is a monovalent vaccine, displaying a single mpox antigen type. In aspects, the vaccine is a polyvalent vaccine, displaying at least two, or at least three, or at least four, or more than four mpox antigen types.

[0029] In aspects, the mpox vaccine composition comprises a non-replicating pseudovirus nanoparticle (PVNP) as described in, for example, U.S. Patent No. 11,833,198 B2, WO 2024 / 102697 Al, U.S. Patent Application Publication No. 2024 / 0131145, and which is further described herein. In brief, many defined neutralizing antigens face a common problem of low immunogenicity for non-replicating vaccine development, due to their small sizes with low valences. This problem can be solved via fusion or conjugation of the antigens to a large, polyvalent protein platform for enhanced immunogenicity. Significantly enhanced immunogenicity of antigens can be achieved after displayed with the NoV S60 particles as a vaccine platform. S60 particles can be easily produced, are stable, and are highly immunogenic toward the displayed antigen. The disclosed viral platform can be assembled as described herein.In short, homotypic interactions of viral capsid proteins are common, driving viral capsid selfformation. By taking advantage of such interactions of the norovirus shell (S) domain that naturally builds the interior shells of norovirus capsids, a 60-valent, icosahedral S60 particle can be obtained using a cell based system. This can been achieved by several modifications to the S domain, for example an R69A mutation to destruct an exposed proteinase cleavage site and triple cysteine mutations (V57C / Q58C / S136C) to establish inter-S domain disulfide bonds for enhanced inter-S domain interactions. The polyvalent S60 particle with 60 exposed S domain C-termini offers an ideal platform for antigen presentation, leading to enhanced immunogenicity to the displayed antigen for vaccine development. A chimeric S60 particle displaying 60 antigenic proteinscan be produced and used to elicit an immunogenic response.

[0030] The PVNP may take the form of an icosahedral particle comprising a norovirus (NoV) shell (S) domain protein (“modified S domain”). In aspects, the PVNP may comprise an aggregate of 60 S modified S domain proteins. In aspects, the modified S domain may comprise or consist of a sequence having at least 90% sequence identity toMKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPW1RNNFV QAPGGEFTVSPANAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTA GKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIK LIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVE (SEQ ID NO: 1).

[0031] The modified S domain sequence of SEQ ID NO: 1 includes a start codon “M.” In certain aspects, the start codon “M” can be omitted. For example, the start codon “M” can be omitted when a signal peptide is appended to the front of the modified S domain sequence of SEQ ID NO: 1.

[0032] In aspects, the non-replicating S60-PVNP comprises a modified S domain having at least 91% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises a modified S domain having at least 92% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises a modified S domain having at least 93% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises a modified S domain having at least 94% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises a modified S domain having at least 95% sequence identity to SEQ ID NO:1. In aspects, the non-replicating S60-PVNP comprises a modified S domain having at least 96% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises a modified S domain having at least 97% sequence identity to SEQ ID NO: 1. In aspects, the nonreplicating S60-PVNP comprises a modified S domain having at least 98% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises a modified S domain having at least 99% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises a modified S domain having 100% sequence identity to SEQ ID NO: 1.

[0033] Disclosed herein are methods and compositions that can be used to form a polyvalent vaccine composition, in particular, using a modified norovirus S particle. The In aspects, the particle is a polyvalent icosahedral composition for antigen presentation. The particle typically has an icosahedral symmetry structure.

[0034] In aspects, the S particle comprises a modified S domain that is wildtype at one or more of positions V57, Q58, or S136, and M140. In aspects, the modified S domain is wildtype at all positions V57, Q58, or S136, and M140.

[0035] The PVNP may comprise (display) one or more mpox antigen types as described herein. In one aspect, the PVNP comprises an LI mpox antigen. In one aspect, the PVNP comprises a J2 mpox antigen. In aspects, the PVNP comprises an mpox LI antigen and is capable of eliciting LI -specific IgA and / or IgG antibodies in an individual administered the mpox vaccine. In aspects, the PVNP comprises an mpox JI antigen and is capable of eliciting JI -specific IgA and / or IgG antibodies in an individual administered the mpox vaccine. In aspects, the PVNP comprises 60 sites for antigen presentation.S60-L1-PVNP

[0036] In one aspect, the non-replicating mpox pseudovirus nanoparticle comprises the LI mpox antigen, in which the PVNP presents an LI mpox antigen on the surface of the nanoparticle (the “S60-L1-PVNP”). In aspects, the S60-L1-PVNP composition induces substantial immunity to mpox infection or reduces the severity or likelihood of at least one symptom thereof when administered to a subject. In this aspect, the S60-L1-PVNP may be formed from the aggregate of fusion proteins, comprising a modified S domain, an mpox LI antigen, and a hinge region.

[0037] In aspects, the mpox LI antigen of the S60-L1-PVNP is that of GenBank ID:WDR5 1051.1, having the following sequence:MGAAASIQTTVNTLSERIS SKLEQEANAS AQTKCDIEIGNFYIRQNHGCNITVKNMC S AD ADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNS SAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQ VAGTGV (SEQ ID NO: 2).

[0038] The LI domain may comprise or consist of a sequence having at least 90% sequence identity to SEQ ID NO: 2. In aspects, the LI domain may comprise or consist of a sequence having at least 95% sequence identity to SEQ ID NO: 2. In aspects, the LI domain may comprise or consist of a sequence having at least 99% sequence identity to SEQ ID NO: 2. In other aspects, the mpox antigen may comprise or consist of a sequence having 100% sequence identity to SEQ ID NO: 2.

[0039] The S60-L1-PVNP may comprise a modified S domain as described herein, for example, a modified S domain having at least 90% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having at least 91% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having at least 92% sequence identity to SEQ ID NO: 1 . In aspects, the non-replicating S60-PVNP comprises an S domain having at least 93% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having at least 94% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having at least 95% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having at least 96% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having at least 97% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having at least 98% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having at least 99% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having 100% sequence identity to SEQ ID NO: 1.

[0040] In aspects, the mpox LI antigen is fused to (operatively linked, or, in other aspects, contiguous with) the NoV S domain protein. In aspects, the mpox LI antigen is operativelyconnected to the NoV S domain protein via a linker, as described herein. In aspects, the mpox antigen LI is operatively connected to the NoV S domain via any amino acid sequence which allows for presentation of the antigen.

[0041] For example, the S60-L1-PVNP fusion protein may comprise a sequence having at least about 90%, 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% sequence identity sequence identity to the following sequence, wherein the modified S domain is in plain text, the hinge region is in bold / italic, the LI region is italicized, and the linker is bold:

[0042] MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDP WIRNNFVQAPGGEFTVSPANAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVIL AGNAFTAGKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYN QSNDSTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLEPPTEEGGGGMGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQ LDA VLSAA TETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSA WDNKLKI QNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGV (SEQ ID NO: 3).

[0043] In a further aspect, the S60-L1 -PVNP fusion protein may further comprise an N- terminal secretory peptide, and may comprise a sequence having at least about 90%, 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% sequence identity sequence identity to the following sequence, wherein MKWVTFISLLFLFSSAYS, SEQ ID NO: 4 is the N-terminal secretory peptide:MKWVTFISLLFLFSSAYSKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVA GQQNVIDPWIRNNFVQAPGGEFTVSPANAPGEILWSAPLGPDLNPYLSHLARMYNGYA GGFEVQVILAGNAFTAGKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDV RNNFYHYNQSNDSTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVE GGGGMGAAASIQTTVNTL SERIS SKLEQEANAS AQTKCDIEIGNF YIRQNHGCNLTVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVK QTCNS S A VVDNKLKIQNVIIDEC YGAPGSPTNLEFINTGS SKGNC AIKALMQLTTK ATTQI APKQVAGTGVHHHHHH (SEQ ID NO: 5)

[0044] It will be understood that antigen sequences used to generate the antigen peptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference sequence, provided that the resulting antigen elicits at least a partial immune response in an individual administered the composition having the antigen.S60-J1-PVNP

[0045] In one aspect, the non-replicating mpox pseudovirus nanoparticle comprises the JI mpox antigen, in which the PVNP presents a JI mpox antigen on the surface of the nanoparticle (the “S60-J1-PVNP”). In aspects, the S60-J1-PVNP composition induces substantial immunity to mpox infection or reduces the severity or likelihood of at least one symptom thereof when administered to a subject. In this aspect, the S60-J1-PVNP may be formed from the aggregate of fusion proteins, comprising a modified S domain, an mpox JI antigen, and a hinge region.

