Single-dose MVA vaccines for protection against orthopoxvirus

Abstract

Provided herein are methods for preventing an mpox virus infection, reducing the effects of an mpox virus infection, or inducing a protective immune response against an mpox virus in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising a modified vaccinia Ankara viral vector encoding programmed cell death protein 1 (PD-1) inhibitory peptide LD10 (MVA-LD10).

Description

[0001] SINGLE-DOSE MV A VACCINES FOR PROTECTION AGAINST ORTHOPOXVIRUS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of U.S. Provisional Application No. 63 / 707,632, filed October 15, 2024. The entirety of each of these applications is hereby incorporated by reference herein for all purposes.

[0004] FIELD OF THE INVENTION

[0005] The invention provides methods of effectively vaccinating a subject, such as a human, against disease caused by orthopoxviruses, including, but not limited to, Monkeypox (mpox) virus, smallpox (variola) virus, borealpox virus, and vaccinia virus (VV), by administering a single dose of a modified vaccinia Ankara (MV A) viral vector and an immune checkpoint inhibitor, for example, a modified vaccinia Ankara (MVA)-LDIO vector encoding the LD10 immune- checkpoint inhibitor peptide in a multimeric conformation, wherein the LD10 immune checkpoint inhibitor peptide is capable of being cleaved and secreted from the cell.

[0006] INCORPORATION BY REFERENCE

[0007] The Sequence Listing XML file named “19101-053W01 ST26” created on October 14, 2025, and having a size of 17,432 bytes, is hereby incorporated by reference.

[0008] BACKGROUND OF THE INVENTION

[0009] Poxviruses have historically caused significant human mortality and continue to pose a serious threat to human health. Currently, there is an ongoing global outbreak of both the Clade I and Clade II mpox virus. Notably, the Clade I mpox global outbreak represents the first ever case of Clade I mpox outside of endemic areas. The WHO recently declared mpox a priority pathogen and its spread a global emergency, with over 17,000 cases and more than 500 deaths reported in 13 countries (WHO News Release - WHO Director-General declares mpox outbreak a public health emergency of international concern. (2024 Aug 14)). The current mpox outbreaks are the largest caseload of orthopoxviruses since smallpox, and the evidence suggests that the measures currently employed are not sufficient to control the outbreak. Furthermore, there is the potential for continued transmission of mpox because of its animal hosts and the genetic potential of mpox to establish in new hosts globally (Tseng, K.K. et al. Viral genomic features predict orthopoxvirus reservoir hosts. bioRxiv. 10.26.564211(2023)). These issues underscore the importance of preparedness and rapid deployment of medical countermeasures.

[0010] In 2019, the United States Food and Drug Administration (FDA) approved the first live, non-replicating vaccine for smallpox and mpox, MVA-BN (Jynneos), a modified vaccina Ankara virus, administered in a 2-dose regimen with each dose administered 4 weeks apart (FDA News Release - FDA approves first live, non-replicating vaccine to prevent smallpox and monkeypox. 2019 Sep 24). MVA-BN, which is related to mpox as a orthopoxvirus, is expected to provide protection across multiple clades of mpox, although real-world effectiveness has only been demonstrated to Clade lib. Following the Clade I mpox outbreak in August 2024, the WHO recommended the non-replicating vaccine MVA-BN, a minimally replicating vaccine LC16m8, and a replicating vaccinia-based vaccine ACAM2000 for use only in immunocompetent nonpregnant individuals (WHO Weekly epidemiological record - Smallpox and mpox (orthopoxviruses) vaccine position paper. 99(34):429-456(2024 Aug 23)).

[0011] ACAM2000 and LC16m8 are both administered through the undesirable route of scarification, and the durability of effectiveness of these vaccines is unknown (Kenner, J. et al. LC16m8: an attenuated smallpox vaccine. Vaccine. 24(47-48):7009-7022(2006 Nov 17)). ACAM2000 is contraindicated for use in several groups including pregnant women. ACAM2000 has demonstrated an increased risk of severe adverse events including myopericarditis and cardiomyopathy (CDC Morbidity and Mortality Weekly Report - Use of JYNNEOS (Smallpox and Monkeypox Vaccine, Live, Nonreplicating) for Preexposure Vaccination of Persons at Risk for Occupational Exposure to Orthopoxviruses: Recommendations of the Advisory Committee on Immunization Practices — United States, 2022. 71(22):734-742(2022 Jun 3)). Accordingly, the widespread use of ACAM2000 is not ideal based on unacceptable safety profiles.

[0012] Findings regarding MVA-BN vaccine durability reveal that MVA-BN elicits a strong antibody response in participants that peaks around 2 weeks after the second dose is administered, and that total IgG and neutralizing antibody titers decline from their peak and return close to baseline levels by the 2-year mark (Priyamvada, L. et al. Serological responses to the MVA-based JYNNEOS monkeypox vaccine in a cohort of participants from the Democratic Republic of Congo. Vaccine. 40:7321-7327(2022)). Accordingly, the two-dose immunization series with MVA-BN (Jynneos) for mpox demonstrates an early decline in neutralizing antibody and breakthrough infections.

[0013] In supply-constrained outbreak situations, WHO recommends “off-label” use of a single dose or intradermal fractional dosing of MVA-BN (WHO Weekly epidemiological record (2024 Aug 23)). A single-dose administration of MVA-BN, however, has been shown to only provide partial protection against Clade lib mpox resulting in breakthrough infections (Deputy, N.P. et al. Vaccine Effectiveness of JYNNEOS against Mpox Disease in the United States. The New Engl J of Med. 388(26):2434-2443(2023 May 18); CDC - Estimated Effectiveness of JYNNEOS Vaccine in Preventing Mpox: AMultijurisdictional Case-Control Study — United States, August 19, 2022- March 31, 2023. Morbidity and Mortality Weekly Report (MMWR). 72(20):553-558(2023 May 19); CDC - Monkeypox Virus Infections After 2 Preexposure Doses of JYNNEOS Vaccine — United States, May 2022-May 2024. Morbidity and Mortality Weekly Report (MMWR). 73(20):460-466(2024 May 23; see also Allard et al., Breakthrough cases of mpox: One-dose vaccination is associated with milder clinical manifestations. Journal of Infection and Public Health 17 (2024); 676-680; Hillus et al., “Safety and effectiveness of MVA-BN vaccination against mpox in at-risk individuals in Germany (SEMVAc and TEMVAc): a combined prospective and retrospective cohort study. Lancet Infec. Dis. 2025; 25:775-87). Moreover, unlike previous generations of smallpox vaccines, MVA-BN induces low humoral immunogenicity and virus neutralization activity against orthopoxviruses, even after a two-dose prime-boost regimen (Cohn, H. et al. Mpox vaccine and infection-driven human immune signatures: an immunological analysis of an observational study. Lancet Infect Dis. 23(11): 1302-1312(2023 Nov)).

[0014] Vaccination noncompliance, especially regarding follow-up for booster doses, is influenced by myriad factors such as lack of access, inadequate knowledge about immunization, and objection to multiple dose administration (Ventola, C L. Immunization in the United States: Recommendations, Barriers, and Measures to Improve Compliance: Part 1 : Childhood Vaccinations. P T. 41(7):426-436(2016 Jul)). In rural, low-income countries, particularly those in Africa and Asia, there exist abundant geographic and economic barriers. Also, the quality of healthcare systems and numerous socioeconomic disparities in these countries often affect vaccine compliance, particularly for second doses. For instance, only 35.2% of Ethiopians and 39.9% South Africans have received at least two doses of the COVID-19 vaccine, highlighting the difficulties in achieving full vaccination compliance in these regions (Arsenault, C. et al. Health system quality and COVID-19 vaccination: a cross-sectional analysis in 14 countries. Lancet Glob Health. 12(l):el56-el65(2024 Jan)). As of January 31, 2023, the estimated MVA-BN vaccination coverage for individuals at risk for MP XV was 36.7% for the first dose and 22.7 % for two doses (CDC - JYNNEOS Vaccination Coverage Among Persons at Risk for Mpox — United States, May 22, 2022-January 31, 2023. Morbidity and Mortality Weekly Report (MMWR). 72( 13) :342- 347(2023 Mar 31)).