[0046] In aspects, the mpox JI antigen of the S60-J1-PVNP is that of GenBank ID: AC#: YP_010377082.1. having the following sequence:

[0047] MDHNQ YLLTMFF ADDD SFFKYF ASQDDES SL SDILQITQ YLDFLLLLLIQ SKN KLEAVGHCYESLSEEYRQLTKFTDSQDFKKLFNKVPIVTDGRVKLNKGYLFDFVISLMR FKKESALATTAIDPVRYIDPRRDIAFSNVMDILKSNKVEK (SEQ ID NO: 6).

[0048] The JI domain may comprise or consist of a sequence having at least 90% sequence identity to JI sequence, SEQ ID NO: 6. In aspects, the JI domain may comprise or consist of a sequence having at least 95% sequence identity to JI sequence, SEQ ID NO: 6. In aspects, the JI domain may comprise or consist of a sequence having at least 99% sequence identity to JI sequence, SEQ ID NO: 6. In other aspects, the mpox antigen may comprise or consist of a sequence having 100% sequence identity to JI sequence, SEQ ID NO: 6.

[0049] In this aspect, the S60-J1-PVNP may comprise a modified S domain as described herein, for example, a modified S domain having at least 90% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60- JI -PVNP comprises an S domain having at least 91% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-PVNP comprises an S domain having at least 92% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-J1-PVNP comprises an S domain having at least 93% sequence identity to SEQ ID NO: 1. Inaspects, the non-replicating S60-J1 -PVNP comprises an S domain having at least 94% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-J1-PVNP comprises an S domain having at least 95% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-J1- PVNP comprises an S domain having at least 96% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-J1-PVNP comprises an S domain having at least 97% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-J1-PVNP comprises an S domain having at least 98% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-J1-PVNP comprises an S domain having at least 99% sequence identity to SEQ ID NO: 1. In aspects, the non-replicating S60-J1-PVNP comprises an S domain having 100% sequence identity to SEQ ID NO: 1. The disclosed compositions may be used for presentation of an antigen, in particular, an mpox antigen. In certain aspects, the antigen may encode for an mpox JI antigen having a size of from 8 amino acids up to about 300 amino acids, or from 8 amino acids up to about 400 amino acids, or from 8 amino acids up to about 500 amino acids.

[0050] In aspects, the mpox JI antigen is fused to (operatively linked, or, in other aspects, contiguous with) the NoV S domain protein. In aspects, the mpox JI antigen is operatively connected to the NoV S domain protein via a linker, as described herein. In aspects, the mpox antigen JI is operatively connected to the NoV S domain via any amino acid sequence which allows for presentation of the mpox JI antigen.

[0051] For example, the S60-J1-PVNP fusion protein may comprise a sequence having at least about 90%, 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% sequence identity sequence identity to the following sequence, wherein the modified S domain is in plain text, the hinge region is in bold / italic, the JI region is italicized, and the linker is bold:

[0052] MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDP WIRNNFVQAPGGEFTVSPANAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVIL AGNAFTAGKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYN QSNDSTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLFPPTEEGGGG MDHNQYLLTMFFADDDSFFKYFASQDDESSLSDILQITQYLDFLLLLLIQSKNKLEAVGHCYESJ EEYRQLTKFTDSQDFKKLFNKVPIVTDGRVKJ.NKGYLFDFVISLMRFKKESALA TTAIDPV RYIDPRRDIAFSNVMDILKSNKVEK (SEQ ID NO: 7).

[0053] It will be understood that antigen sequences used to generate the antigen peptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference sequence, provided that the resulting antigen elicits at least a partial immune response in an individual administered the composition having the antigen.

[0054] In aspects, the PVNP is a monovalent S60-PVNP displaying an mpox antigen, for example the LI or JI antigen. In another aspect, the PVNP may be a bivalent or polyvalent PVNP displaying more than one variant of the LI and / or JI antigen, in which the more than one antigen comprises at least two, or at least three, or at least four, or more than four different mpox antigens. In aspects, the different mpox antigens may be variants of the same antigen, for example an LI and / or a JI antigen in which one or more amino acids are different.Fusion Proteins

[0055] Further disclosed are fusion proteins, which may comprise an mpox antigen, a linker region, a hinge region, and an S-domain. In aspects, the fusion proteins are recombinant fusion proteins that form the basis of the polyvalent icosahedral (PVNP) compositions. The fusion protein may comprise a norovirus (NoV) S domain protein having a mutation to the trypsin site as described above; a linker protein domain operatively connected to the modified S domain having one or more of the aforementioned mutations; and an antigen domain operatively connected to the linker. Expression of the fusion proteins may be carried out using methods known in the art and as disclosed herein.

[0056] The PVNP may be formed from an aggregate of fusion proteins as described herein, the aggregate of fusion proteins forming an icosahedral structure and a nanoparticle shell. In this aspect, the fusion protein may comprise: a modified S domain protein; an mpox antigen; anda peptide linker, the peptide linker connecting the C-terminus of the modified S domain to the mpox antigen; wherein the modified S domain protein forms the interior of the nanoparticle shell of the PVNP composition; wherein the nanoparticle shell displays 60 exposed C-termini of the S domain in a triangular pattern, each S domain thereby displaying an mpox antigen on the surface of the nanoparticle shell, the nanoparticle shell thereby displaying 60 mpox antigens on the surface of the nanoparticle shell.

[0057] For example, the fusion proteins may comprise the S-domain, which comprises the hinge region of norovirus, a linker, and an mpox antigen sequence (e.g., the LI and / or JI antigen). In aspects, the hinge region comprises or consists of the sequence FLVPPTVE (SEQ ID NO: 8).

[0058] In general, the fusion protein may comprise, from the N-terminus to the C-terminus direction, an S domain, a linker, and an mpox antigen sequence.Polyvalent Particles

[0059] As described above, recombinant fusion protein comprising a norovirus (NoV) S domain protein, a linker protein domain operatively connected to the norovirus S domain protein, and an antigen protein domain operatively connected to the linker is disclosed. The fusion proteins aggregate to form a PVNP, which may have an icosahedral symmetry structure. In one aspect, the PVNP comprises 60 sites for antigen (mpox LI and / or JI) presentation. In one aspect, the norovirus S domain protein is that of a calicivirus. The calicivirus may be characterized by having 180 copies of a single capsid protein. In one aspect, the PVNP may be tag-free. In a further aspect, the PVNP may comprise a tag, for example, a HIS tag. The HIS tag may be useful, for example, for manufacturing and purifying the PVNP composition. In one aspect, the PVNP may have a diameter of between about 20 and about 40 nm.

[0060] In aspects, the norovirus S domain protein comprises a mutation in a proteinase cleavage site of the NoV S domain protein, wherein the mutation renders the site resistant to trypsin cleavage. One or more mutations may be made to the site, provided the mutation effectively destroys the trypsin cleavage site. Modifications to the site that achieve such effect may include amutation at position 69 or position 70. In aspects, the mutation occurs at position R69. In aspects, the mutation may be a change to any amino acid other than K (lysine), which is sufficient to destroy the proteinase cleavage site. In aspects, the mutation is R69A. In aspects, the mutation may occur at position N70, for example, the mutation may be any amino acid other than P (proline) sufficient to destroy the proteinase cleavage site. In one aspect, the norovirus S domain protein may comprise a mutation sufficient to provide a non-native disulfide bond binding site. The norovirus S domain protein may comprise a mutation of at least two amino acids (that are sterically close to each other) to cysteine residues sufficient to provide at least one non-native disulfide bond binding site, or, in other aspects, at least two non-native disulfide bond binding sites, or at least three non-native disulfide bond binding sites between neighboring S domain proteins of the polyvalent icosahedral S particle. In certain aspects, the mutation may be selected from V57C, Q58C, S136C, M140C, or a combination thereof.

[0061] In aspects, the norovirus S domain protein may comprise a wildtype sequence at one more, or two or more, or three or more, or all four of the amino acids at position 57, 58, 136, and 140, with reference to the wild type P Domain sequence, SEQ ID NO: 9.SRTKPFTVPILTVEEMSNSRFPIPLEKLYTGPSSAFVVQPQNGRCTTDGVLLGTTQLSAVN ICTFRGDVTHIAGSHDYIMNLASQNWNNYDPTEEIPAPLGTPDFVGKIQGMLTQTTREDG STRAHKATVSTGSVHFTPKLGSVQYTTDTNNDLQTGQNTKFTPVGVIQDGNNHQNEPQ QWVLPNYSGRTGHNVHLAPAVAPTFPGEQLLFFRSTMPGCSGYPNMNLDCLLPQEWVQ HFYQEAAPAQSDVALLRFVNPDTGRVLFECKLHKSGYVTVAHTGPHDLVIPPNGYFRFD SWVNQFYTLAPMGNGAGRRRAL (SEQ ID NO: 9).Linker

[0062] In aspects, the linker may comprise an amino acid sequence of a length sufficient to provide space and certain flexibility between the S domain protein particle and the displayed antigens. The linker is typically a short peptide of one to ten amino acid units, or three to six amino acids, that connect the C-terminus of the S domain to the displayed antigens. The linker provides space and certain flexibility between the S60 particle and the displayed antigens, which helps the independent folding of the S domain and the displayed antigens. A longer linker may be used as necessary. The amino acid length of the linker should be sufficient to allow flexibility of the proteindomains to form the claimed compositions. In one aspect, the non-replicating S60-PVNP may comprise a linker having a sequence selected from HHHH (SEQ ID NO: 10), GGGG (SEQ ID NO: 11), GSGS (SEQ ID NO: 12), HHHHHH (SEQ ID NO: 13) and SSRVDGGGGG (SEQ ID NO: 14). In one aspect, the linker may comprise the sequence GGGG (SEQ ID NO: 11). In one aspect, the linker may comprise the sequence SSRVDGGGGG (SEQ ID NO: 14). In aspects, the linker comprises or consists of GGGG (SEQ ID NO: 11). In aspects, the linker comprises or consists of SSRVDGGGGG (SEQ ID NO: 14).Vaccine Compositions

[0063] In aspects, the vaccine compositions may further comprise one or more pharmaceutically-acceptable carriers, for example, a solvent, dispersion media, coating, stabilizing agent, diluent, preservative, antibacterial and / or antifungal agent, isotonic agent, adsorption delaying agent, adjuvant, or combinations thereof. In one aspect, the vaccine composition comprises physiological saline. Optionally, a protectant may be included, for example, an anti- microbiological active agent, such as for example Gentamycin, Merthiolate, and the like. Stabilizing agents which may be used include saccharides, trehalose, mannitol, saccharose and the like, which may be added in an amount sufficient to increase and / or maintain product shelf-life. The disclosed herein may include known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as, for example, saline or a corresponding plasma protein solution may be used. Exemplary diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Exemplary isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Exemplary stabilizers may include albumin and alkali salts of ethylenediaminetetraacetic acid, among others.

[0064] In one aspect, disclosed is a container, for example a container suited for delivery to an individual in need thereof, for example a capsule, a vial, or a syringe, comprising at least one dose of the immunogenic composition as disclosed herein. The container may comprise, for example, 1 to 250 doses of the immunogenic composition, or in other aspects, 1, 10, 25, 50, 100, 150, 200, or 250 doses of the immunogenic composition. In one aspect, each of the containers may comprise a single dose of the vaccine composition, or more than one dose of the vaccine composition andmay further comprises an anti-microbiological active agent. Those agents may include, for example, antibiotics such as Gentamicin and Merthiolate and the like.Kits

[0065] A further aspect relates to a kit. The kit may comprise any of the containers described above and an instruction manual, including the information for the delivery of the immunogenic composition disclosed above. For example, instructions related to intramuscular application of at least one dose may be provided for lessening the severity of clinical symptoms associated with an infection of an antigen as disclosed here. The kits and / or compositions may further include an immune stimulant such as keyhole limpet hemocyanin (KLH), or incomplete Freund's adjuvant (KLH / ICFA). Any other immune stimulant known to a person skilled in the art may also be used. In one aspect, the adjuvant is aluminum hydroxide.Methods of Making

[0066] In one aspect, a method of making the disclosed polyvalent icosahedral structures is disclosed. The method may comprise the steps of a) making a first region comprising a modified NoV S domain protein, wherein the modification comprises a mutation sufficient to destruct an exposed protease cleavage site (wherein the mutation prevents protein degradation), preferably an R69A mutation, and b) recombinantly expressing the first region having a modified NoV S domain protein with a linker and an antigen. In certain aspects, the PVNP composition may be effectively produced in E.coli. In aspects, the PVNP composition is produced via a prokaryotic system. In aspects, the PVNP composition is produced via a eukaryotic system. In aspects, the PVNP composition is produced via both a prokaryotic system and a eukaryotic system.Method of Immunizing an Individual

[0067] In one aspect, a method of immunizing an individual in need thereof against an mpox infection is disclosed. For example, in one aspect, a method of inducing an antigen specific immune response in an individual is disclosed, the method comprising administering any of the vaccine compositions described herein to an individual in an amount effective to produce an antigen specific immune response. In some aspects, the antigen specific immune response may comprise a T cell response. In some aspects, the antigen specific immune response may comprisea B cell response. In some aspects, the method may comprise a single administration of the vaccine composition. In some aspects, the method may further comprise administering a booster dose of the vaccine. In some aspects, the vaccine composition may be administered to the subject by intradermal or intramuscular injection. The method may comprise administering a RV-PNVP or vaccine composition containing an RV-PVNP as disclosed here. In one aspect, the administration may be sufficient to induce a neutralizing antibody titer against the antigen. The disclosed RV- PVNPs may be used to elicit an immune response in an individual in need thereof, comprising administering a RV-PVNP or composition disclosed herein, to the individual. In some aspects, the vaccine is administered to the individual via a route selected from intramuscular administration, intradermal administration and subcutaneous administration. In some aspects, the administering step comprises contacting a muscle tissue of the individual with a device suitable for injection of the composition. In some aspects, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition in combination with electroporation.

[0068] In aspects, the method of reducing the severity of an mpox infection and / or eliciting an immune response in a subject against mpox in an individual in need thereof is disclosed, the method comprising administering an mpox vaccine as disclosed herein to the individual. In aspects, the administration comprises one dose, or two doses, or three doses of the mpox vaccine. In aspects, the composition is administered to an individual via a route selected from one or more of intradermal injection, intramuscular injection, or by intranasal administration. In aspects, the administered composition comprises a pharmaceutically acceptable carrier. In aspects, the administered composition comprises one or more of an adjuvant and a preservative.Dosing

[0069] In one aspect, provided are methods comprising administering the vaccine compositions to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.

[0070] In certain aspects, the compositions may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic or prophylactic effect. The dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain aspects, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens may be used. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.Dosage Forms

[0071] The vaccine composition described herein may be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).

[0072] Liquid Dosage Forms

[0073] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (inparticular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain aspects for parenteral administration, compositions may be mixed with solubilizing agents, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0074] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated and may include suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0075] Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.Pulmonary

[0076] Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation may be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

[0077] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w / w) and as much as 100% (w / w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets and / or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w / w) active ingredient, where the balance may comprise an orally dissolvable and / or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.Coatings or Shells

[0078] Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.Multi-Dose and Repeat-Dose Administration

[0079] In some aspects, the vaccine compositions may be administered in two or more doses (referred to herein as “multi-dose administration”). Such doses may comprise the same components or may comprise components not included in a previous dose. Such doses may comprise the same mass and / or volume of components or an altered mass and / or volume of components in comparison to a previous dose. In some aspects, multi-dose administration may comprise repeat-dose administration. As used herein, the term “repeat-dose administration” refers to two or more doses administered consecutively or within a regimen of repeat doses comprising substantially the same components provided at substantially the same mass and / or volume. In some aspects, subjects may display a repeat-dose response. As used herein, the term “repeat-dose response” refers to a response in a subject to a repeat-dose that differs from that of another doseadministered within a repeat-dose administration regimen. In some aspects, such a response may be the expression of a protein in response to a repeat-dose comprising the vaccine composition.

[0080] In one aspect, a method of eliciting an immune response, in particular, an immune response to an mpox antigen, sufficient to deter, prevent, decrease the likelihood of obtaining, reduce the severity of, and / or mitigate an mpox infection, in an individual in need thereof is disclosed. In this aspect, the method may include the step of administering a vaccine composition as disclosed above to an individual in need thereof. The disclosed compositions may be administered to an individual according to any method known in the art. The vaccine compositions may be administered prophylactically to an individual suspected of having a future exposure to the antigen incorporated into the vaccine composition. In certain aspects, provided is a method of providing an immune response that protects an individual receiving the composition from infection, or reduces or lessens the severity of the clinical symptoms associated from an infection. Dosage regimen may be a single dose schedule or a multiple dose schedule (e.g., including booster doses) with a unit dosage form of the composition administered at different times. The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the antigenic compositions disclosed herein in an amount sufficient to produce the desired effect, which compositions are provided in association with a pharmaceutically acceptable excipient (e.g., pharmaceutically acceptable diluent, carrier or vehicle). The vaccine may be administered in conjunction with other immunoregulatory agents.