[0015] Further still, neutralizing antibody titers are proposed as a potential correlate of protection against lethal mpox in animal models (Edghill-Smith, Y. et al. Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus. Nat Med. 11(7):740-747(2005 Jul)). The effectiveness of antibodies binding to vaccinia virus may vary for mpox due to inconsistent cross recognition influenced by exposure history (Zaeck, L.M. et al. Low levels of monkeypox virus-neutralizing antibodies after MVA-BN vaccination in healthy individuals. Nat Med. 29(l):270-278(2023 Jan)), whereas a vaccine which promotes T-cell-based protection may offer improved protection based on more conserved epitopes. While data on neutralizing antibodies in mpox is limited, there is even less information on cellular immunity. T cells have the potential to offer broader, longer-lasting protection and are recognized to be less affected by antibody-evading mutations, as seen in other viral infections like smallpox.

[0016] Accordingly, improved orthopoxvirus vaccination strategies that provide safe and effective immunization are needed.

[0017] SUMMARY OF THE INVENTION

[0018] Provided herein are methods of preventing an orthopoxvirus infection, reducing the effects of an orthopoxvirus infection, and / or inducing a protective immune response against an orthopoxvirus, wherein the orthopoxvirus is an mpox virus, a smallpox virus, a borealpox virus, vaccinia virus, or other disease causing orthopoxvirus, in a subject, such as a human, comprising administering to the subject a modified vaccinia Ankara (MV A) viral vector and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody, antibody fragment, antigen binding fragment, or peptide, wherein the immune checkpoint inhibitor inhibits programmed-cell death protein 1 (PD-1), programed cell death ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In some embodiments, the immune checkpoint inhibitor is encoded by the MVA vector. In some embodiments, the MVA vector encoding a checkpoint inhibitor is MVA-LD10. In some embodiments, the MVA vector encoding a checkpoint inhibitor is MVA-LD01. In some embodiments, the MVA vector and immune checkpoint inhibitor are administered only once. In some embodiments, the subject has not been exposed to, or has no known exposure to, an orthopoxvirus, for example, an mpox virus, a smallpox virus, a borealpox virus, vaccinia virus, and / or other disease causing orthopoxvirus.

[0019] The use of an MVA vector in combination with an immune checkpoint inhibitor, for example, MVA-LD10, results in enhanced immunity to an orthopoxvirus via the development of a cellular immune response through an augmented CD4+ T-cell response to MVA (see FIG. 4A) and a significant increase in activated CD8+ T cells (see FIG. 4B) due, in significant part, to the expression of the PD-1 inhibitory peptide LD10 (see, e.g., Example 2). The induction of both a humoral and cellular immune response, as exemplified herein, results in a more robust, longer- lasting immune protection against vaccinia virus infection lethal challenges in mice. Furthermore, the induction of both a humoral and cellular immune response results in a single-dose administration of MVA-LD10 providing sufficiently protective immunity to vaccinia virus viral infection (see, e.g., FIG. 3C, 5D, and 7D). Accordingly, the use of a single-dose administration of MVA-LD10 to provide protective immunity to an orthopoxvirus addresses the challenges of the current standard two-dose vaccination series required by, for example, MVA-BN, while also achieving superior, longer-lasting immune protection against an orthopoxvirus infection, for example, an mpox infection.

[0020] MVA-LD10 is an engineered recombinant MVA encoding the PD-1 inhibitory peptide LD 10 with sequence additions to allow for a novel secreted 5 copy multimer design (see, e.g., SEQ ID NO: 6). Dot blot analysis of infected cell culture supernatant demonstrates efficient LD10 peptide secretion from a host cell following MVA vector infection (see, e.g., FIG. 2 of Example

[0021] 1). Without wishing to be bound to any particular theory, the remarkably rapid peptide activation of the T cell compartment to MVA-LD10 may be due to the inhibitory effects of LD10 on the rapid upregulation of PD-1 upon activation of naive CD8+ T cells (see, e.g., FIG. 4B of Example

[0022] 2). Modulation of PD-1 signaling occurs shortly after vaccination, and LD10 likely acts on the early activated T cells. As dendritic cells overexpressing PD-L1 are capable of promoting Treg generation in naive CD4+ T cells, PD-L1 blockade provided by the secretion of LD10 may result in reduced Treg expansion. Thus, it is possible that LD10 binds to the basal PD-1 on Tregs and thereby decreases expansion or inhibitory function, allowing for a greater number of vaccine- induced, MVA antigen-specific CD8+ T cells. Likewise, targeting conserved T cell epitopes on MVA is a strategy that offers a solution to the rapid evolution of mpox virus escape to neutralizing antibodies. Sole focus on neutralizing antibodies presents a new problem of the need to continually update vaccines to match highly mutable regions targeted by neutralizing antibodies.