[0081] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1

[0082] The recent worldwide incidence of mpox infection and concerns about future emerging variants of mpox viruses highlight the need for the development of a new generation of mpox vaccines. To achieve this goal, Applicant utilized a norovirus S nanoparticle vaccine platform to produce and evaluate two pseudovirus nanoparticles (PVNPs), S-Ll and S-JL These PVNPs displayed the LI neutralizing antigen target of the vaccinia virus and a yet untested JI antigen of the mpox virus, respectively, with the aim of creating an effective nanoparticle-based mpox vaccine. Each self-assembled PVNP consists of an inner shell resembling the interior layer of the norovirus capsid and multiple LI or JI antigens on the surface. The PVNPs improved the antibody responses toward the displayed LI or JI antigens in mice, resulting in significantly greater Ll / Jl- specific IgG and IgA titers than those elicited by the corresponding free LI or JI antigens. After immunization with the S-Ll PVNPs, the mouse sera exhibited high neutralizing antibody titers against the vaccinia virus, and the S-Ll PVNPs provided mice with 100% protection against mortality caused by vaccinia virus challenge. In contrast, the S-J 1 PVNPs induced low neutralizing antibody titers and conferred mice weak protective immunity. These data confirm that the LI protein is an excellent vaccine target and that the readily available S-Ll PVNPs are a promising mpox vaccine candidate worthy of further development.

[0083] The recent mpox outbreak around the globe and the relatively high rate of adverse events of the current live vaccinia virus-based vaccines call for a new generation of mpox vaccine. Applicant developed two pseudovirus nanoparticles (PVNPs), named S-Ll and S-Jl, that displayed the LI neutralizing antigen and a yet untested J 1 antigen, respectively, aiming to develop an effective nanoparticle based mpox vaccine. Each self-assembled PVNP comprises an inner shell resembled the interior layer of norovirus capsid and multiple surface displayed LI or JI antigens. Immunization studies demonstrate that the PVNPs improved the antibody responses toward the displayed LI or JI antigens in mice, resulting in significantly higher Ll / Jl specific IgG and IgA titers compared with those elicited by the corresponding free LI or JI antigens. Further investigation revealed that the S-Ll PVNP immunized mouse sera neutralized against vaccinia virus replication at high (2400) titer, whereas the mouse sera after vaccination with the S-J 1 PVNP exhibited low (53.5) neutralization titer. These data indicated that the LI protein is a better vaccine target than the JI protein, and the S-Ll PVNP is a promising mpox vaccine.

[0084] Applicant developed a norovirus inner shell (S)-based nanoparticle as a vaccine platform to create pseudovirus nanoparticles (PVNPs) displaying various antigens in polyvalent formats, aiming to enhance immune responses. When the norovirus S domain (24.6 kDa) is produced using a protein expressing system, the S protein self-assembles into 22 nm S nanoparticles (SNPs) with T=1 icosahedral symmetry, consisting of 60 S proteins. Each SNP has 60 exposed, flexible S domain termini on the surface, making the SNP an excellent platform for displaying antigens of other pathogens. Applicant generated two self-assembled PVNPs, each of which displayed the LI antigen of vaccinia virus or the JI antigen of mpox virus, and evaluated them as vaccine candidates. LI is a myristoylated peripheral protein of vaccinia virus that plays a key role in facilitating viral-host cell membrane fusion and viral entry, two critical initial steps in mpox / vaccinia virus infection. Thus, LI is a major neutralizing antigen and an ideal target for mpox vaccine development. On the other hand, JI is a membrane protein essential for virion morphogenesis, growth, and plaque formation. Unlike the LI protein, which has been proven to be an excellent vaccine target among several DNA / subunit vaccine candidates, J 1 has not yet been studied as a vaccine target. Both the LI and JI proteins are highly conserved among orthopoxviruses, with amino acid sequences nearly identical between mpox and vaccinia viruses.

[0085] Both types of PVNPs self-assemble after recombinant S-Ll and S-Jl fusion proteins are generated using mammalian CHO (Chinese hamster ovary) cell expression system. While both types of PVNPs were immunogenic in mice, eliciting high antibody titers toward the displayed LI or JI antigens, only the sera from mice immunized with the S-Ll PVNPs demonstrated a high neutralizing antibody titer against vaccinia virus replication. Additionally, the S-Ll PVNPs provided mice with 100% protection against mortality caused by vaccinia virus challenge.Materials and methods

[0086] Plasmids for recombinant protein expression. A DNA fragment was designed to encode the S-Ll fusion protein, which comprises the following sequence components: 1) an N- terminal secretory signal peptide (MKWVTFISLLFLFSSAYS, SEQ ID NO: 4); 2) a GII.4 norovirus S domain with an R69A mutation and a hinge as described previously (Xia M, et al., Bioengineered Norovirus S60 Nanoparticles as a Multifunctional Vaccine Platform. ACS Nano. 2018;12(l l):10665-82. Epub 2018 / 09 / 21. doi: 10.1021 / acsnano.8b02776. PubMed PMID:30234973; PMCID: PMC6261714 and Xia M et al., Bioengineered pseudovirus nanoparticles displaying the HA1 antigens of influenza viruses for enhanced immunogenicity. Nano Res. 2022: 1-10. Epub 2022 / 02 / 03. doi: 10.1007 / s 12274-021 -4011-x. PubMed PMID: 35106126; PMCID: PMC8795936.); 3) a four-glycine linker (GGGG, SEQ ID NO: 11); 4) a polypeptide containing the first 184 amino acids of the LI protein of vaccinia virus (Western Reserve strain, PDB ID: 4U6H) (SEQ ID NO. 2); and 5) a C-terminal Hisx6 tag for purification purposes. Another DNA fragment encoding the S-Jl fusion protein was designed similarly, replacing the LI -encoding sequence with the JI protein-encoding sequence (152 residues)(SEQ ID NO. 6 of an mpox virus (GenBank AC#: YP_010377082.1). Both DNA fragments were synthesized by GenScript (Piscataway) and inserted into the pcDNATM3.4 vector (Thermo Fisher Scientific) for protein expression in the CHO cell system. Additionally, the DNA sequences encoding the LI and JI proteins were inserted into the pET24b vector, respectively, for protein expression using the Escherichia coli system.

[0087] Recombinant protein expression and purification. Recombinant S-Ll and S-Jl fusion proteins were expressed using the ExpiCHO Expression System (Gibco / Thermo Fisher Scientific) through transient transfection. Briefly, ExpiCHO-S cells, prepared according to the manufacturer’s instructions at 6xl06cells / mL, were transfected with the respective plasmids (see above) and incubated at 37°C on an orbital shaker. At 24 hours post transfection, the ExpiFectamine CHO Enhancer and ExpiCHO Feed were added. The cell cultures were harvested 10 days post transfection by centrifugation at 3450 x g (5000 rpm) for 20 min at 4°C using an Avanti J-26XP centrifuge (Beckman Coulter) with a JA 14 rotor, after which the cells / cell debris were separated from the culture medium (supernatant). The culture supernatant was mixed with Hisx6 tag binding cobalt resin (Thermo Fisher Scientific) for three hours, after which the mixture was loaded into an empty column to collect the resin. After washing three to four times with 10 resin volumes of washing buffer (20 mM of sodium phosphate, 0.5 M of NaCl, pH 7.4), the bound target proteins were eluted using elution buffer (20 mM of sodium phosphate, 0.5 M of NaCl, 500 mM imidazole, pH 7.4). Additionally, the S-Ll and S-Jl fusion proteins were produced using the CHO cell expression system through the protein expression service provided by GenScript.

[0088] In addition, C-terminally His-tagged LI and JI proteins were produced by the E. coli expression system using the BL21 / DE3 Arctic strain and purified through a denaturing protocolfollowing the instructions outlined in the HisPurTM Cobalt Resin Manual (Thermo Fisher Scientific), as described previously. Briefly, after overnight induction with 0.4 mM of IPTG (isopropyl B-D-l-thiogalactopyranoside), the bacteria were harvested and mixed with denaturing buffer (6 M of guanidine hydrochloride, 200 mM of tributylphosphine, 0.5 M of iodoacetamide, 100 mM of ammonium bicarbonate, 0.2 mg / ml of Proteomics Grade Trypsin; pH 8.5). Following sonication, the denatured target proteins were isolated using cobalt resin. The purified proteins were then serially dialyzed overnight against phosphate buffer (50 mM of sodium phosphate, 300 mM of NaCl, pH 7.4) containing 6 M, 4 M, 2 M or 1 M urea to remove the guanidine hydrochloride and reduce the concentration of urea. Finally, the proteins in phosphate buffer with 1 M urea were dialyzed against redox buffer (100 mM of Tris, 400 mM of L-arginine, 2 mM of EDTA, 5 mM of reduced glutathione, and 1 mM of oxidized glutathione, pH 8.0) overnight to remove the urea for protein refolding.