[0023] As shown herein, the use of MVA-LD10 offers superior protection from lethal orthopoxvirus challenge compared to one and two doses of MVA alone and promotes enhanced cellular immunity (see, e.g., Examples 2-3; FIGS. 3C, 5D, and 7D). Following a single-dose immunization of MVA-LD10, durable protection from morbidity and mortality was also demonstrated with a delayed vaccinia virus challenge (see, e.g., Example 2, FIG. 3C). Analysis of post-vaccination, pre-challenge cellular immunity revealed a significant increase of functional antigen-specific CD8+ T cells 17 weeks following vaccination from single dose MVA-LD10 as compared to one dose of MVA (see, e.g., Example 2, FIG. 4B). Evaluation of neutralizing antibodies to vaccinia virus at late times post-vaccination reveals similar titers between one dose or two doses of MVA and one dose of MVA-LD10. This indicates a strong, durable T cell memory response formed from a single dose of MVA-LD10. Vaccine efficacy was determined using an established vaccinia virus strain WR challenge model. Mice were administered a lethal, intranasal dose 106PFU / ml VV at 17 weeks (day 150) (see, e.g., Example 2) or a higher intranasal dose of 107PFU / ml VV at the earlier time points of 4 weeks (day 56) and 9 weeks (day 90) (see, e.g., Example 3) post-vaccination. Strikingly, vaccination with a single dose of MVA-LD10 offered the greatest protection from weight loss and disease of all vaccine groups tested at each vaccinia virus challenge timepoint (see, e.g., Examples 2-3; FIGS. 3A-3C, 5A-5D, and 7A-7F)). At the late mpox challenge timepoint, over 17 weeks following vaccination, MVA-LD10 demonstrated the highest degree of protection from disease as observed by minimal weight loss and disease scores (see, e.g., Example 2, FIG. 3A-3C). The challenge timepoint of 4 weeks (day 56) post-vaccination revealed that one dose of MVA-LD10 offered complete protection from death similar to two doses of MVA, whereas by comparison, one dose of MVA alone was not fully protective (see, e.g., Example 3, FIG. 5D). Evaluation of viremia 3 days following the Day 56 challenge revealed a significant reduction in viral load in sera of mice immunized with MVA-LD10 as compared to MVA (see, e.g., Example 3, FIG. 6). Following day 90 challenge, MVA-LD10 offered a higher degree of protection from death and disease than two doses of MVA (see, e.g., Example 3, FIG. 7D). Likewise at day 90, the enhanced protection with MVA-LD10 single-dose is evident as no mice died following challenge compared to the MVA single-dose group where 4 of 6 mice succumbed to challenge. Together, this data demonstrates that MVA-LD10 offers superior protection from orthopoxvirus challenge than one or two doses of MVA and disease protection correlated with cellular and not humoral immunity.

[0024] In one aspect, provided herein is a method for inducing a protective immune response against an orthopoxvirus, including, but not limited to, an mpox virus, smallpox virus, borealpox virus, vaccinia virus, or other disease causing orthopoxvirus in a subject, such as a human, comprising administering to the subject an MVA vector and an immune checkpoint inhibitor. In some embodiments, the MVA vector and the immune checkpoint inhibitor are administered in a prophylactically therapeutically effective amount, wherein the MVA and immune checkpoint inhibitor are administered in a single dose. In some embodiments, the immune checkpoint inhibitor is an antibody, antibody fragment, antigen binding fragment, or peptide. In some embodiments, the immune checkpoint inhibitor inhibits programmed-cell death protein 1 (PD-1), programed cell death ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD- 1 inhibitor is the peptide LD10. In some embodiments, the immune checkpoint inhibitor is encoded by the MVA vector. In some embodiments, the MVA vector is MVA-LD10. In some embodiments, the PD-1 inhibitor is the peptide LD01. In some embodiments, the MVA vector is MVA-LD01. In some embodiments, the subject has not been exposed to, or has no known exposure to, an orthopoxvirus, for example, an mpox virus, a smallpox virus, a borealpox virus, vaccinia virus, and / or other disease causing orthopoxvirus.

[0025] In one aspect, provided herein is a method for reducing the effects of a orthopoxvirus, including, but not limited to, an mpox virus, smallpox virus, borealpox virus, vaccinia virus, or other disease causing orthopoxvirus in a subject, such as a human, comprising administering to the subject a an MVA vector and an immune checkpoint inhibitor. In some embodiments, the MVA vector and the immune checkpoint inhibitor are administered in a therapeutically effective amount, wherein the MVA vector and immune checkpoint inhibitor are administered in a single dose. In some embodiments, the immune checkpoint inhibitor is an antibody, antibody fragment, antigen binding fragment, or peptide. In some embodiments, the immune checkpoint inhibitor inhibits programmed-cell death protein 1 (PD-1), programed cell death ligand 1 (PD-L1), or cytotoxic T- lymphocyte-associated protein 4 (CTLA-4). In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is the peptide LD10. In some embodiments, the immune checkpoint inhibitor is encoded by the MVA vector. In some embodiments, the MVA vector is MVA-LD10. In some embodiments, the PD-1 inhibitor is the peptide LD01. In some embodiments, the MVA vector is MVA-LD01. In some embodiments, the subject has not been exposed to, or has no known exposure to, an orthopoxvirus, for example, an mpox virus, a smallpox virus, a borealpox virus, vaccinia virus, and / or other disease causing orthopoxvirus.

[0026] In one aspect, provided herein is a method of preventing an mpox virus infection in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising an MVA vector and an immune checkpoint inhibitor. In some embodiments, the pharmaceutical composition comprising the MVA and the immune checkpoint inhibitor are administered in a therapeutically effective amount in a single dose. In some embodiments, the immune checkpoint inhibitor is an antibody, antibody fragment, antigen binding fragment, or peptide. In some embodiments, the immune checkpoint inhibitor inhibits programmed-cell death protein 1 (PD-1), programed cell death ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is the peptide LD10. In one alternative embodiment, the PD-1 inhibitor is the peptide LD01. In some embodiments, the immune checkpoint inhibitor is encoded by the MVA vector. In some embodiments, the MVA vector is MVA-LD10. In some embodiments, the MVA vector is MVA-LD01. In some embodiments, the subject has not been exposed to, or has no known exposure to, an mpox virus.

[0027] Accordingly, in another aspect, provided herein is a method for inducing a protective immune response against an mpox virus in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising MVA-LD10. In some embodiments, the MVA-LD10 is administered in a therapeutically effective amount in a single dose. In some embodiments, the induced protective immunity is to mpox Clade I. Tn some embodiments, the induced protective immunity is to mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD10 is administered in a therapeutically effective amount in two doses during a prime boost regimen. In some embodiments, the subject has not been exposed to, or has no known exposure to, an mpox virus.

[0028] In one aspect, provided herein is a method of reducing the effects of an mpox virus infection in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising MVA-LD10. In some embodiments, the MVA-LD10 is administered in a therapeutically effective amount in a single dose. In some embodiments, the mpox virus is mpox Clade I. In some embodiments, the mpox virus is mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD10 is administered in a therapeutically effective amount in two doses during a prime boost regimen. In some embodiments, the subject has not been exposed to, or has no known exposure to, an mpox virus.

[0029] In one aspect, provided herein is a method of preventing an mpox virus infection in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising MVA-LD10. In some embodiments, the MVA-LD10 is administered in a therapeutically effective amount in a single dose. In some embodiments, the mpox virus is mpox Clade I. In some embodiments, the mpox virus is mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD10 is administered in a therapeutically effective amount in two doses during a prime boost regimen. In some embodiments, the subject has not been exposed to, or has no known exposure to, an mpox virus.

[0030] In an alternative aspect, provided herein is a method for inducing a protective immune response against an mpox virus in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising MVA-LD01. In some embodiments, the MVA- LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the induced protective immunity is to mpox Clade I. In some embodiments, the induced protective immunity is to mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen. In some embodiments, the subject has not been exposed to, or has no known exposure to, an mpox virus. In one alternative aspect, provided herein is a method of reducing the effects of an mpox virus infection in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising MVA-LD01. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the mpox virus is mpox Clade I. In some embodiments, the mpox virus is mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen. In some embodiments, the subject has not been exposed to, or has no known exposure to, an mpox virus.