[0089] SDS-PAGE, protein quantitation, and Western blotting. The quality of the purified recombinant proteins was evaluated through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using 10% to 12% separating gels. Proteins were quantitated by SDS-PAGE using serially diluted bovine serum albumin (BSA; Bio-Rad) at known concentrations as standards on the same gels. The determined protein concentrations were cross-referenced with the absorbance at 280 nm using a NanoDrop Microvolume Spectrophotometer (Thermo Fisher Scientific). Western blotting was performed as described previously using guinea pig hyperimmune sera against norovirus VLPs containing the S domain. The secondary antibody used was a fluorescence-labeled donkey antibody against guinea pig IgG (IRdye 680RD, LI-COR Biosciences). Specific signals for the S-Ll and S-Jl proteins were documented using an Odyssey CLx imager (LI-COR Biosciences).

[0090] Transmission electron microscopy (TEM). The formation of PVNPs by the S-Ll or S-Jl fusion protein was examined using TEM, as described previously. The purified S-Ll and S- J1 proteins were applied to grids (TED PELLA, Inc.) and stained with 1% ammonium molybdate. The air-dried grids were observed using an EM10 C2 electron microscope (Zeiss, Germany) at 80 kV with magnifications ranging from 15,000* to 40,000*.

[0091] Dynamic light scattering (DLS). DLS was used to measure the size distribution of the S-Ll and S-Jl PVNPs following previously described methods. In brief, the purified S-Ll and S- J1 proteins (200 pL each) were loaded into separate wells of a 96-well microplate with a clear bottom (Greiner Bio-One) and analyzed using a DynaPro Plate Reader III DLS instrument (Wyatt Technology). The recorded data were then analyzed using the software DYNAMICS (Wyatt Technology).

[0092] Structural modeling of the S-Ll PVNPs. UCSF ChimeraX software (version 1.4) (48) was used to construct 3-dimensional (3D) structural models of S-Ll PVNPs utilizing the cryoEM (cryogenic electron microscopy) density map of the S60-VP8* nanoparticles as a template. The crystal structures of the inner shells of the 60-valent VLPs of feline calicivirus (FCV) (PDB code: 4PB6) or GII.4 NoV, and the LI protein of vaccinia virus (PDB code: lypy) (22) were incorporated into the corresponding regions of the template. UCSF ChimeraX software was also used for structural analysis of the S-Ll model and for creation of the final images.

[0093] Mouse immunization for Ll / Jl-specific antibody response. Forty pathogen-free BALB / c mice aged ~6 weeks were randomly divided into five groups of 8 mice each (n = 8 mice / group). The mice in each group were immunized with one of the following immunogens at 10 pg / mouse three times at two-week intervals: 1) S-Ll PVNP (S-Ll); 2) S-Jl PVNP (S-Jl); 3) free LI protein (LI); 4) free II protein (JI); and 5) S60 nanoparticle (S) as a negative control. All immunogens were adjuvanted with aluminum salt (Imject Alum, Thermo Fisher Scientific) at 25 pl / dose (20 pg / mouse / dose). Immunogens were administered intramuscularly (IM) in the thigh muscle three times in an 80 pl volume. Small amounts of blood samples were taken two weeks after the second immunization through the tail vein, while blood was collected two weeks after the third immunization via heart puncture. Sera were prepared from blood samples through an established protocol. All mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were maintained at the Division of Veterinary Services of Cincinnati Children’s Hospital Medical Center (CCHMC) during the experiments.

[0094] Determination of serum antibody titers specific to LI or JI antigen. LI- and Jl- specific IgG and IgA titers were measured using an enzyme immunoassay (EIA). Briefly, 96-well microtiter plates were coated with bacterially expressed and purified LI or JI protein at aconcentration of 5 pg / mL as capture antigens. After blocking with 5% nonfat milk, mouse sera were serially diluted and incubated with the coated antigens. The detection of bound IgG was performed using a goat-anti -mouse IgG-HRP (horseradish peroxidase) conjugate (1 :5000, MP Biomedicals) for Ll / Jl -specific IgG. For Ll / Jl-specific IgA, a goat-anti-mouse IgA-HRP conjugate (1 :2000, Invitrogen) was used. The Ll / Jl-specific IgG / IgA titers were defined as the maximum dilutions of sera that produced positive signals (OD450 > 0.2).

[0095] Serum neutralizing antibody titers against vaccinia virus replication. These titers were determined by the plaque reduction neutralization test (PRNT) as described in Kaever T, et al., Potent neutralization of vaccinia virus by divergent murine antibodies targeting a common site of vulnerability in LI protein. J Virol. 2014;88(19): 11339-55. Epub 2014 / 07 / 18. doi: 10.1128 / JVI.01491-14. PubMed PMID: 25031354; PMCID: PMC4178804. Briefly, Vero E6 cells were grown in 6-well culture plates until confluence. Vaccinia virus Western Reserve (WR) strain (kindly provided by Dr. Koi chi Araki at Cincinnati Children’s Hospital Medical Center) at -150 PFU in Dulbecco's modified Eagle's medium (DMEM) was treated with 2-fold serially diluted mouse serum for 30 min at 37° C in a total volume of 0.5 mL. The virus / serum mixture was subsequently added to the cells. Wells with cells and vaccinia virus without serum treatment served as positive controls, while wells with cells and medium only without virus / serum served as negative controls. After the plates were shaken gently for 1.5 h at 37°C, the virus / serum mixture was removed, and the cells were overlaid with 2 mL of DMEM containing 2% FBS. After 4 days of incubation at 37°C, the cells were fixed with 3.7% formaldehyde and stained with a 0.5% crystal violet solution for plaque counting. Serum neutralizing titers were defined as the maximum dilutions of the sera that showed at least 50% plaque reduction compared with the positive control wells.

[0096] Mouse vaccination and vaccinia virus challenge. Twenty -four female BALB / c mice at 6 weeks of age were randomly divided into three groups (n=8 mice / group). The mice in each group were immunized intramuscularly with one of the following immunogens at 10 pg / mouse three times at two-week intervals: 1) S-Ll PVNP (S-Ll), 2) S-Jl PVNP (S-Jl), or 3) S60 nanoparticle (S). Two weeks after the third immunization, mouse blood samples were collected via the tail vein for Ll / Jl-specific antibody titer determination. Sixteen days after the third immunization, the mice were intranasally challenged with 4 times the median lethal dose(equivalent to 1.6* 105PFU) of vaccinia virus (WR strain). The weight changes, disease progression, and survival rates of the challenged mice were monitored daily for 14 days. As required by IACUC guidelines, the challenged mice were euthanized when they lost more than 25% of their original body weight. The experiment was terminated after 14 days.

[0097] Statistical analysis. Statistical differences between two data groups were calculated by GraphPad Prism 9.0 (GraphPad Software, Inc.) via an unpaired t test. Differences in survival curves were analyzed employing the log-rank (Mantel-Cox) test (chi-square test). The statistical significance of the differences was determined as follows: nonsignificant (marked as “ns”) for P values > 0.05, significant (marked as “*”) for p values < 0.05, highly significant (marked as “**”) for P values < 0.01, and extremely significant (marked as “***”) for P values < 0.001, or (marked as “****”) for p values < 0.0001.Results

[0098] Generation and characterization of the S-Ll and S-Jl fusion proteins. C- terminally Hisx6-tagged, soluble S-Ll and S-Jl proteins (FIG. 1, A) were produced using the mammalian CHO cell expression system and purified with Cobalt resin at a yield of -20 mg / 100 mL of CHO cell culture for the S-Ll protein and -10 mg / 100 mL of CHO cell culture for the S-Jl protein. SDS-PAGE analysis of the two purified proteins revealed a single major band each at a molecular weight (MW) of -52 kDa (for S-Ll) and ~45 kDa (for S-Jl) (FIG. 1, B), which were larger than their calculated MWs of -44.4 (for S-Ll) and 41.7 (for S-Ll) kDa, respectively. To prove the identities of the two proteins, a Western blot analysis was performed using a previously prepared antibody against norovirus VLPs that contain the S domain protein. The specific signals to the purified S-Ll and S-Jl proteins (FIG. 1, C) verified their identities. The observed mobility shifts of the two proteins in the SDS-PAGE analysis may represent their posttranslational modifications, including the known myristoylation of the LI protein. The smeared signals around the two major bands (FIG. 1, B and C) may represent protein molecules with more or fewer modifications than the majority of the proteins.