[0031] In one alternative aspect, provided herein is a method of preventing an mpox virus infection in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising MVA-LD01. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the mpox virus is mpox Clade I. In some embodiments, the mpox virus is mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen. In some embodiments, the subject has not been exposed to, or has no known exposure to, an mpox virus.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is a schematic of the MVA-LD10 vector illustrating the design of nucleic acid sequences, which encode for the LD10 peptide, respectively, inserted into the MVA genome between two essential genes under control of an MVA specific promoter. The LD10 encoding nucleic acid sequence is preceded by a signal sequence routing the LD10 peptide for secretion and followed by a cleavage site to separate multimeric peptides. The secretion signal, peptide-encoding sequence, and cleavage site is repeated 5 times and then transcription is terminated with a stop codon.

[0034] FIG. 2 is a dot blot showing the expression of recombinant LD10 peptide in DF-1 cells infected with MVA-LD10. DF1 cells were infected with parental modified vaccinia Ankara (MVA, Space 6) or MVA encoding LD10 (MVA-LD10, Space 5). Uninfected cells (Space 7) and blank aliquots (Spaces 1, 2, 3, 8) were included as negative controls. Separately, twenty micrograms of LD10 peptide (Space 4) was also loaded onto the membrane as a positive control. The membrane was probed with LD10 antibody, demonstrating signal in the MVA-LD10 and LD10 peptide samples.

[0035] FIG. 3 A shows protection of MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 150. Weight changes in percent, as represented on the y-axis, were measured daily for 14 days post-challenge, as represented on the x-axis. Lines demonstrate median weight loss values for treatment groups ACAM2000 (Group B) MVA Prime (Group C), MVA Prime- Boost (Group D), and MVA-LD10 Prime (Group E), as compared to control (Group A). Error bars indicate standard deviation.

[0036] FIG. 3B shows improved Clinical Scores in MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 150. Lines demonstrate Clinical Score, as represented on the y-axis, over the days following challenge, as represented on the x-axis, for treatment groups ACAM2000 (Group B) MVA Prime (Group C), MVA Prime-Boost (Group D), and MVA-LD10 Prime (Group E), as compared to control (Group A). Clinical score is generated by combining parameters assessing changes in body temperature and visually inspected clinical symptoms such as ruffled fur, hunched posture, respiratory distress, and lethargy, with poorer clinical symptoms yielding higher clinical scores.

[0037] FIG. 3C shows improved survival of MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 150. Lines demonstrate survival probabilities, as represented on the y-axis, over the days following challenge, as represented on the x-axis, for treatment groups ACAM2000 (Group B) MVA Prime (Group C), MVA Prime-Boost (Group D), and MVA-LD10 Prime (Group E), as compared to control (Group A).

[0038] FIG. 4A shows superior MVA-LD10 vaccine cellular immunity administered at both a prime and prime-boost regimen. Immunized mice were evaluated for CD4+ T cell functionality by re-stimulation of splenocytes with MVA and intracellular cytokine staining followed by flow cytometry.

[0039] FIG. 4B shows superior MVA-LD10 vaccine cellular immunity administered at both a prime and prime-boost regimen. Immunized mice were evaluated for CD8+ T cell functionality by re-stimulation of splenocytes with MVA and intracellular cytokine staining followed by flow cytometry. A significant increase in % CD8+ / TFNg+ T cells was observed in mice receiving MVA- LD10 prime versus MVA prime regimen (*, p < 0.05).

[0040] FIG. 5 A shows protection ofMVA-LDlO prime-vaccinated mice from lethal vaccinia virus challenge on Day 55. Weight changes in percent, as represented on the y-axis, were measured daily for 14 days post-challenge, as represented on the x-axis. Lines demonstrate median weight loss values for treatment groups MVA Prime (Group B), MVA Prime-Boost (Group C), and MVA- LD 10 Prime (Group D), as compared to control (Group A). Error bars indicate standard deviation.