[0099] Applicant also prepared the free LI and JI proteins without the S domain using the bacterial expression system at yields of 15 to 20 mg / L of bacterial culture. SDS-PAGE showed that the MWs were as expected at 19.5 (LI) and 17.3 (JI) kDa, respectively (FIG. 1, D). Since theE. coli system lacks many posttranslational modifications, the less smear bands corresponding to the Ll / Jl proteins on the SDS-PAGE gel further support that the mammalian cell-expressed S-Ll and S-Jl proteins retained their posttranslational modifications.

[0100] Self-assembly of the S-Ll and S-Jl PVNPs. Inspection of the purified S-Ll and S-Jl protein samples via TEM after negative staining revealed numerous nanoparticles, the majority of which were approximately 26 nm in diameter. This indicated that the S-Ll and S-Jl proteins selfassembled into PVNPs (FIG. 1, E and F), apparently due to the tendency of the norovirus S domain to form shell-like nanoparticles. It was observed that some of the PVNPs were larger or smaller than 26 nm. Norovirus VLPs are known to be organized in T=l, T=3, and / or T=4 icosahedral symmetry with different VLP sizes that are composed of 60, 180, and 240 VP1 proteins, respectively. Thus, the larger PVNPs may represent those with T=3 and T=4 icosahedral symmetries, comprising 180 and 240 fusion proteins respectively, while the symmetries of those smaller ones remain to be determined. The size distributions of the PVNPs were also analyzed by DLS, revealing that the particle sizes of the S-Ll PVNPs ranged from 10 to 40 nm, while those of the S-Jl PVNPs ranged from 7 to 40 nm (FIG. 1, G and H), consistent with the particle sizes observed by TEM.

[0101] 3D structural modeling of the S60-L1 PVNP. The major population of the S-Ll and S-Jl PVNPs, ~26 nm in diameter, resembled that of the S60-VP8* PVNPs, whose structure with T-l icosahedral symmetry has been elucidated by cryoEM. This strongly suggested that the majority of the S-Ll and S-Jl PVNPs shared the same T=1 icosahedral symmetry and consisted of 60 S-L1 / S-J1 proteins. Since the crystal structure of the LI protein has been solved (22), Applicant utilized the cryoEM structure of S60-VP8* PVNP as a template to generate a 3D structural model of the S60-L1 PVNP for a deeper understanding of its structural features. Specifically, the S60-L1 PVNP model was created (FIG. 2) using UCSF ChimeraX software by docking the N-terminal ends of the 60 LI structures (PDB code: lypy) to the 60 exposed C- terminal ends of the S domains of the S60 nanoparticle (FIG. 2, A) based on the cryoEM density map of the S60-VP8* PVNP.

[0102] Based on this model, images representing the full and sectional views of the S60-L1 PVNP in the surface representations (FIG. 2, A to C), cartoon representations (FIG. 2, D to F), andtransparent surface models showing the cartoon representations (FIG. 2, G to I), were generated. These image analyses indicated that 60 LI proteins stand upright on the surface of the S60 nanoparticle, forming the S60-L1 PVNP. The previously identified conformational neutralizing epitope centering at ASP35 (D35) is located at the distal end of each LI protein (FIG. 2, J). This model also indicates that the PVNPs appear differently at various viewing angles. While it is round-shaped at the five-fold axis (FIG. 2, B, C, E, F, H, and I), it is more angular-shaped with a round corner (FIG. 2, K) at the two-fold axis and a pentagon with the top corner snipped (FIG. 2, L) at the three-fold axis. This difference may explain the difference in the morphologies of the PVNPs observed in the TEM micrographs (FIG. 1, E and F). A 3D structural model of the S-Jl PVNPs was not generated due to the lack of the J 1 structure.

[0103] Enhanced immune responses to the PVNP-displayed LI or JI antigens. To demonstrate the enhanced immune responses against the presented LI and JI antigens by polyvalent PVNPs, BALB / c mice were immunized with the S-Ll and S-Jl PVNPs, with free LI and JI proteins used as controls for comparison. EIA assays using the bacterially expressed and purified LI or JI protein as capture antigens revealed that after two or three immunizations, the S- L1 and S-J 1 PVNPs induced significantly greater LI- or J 1-specific IgG titers in mice compared to those elicited by the free LI or JI proteins, respectively (Ps<0.05; FIG. 3, A and B). Specifically, after three immunizations, the Ll-specific titer elicited by the S-Ll PVNPs reached 31,468, and the Jl -specific titer induced by the S-Jl PVNPs was 31,400, representing 5.3-fold and 3.8-fold increases, respectively, compared to those induced by the free LI and JI proteins, respectively. Serum IgA responses were also determined by EIAs, revealing a similar enhancement in LI- or Jl-specific IgA responses by the S-Ll or S-Jl PVNPs compared to those elicited by the free LI or JI proteins. As a negative control, S60 nanoparticles did not elicit detectable LI- or Jl-specific IgG or IgA titers. These data demonstrated that the S-Ll and S-Jl PVNPs substantially improved the antibody responses toward the displayed LI and JI antigens, respectively.

[0104] High neutralizing antibody titers of the S-Ll PVNP-immunized mouse sera. A plaque reduction neutralization test (PRNT) was used to determine the neutralizing antibody titers of the mouse sera after two or three immunizations with various immunogens (see above) against replications of the vaccinia virus WR strain in cell culture. The results (FIG. 4) demonstrated that the sera obtained from mice immunized with the S-Ll PVNPs exhibited high neutralizing antibodytiters, reaching 250 after two immunizations or 2400 after three immunizations. These neutralizing antibody titers were 45 folds higher than that of the mouse sera after three immunizations with the S-Jl PVNPs, although the two groups of mouse sera exhibited similar high IgG and IgA titers (FIG. 3). The neutralizing antibody titers induced by the S-Ll PVNPs were significantly greater than those elicited by the free LI antigen, confirming the enhanced immunogenicity of the LI antigen induced by the polyvalent PVNPs. On the other hand, the low neutralizing antibody titers induced by the S-Jl PVNPs and the free JI antigen indicate that the JI protein is not an effective neutralizing antigen. As expected, the mouse sera after immunization with the S60 nanoparticles did not show detectable neutralization (FIG. 4).

[0105] Protection of the S-Ll PVNPs against body weight loss and mortality caused by vaccinia virus challenge. Next, Applicant determined the protective efficacy of the PVNP vaccines. After three immunizations, both the S-Ll PVNPs and the S-Jl PVNPs induced high Ll / Jl -specific serum IgG titers (FIG. 5, A), among which the S-J 1 PVNPs elicited an even greater J 1 IgG titer than the LI IgG titer induced by the S-Ll PVNPs. After the vaccinia virus (WR strain) challenge, the mice vaccinated with the S60 nanoparticles began to die on day 6 post challenge (DPC 6), and all the mice died after 10 DPC (FIG. 5, B, green line). In contrast, all the mice immunized with the S-Ll PVNPs survived the viral challenge, indicating 100% protection against mortality caused by the vaccinia virus challenge (FIG. 5, B, blue line). However, the protection of the mice vaccinated with the S-Jl PVNPs was low at 25%, as only two out of the eight mice survived the viral challenge (FIG. 5, B, red line).

[0106] Accordingly, mouse body weight change curves indicated that the mice immunized with the S60 nanoparticles began to lose weight on DPC 3 and did not recover (FIG. 5, C, green line). In contrast, the mice immunized with the S-Ll PVNPs started losing weight on DPC 5, reached a maximal weight loss of 13.1% on DPC 7, and then steadily regained weight afterward (FIG. 5, C, blue line). The mice immunized with the S-Jl PVNPs began losing weight on DPC 3, reaching a maximal weight loss on DP7. Subsequently, this mouse group experienced a gradual increase in average body weight (FIG. 5, C, red line), which was partially attributed to the removal of deceased or euthanized animals that reached the 25% weight loss threshold. Taken together, Applicant’s study demonstrated that LI antigen is an excellent vaccine target that is superior tothe JI antigen, and S-Ll PVNPs are a promising vaccine candidate for both vaccinia virus and mpox virus.Discussion

[0107] In this study, Applicant designed and generated two PVNPs displaying the surface antigens LI and a membrane-associated protein JI of vaccinia or mpox virus. Applicant then evaluated the biochemical, biophysical, immune responses, and protective immunity of these PVNPs, with the aim to develop a useful, nanoparticle-based mpox vaccine. The two PVNPs selfassembled from the corresponding fusion protein, S-Ll or S-Jl, which were produced at high yields through the use of a mammalian CHO cell expression system. Like other known S60 nanoparticle-based PVNPs constructed previously, the S-Ll and S-Jl PVNPs are expected to be composed of an inner shell formed by the S domains, resembling the interior layer of the norovirus capsid, and multiple LI or JI antigens displayed on the surface. The polyvalent PVNPs substantially increased the immune responses toward the displayed LI or JI antigen, eliciting significantly greater Ll / Jl-specific antibody titers than those induced by the free Ll / Jl antigens. Further investigation of mouse sera after two or three immunizations with the S-Ll PVNPs revealed significantly greater serum neutralizing antibody titers against replication of the vaccinia virus in cell culture compared with those of mouse sera after immunization with the S-Jl PVNPs. Consequently, the S-Ll PVNPs provided mice with 100% protection against mortality caused by the vaccinia virus challenge. These data support the conclusion that the readily available S-Ll PVNPs are a promising mpox vaccine candidate.

[0108] The significantly higher immune response toward the Ll / Jl antigens on the S-L1 / S-J1 PVNPs, compared to that induced by the free Ll / Jl antigens, may be attributed to the polyvalent presentation of the Ll / Jl antigens by the PVNPs. It was noted that both LI and JI were immunogenic, either as free antigens or on PVNPs. However, the high neutralizing antibody titers (up to 2,400) induced by the S-Ll PVNPs and the high protective efficacy (100%) conferred by the S-Ll PVNPs, compared to the low neutralizing antibody titers (up to 53.5) and low protective efficacy (25%) conferred by the S-Jl PVNPs, indicate that the LI protein is an excellent vaccine target for mpox vaccine development, while the JI protein is not. LI is known as an important surface protein in the life cycle of an orthopoxvirus that plays important roles in the early steps ofviral infection, including viral entry through viral-host cell membrane fusion. By binding to the LI protein, LI -specific antibodies can block the functions of the LI protein, thus inhibiting viral infection. Consequently, the LI antigen has been incorporated into several vaccine candidates against orthopoxviruses in the form of DNA vaccines or recombinant protein-based vaccines. A further study mapped the binding region of a neutralizing antibody to a conformational epitope, with Asp35 identified as the key residue (FIG. 2, J). The study also showed that this epitope is a common site of vulnerability for potent neutralization by a divergent group of antibodies. On the other hand, JI is known for its roles in virion morphogenesis and plaque formation, and these roles may not be required for viral infection.

[0109] In addition to the LI antigen, three additional virion surface proteins of vaccinia virus, namely A27 of IMV, as well as A33 and B5 of EEV, have also been demonstrated to be good vaccine targets against orthopoxviruses, see, e.g., Buchman GW et al., A protein-based smallpox vaccine protects non-human primates from a lethal monkeypox virus challenge. Vaccine. 2010;28(40):6627-36. Epub 2010 / 07 / 28. doi: 10.1016 / j.vaccine.2010.07.030. PubMed PMID: 20659519; PMCID: PMC2939220, Fogg CN et al., Adjuvant-enhanced antibody responses to recombinant proteins correlates with protection of mice and monkeys to orthopoxvirus challenges. Vaccine. 2007;25(15):2787-99. Epub 2007 / 01 / 19. doi: 10.1016 / j.vaccine.2006.12.037. PubMed PMID: 17229505; PMCID: PMC1952660, Hooper JW et al., Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox. J Virol. 2004;78(9):4433-43. Epub 2004 / 04 / 14. doi: 10.1128 / jvi.78.9.4433-4443.2004. PubMed PMID: 15078924; PMCID: PMC387704, and Xiao Y et al, A protein-based smallpox vaccine protects mice from vaccinia and ectromelia virus challenges when given as a prime and single boost. Vaccine. 2007;25(7): 1214-24. Epub 2006 / 11 / 14. doi: 10.1016 / j. vaccine.2006.10.009. PubMed PMID: 17098336; PMCID: PMC 1857298. This is evidenced by several multicomponent DNA and recombinant protein vaccine candidates, revealing high neutralization and protection. To further enhance the protective efficacy of the nanoparticle mpox vaccine candidates described herein, additional PVNPs that display the A27, A33, or B5 protein can be combined with the readily available S-Ll PVNPs as a cocktail or multivalent vaccine candidate.

[0110] Molecular weight and valence are two factors affecting the immunogenicity of an antigen. Thus, the relatively low IgG and IgA responses induced by the free LI and JI proteins,compared with their S-L1 / S-J1 PVNP counterparts, are most likely attributed to their single valence and small molecular weights (<20 kDa), in contrast to the polyvalence of the PVNPs with a much greater molecular weight (>2 MDa), highlighting the advantage of the S-Ll PVNP as a vaccine candidate. Applicant observed that the neutralizing antibody titers induced by the free LI antigen were very low. This difference may also be attributed to possible misfolding and / or the lack of posttranslational modifications, such as myristoylation, in the E. coli- xo xcQ recombinant LI protein, as myristoylation is known to occur in the authentic LI protein. CHO cell-expressed proteins can undergo glycosylation, which might explain the smeared protein bands observed on the SDS-PAGE and western blot. However, because poxvirus proteins do not undergo the typical secretory trafficking that eukaryotic membrane proteins do, glycosylation should not occur on vaccinia virus-expressed LI protein. In general, a eukaryotic expression system capable of providing better folding environment and post-translational modifications to the target protein may be required to produce S-Ll PVNPs. It is noted that despite 100% protection against mortality caused by the challenge with vaccinia virus, the S-Ll PVNP vaccinated mice underwent up to 12% weight loss on DPC 7, indicating that the vaccinated mice are infected by the challenged vaccinia virus. A tri- or quadrivalent PVNP vaccine covering additional A27, A33, and B5 antigens that have also been shown to be good vaccine targets may be used.Conclusion

[0111] Using the norovirus S nanoparticle as a platform, Applicant generated two pseudovirus nanoparticles (PVNPs) that display the LI antigen of the vaccinia virus and the JI antigen of the mpox virus, respectively, and evaluated them as an mpox vaccine candidate. Applicant’s results demonstrated that the S-Ll PVNPs elicited high neutralizing antibody titers in mice against the vaccinia virus and provided 100% protection against mortality caused by vaccinia virus challenge. In contrast, the S-J 1 PVNPs induced low neutralizing antibody titers and conferred weak protective immunity in mice. Thus, the readily available S-Ll PVNPs are a promising mpox vaccine candidate worthy of further development.

[0112] All percentages and ratios are calculated by weight unless otherwise indicated.

[0113] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0114] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0115] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[0116] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNTPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

[0117] Exemplary Combinations

[0118] The following examples relate to various non-exhaustive ways in which the teachings herein may be combined or applied. The following examples are not intended to restrict the coverage of any claims that may be presented at any time in this application or in subsequent filings of this application. No disclaimer is intended. The following examples are being provided for nothing more than merely illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the below examples. Therefore, none of the aspects or features referred to below should be deemed critical unless otherwise explicitly indicated as such at a later date by the inventors or by a successor in interest to the inventors. If any claims are presented in this application or in subsequent filings related to this application that include additional features beyond those referred to below, those additional features shall not be presumed to have been added for any reason relating to patentability.

[0119] Example 1

[0120] An mpox vaccine composition comprising a non-replicating pseudovirus nanoparticle (PVNP), the PVNP displaying an LI antigen.

[0121] Example 2

[0122] The mpox vaccine of example 1, wherein the PVNP comprises an icosahedral particle comprising a norovirus shell domain protein (“S domain protein”).

[0123] Example 3

[0124] The mpox vaccine of example 2, the S domain protein comprising 60 S domain proteins.

[0125] Example 4

[0126] The mpox vaccine of example 2, the S domain protein comprising a sequence having at least 90% sequence identity to SEQ ID NO: 1.

[0127] Example 5

[0128] The mpox vaccine of example 2, the S domain protein comprising a sequence having at least 95% sequence identity to SEQ ID NO: 1, wherein the S domain comprises the mutation R69A.

[0129] Example 6

[0130] The mpox vaccine of example 2, the S domain protein comprising a sequence having at least 99% sequence identity to SEQ ID NO: 1, wherein the S domain comprises the mutation R69A.

[0131] Example 7

[0132] The mpox vaccine of example 2, the S domain protein comprising SEQ ID NO: 1.

[0133] Example 8

[0134] The mpox vaccine of example 1, wherein the mpox vaccine elicits LI specific IgG and / or IgA specific antibodies in an individual administered the mpox vaccine.

[0135] Example 9

[0136] The mpox vaccine of example 1, the mpox vaccine being produced via a prokaryotic system.

[0137] Example 10

[0138] The mpox vaccine of example 1, the mpox vaccine being produced via a eukaryotic system.

[0139] Example 11

[0140] A pseudovirus nanoparticle (PVNP) comprising, consisting of, or consisting essentially of an aggregate of fusion proteins, the fusion proteins forming an icosahedral structure and a nanoparticle shell, the fusion protein comprising a) a secretory signal peptide;b) a modified norovirus S domain protein (“S domain protein”), the S domain protein forming an interior nanoparticle shell of the PVNP composition; c) an LI antigen from mpox virus; and d) a peptide linker, the peptide linker connecting the C-terminus of the S domain protein to the LI antigen and the S domain protein; wherein: the inner nanoparticle shell displays 60 LI antigens on a surface of the nanoparticle shell via exposed S domain C-termini.

[0141] Example 12

[0142] The PVNP composition of example 11, wherein the secretory signal peptide has at least about 80%, or at least about 90%, or at least about 95%, or 100% sequence identity to MKWVTFISLLFLFSSAYS (SEQ ID NO: 4).

[0143] Example 13

[0144] The PVNP composition of example 11, the S domain comprising or consisting of SEQ ID NO: 1.

[0145] Example 14

[0146] The PVNP composition of example 11, wherein the LI antigen has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 2.

[0147] Example 15

[0148] The PVNP composition of example 11, the peptide linker comprising or consisting of SEQ ID NO: 11.

[0149] Example 16

[0150] The PVNP composition of example 11, the LI antigen comprising Asp35.

[0151] Example 17

[0152] The PVNP composition of example 11, the LI antigen comprising a His tag fused to the C-terminus of the S-Ll antigen.

[0153] Example 18

[0154] The PVNP composition of example 11, the LI antigen comprising a His tag fused to the C-terminus of the S domain protein, the His tag comprising SEQ ID NO: 13.

[0155] Example 19

[0156] The PVNP composition of example 1 Iwherein the NoV S domain has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identity to SEQ ID NO: 1.

[0157] Example 20

[0158] The PVNP composition of example 19, wherein the S domain protein is wildtype at one or more of positions V57, Q58, or S136, and M140.

[0159] Example 21

[0160] The PVNP composition of example 19, wherein the S domain protein is wildtype at least two of positions V57, Q58, or SI 36, and M140.

[0161] Example 22

[0162] The PVNP composition of example 19, wherein the S domain protein is wildtype at least three of positions V57, Q58, or S 136, and M140.

[0163] Example 23

[0164] The PVNP composition of example 19, wherein the S domain protein is wildtype at each of positions V57, Q58, or S136, and M140.

[0165] Example 24

[0166] The PVNP composition of example 11, wherein the PVNP composition elicits a specific IgG and IgA titer against the LI antigen in an individual administered the PVNP composition.

[0167] Example 25

[0168] A method of eliciting an immune response against an mpox virus in an individual in need thereof, comprising administering the composition of example 1 in an amount effective to produce an antigen-specific immune response in the individual.

[0169] Example 26

[0170] An S-Ll fusion protein comprising a sequence having at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 5.

[0171] Example 27

[0172] A cell system for producing a PVNP composition comprising Chinese Hamster Ovary (CHO) cells capable of expressing a fusion protein comprising a modified S domain protein and an LI antigen.

[0173] Example 28

[0174] The cell system of example 27, said fusion protein comprising MKWVTFISLLFLF S S AYSKMASND ASP SDGST ANLVPEVNNEVMALEP VVGAAIAAP VA GQQNVIDPWIRNNFVQAPGGEFTVSPANAPGEILWSAPLGPDLNPYLSHLARMYNGYA GGFEVQVILAGNAFTAGKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDV RNNFYHYNQSNDSTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVE GGGGMGAAASIQTTVNTL SERIS SKLEQEANAS AQTKCDIEIGNF YIRQNHGCNLTVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVK QTCNS S AVVDNKLKIQNVIIDEC YGAPGSPTNLEFINTGS SKGNC AIKALMQLTTKATTQI APKQVAGTGVHHHHHH (SEQ ID NO: 5).

Claims

CLAIMSWhat is claimed is:

1. An mpox vaccine composition comprising a non-replicating pseudovirus nanoparticle (PVNP), the PVNP displaying an LI antigen.

2. The mpox vaccine of claim 1, wherein the PVNP comprises an icosahedral particle comprising a norovirus shell domain protein (“S domain protein”).

3. The mpox vaccine of claim 2, the S domain protein comprising 60 S domain proteins.

4. The mpox vaccine of claim 2, the S domain protein comprising a sequence having at least 90% sequence identity to SEQ ID NO: 1.

5. The mpox vaccine of claim 2, the S domain protein comprising a sequence having at least 95% sequence identity to SEQ ID NO: 1, wherein the S domain protein comprises a R69A mutation.

6. The mpox vaccine of claim 2, the S domain protein comprising a sequence having at least 99% sequence identity to SEQ ID NO: 1, wherein the S domain protein comprises a R69A mutation.

7. The mpox vaccine of claim 2, the S domain protein comprising SEQ ID NO: 1.

8. The mpox vaccine of claim 1, wherein the mpox vaccine elicits LI specific IgG and / or IgA specific antibodies in an individual administered the mpox vaccine.

9. The mpox vaccine of claim 1, the mpox vaccine being produced via a prokaryotic system.

10. The mpox vaccine of claim 1, the mpox vaccine being produced via a eukaryotic system.

11. A pseudovirus nanoparticle (PVNP) comprising, consisting of, or consisting essentially of an aggregate of fusion proteins, the fusion proteins forming an icosahedral structure and a nanoparticle shell, the fusion protein comprising a) a secretory signal peptide;b) a modified norovirus S domain protein (“S domain protein”), the S domain protein forming an interior nanoparticle shell of the PVNP; c) an LI antigen from mpox virus; and d) a peptide linker, the peptide linker connecting the C-terminus of the S domain protein to the LI antigen and the S domain protein; wherein: an inner nanoparticle shell displays 60 LI antigens on a surface of the nanoparticle shell via exposed S domain C-termini.

12. The PVNP composition of claim 11, wherein the secretory signal peptide has at least about 80%, or at least about 90%, or at least about 95%, or 100% sequence identity to MKWVTFISLLFLFSSAYS (SEQ ID NO: 4).

13. The PVNP composition of claim 11, the S domain comprising or consisting of SEQ ID NO: 1.

14. The PVNP composition of claim 11, wherein the LI antigen has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 2.

15. The PVNP composition of claim 11, the peptide linker comprising or consisting of SEQ ID NO: 11.

16. The PVNP composition of claim 11, the LI antigen comprising Asp35.

17. The PVNP composition of claim 11, the LI antigen comprising a His tag fused to the C- terminus of an S-L l antigen.

18. The PVNP composition of claim 11, the LI antigen comprising a His tag fused to the C- terminus of the S domain protein, the His tag comprising SEQ ID NO: 13.

19. The PVNP composition of claim 11 , wherein the NoV S domain has at least about 90%, or at least about 91%, or at least about 92% or at least about 93%, or at least about 94%, or at least about 95% or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identity to SEQ ID NO: 1.

20. The PVNP composition of claim 19, wherein the S domain protein is wildtype at one or more of positions V57, Q58, or S136, and M140.

21. The PVNP composition of claim 19, wherein the S domain protein is wildtype at least two of positions V57, Q58, or S136, and M140.

22. The PVNP composition of claim 19, wherein the S domain protein is wildtype at least three of positions V57, Q58, or S136, and M140.

23. The PVNP composition of claim 19, wherein the S domain protein is wildtype at each of positions V57, Q58, or S136, and M140.

24. The PVNP composition of claim 11, wherein the PVNP composition elicits a specific IgG and IgA titer against the LI antigen in an individual administered the PVNP composition.

25. A method of eliciting an immune response against an mpox virus in an individual in need thereof, comprising administering the mpox vaccine composition of claim 1 in an amount effective to produce an antigen-specific immune response in the individual.

26. An S-Ll fusion protein comprising a sequence having at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to SEQ ID NO: 5.

27. A cell system for producing a PVNP composition comprising Chinese Hamster Ovary (CHO) cells capable of expressing a fusion protein comprising a modified S domain protein and an LI antigen.

28. The cell system of claim 27, said fusion protein comprising SEQ ID NO: 5.

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