[0041] FIG. 5B shows improved Clinical Scores in MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 55. Lines demonstrate Clinical Score, as represented on the y-axis, over the days following challenge, as represented on the x-axis, for treatment groups MVA Prime (Group B), MVA Prime-Boost (Group C), and MVA-LD10 Prime (Group D), as compared to control (Group A). Clinical score is generated by combining parameters assessing changes in body temperature and visually inspected clinical symptoms such as ruffled fur, hunched posture, respiratory distress, and lethargy, with poorer clinical symptoms yielding higher clinical scores.

[0042] FIG. 5C shows improved Clinical Scores in MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 55 as measured at Day 4 (D4) post challenge. Clinical Score is represented on the y-axis for treatment groups MVA Prime (Group B), MVA Prime-Boost (Group C), and MVA-LD10 Prime (Group D), as compared to control (Group A). Clinical score is generated by combining parameters assessing changes in body temperature and visually inspected clinical symptoms such as ruffled fur, hunched posture, respiratory distress, and lethargy, with poorer clinical symptoms yielding higher clinical scores.

[0043] FIG. 5D shows improved survival of MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 55. Lines demonstrate survival probabilities, as represented on the y-axis, over the days following challenge, as represented on the x-axis, for treatment groups MVA Prime (Group B), MVA Prime-Boost (Group C), and MVA-LD10 Prime (Group D), as compared to control (Group A).

[0044] FIG. 6 shows viremia at 3 days following Day 55 challenge. The level of vaccinia specific E3L RNA in sera 3 days following challenge was determined by qPCR.

[0045] FIG. 7A shows protection of MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 90. Weight changes in percent, as represented on the y-axis, were measured daily for 14 days post-challenge, as represented on the x-axis. Lines demonstrate median weight loss values for treatment groups MVA Prime (Group B), MVA Prime-Boost (Group C), and MVA- LD 10 Prime (Group D), as compared to control (Group A). Error bars indicate standard deviation.

[0046] FIG. 7B shows improved Clinical Scores in MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 90. Lines demonstrate Clinical Score, as represented on the y-axis, over the days following challenge, as represented on the x-axis, for treatment groups MVA Prime (Group B), MVA Prime-Boost (Group C), and MVA-LD10 Prime (Group D), as compared to control (Group A). Clinical score is generated by combining parameters assessing changes in body temperature and visually inspected clinical symptoms such as ruffled fur, hunched posture, respiratory distress, and lethargy, with poorer clinical symptoms yielding higher clinical scores.

[0047] FIG. 7C shows improved Clinical Scores in MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 90 as measured at Day 5 (D5) post challenge. Clinical Score is represented on the y-axis for treatment groups MVA Prime (Group B), MVA Prime-Boost (Group C), and MVA-LD10 Prime (Group D), as compared to control (Group A). Clinical score is generated by combining parameters assessing changes in body temperature and visually inspected clinical symptoms such as ruffled fur, hunched posture, respiratory distress, and lethargy, with poorer clinical symptoms yielding higher clinical scores.

[0048] FIG. 7D shows improved survival of MVA-LD10 prime-vaccinated mice from lethal vaccinia virus challenge on Day 90. Lines demonstrate survival probabilities, as represented on the y-axis, over the days following challenge, as represented on the x-axis, for treatment groups MVA Prime (Group B), MVA Prime-Boost (Group C), and MVA-LD10 Prime (Group D), as compared to control (Group A).

[0049] FIG. 7E shows comparable suppression of viral replication at the site of infection between the MVA / MVA and single-dose MVA-LD10 groups following lethal vaccinia virus challenge. VACV A12L gene levels in lung homogenates were measured by qPCR on day 6 post-intranasal challenge with 1 * 107PFU VACV-WR at Day 90 post-prime. Each point represents one mouse (n = 6 / group); bars indicate group means. Data are logw-transformed 2A-AACt values normalized to P-actin (offset +5). Mice receiving two-dose MVA or single-dose MVA-LD10 exhibited significantly lower lung viral DNA levels compared to mock and single-dose MVA controls (P < 0.0001, one-way ANOVA with Tukey’s test). FIG. 7F shows significant reduction of viral burden in lung homogenates of MVA / MVA and single-dose MVA-LD10 groups following lethal vaccinia virus challenge. Mice (n = 6 per group) were intranasally challenged with 1 * 107PFU VACV-WR at day 90 post-prime, and lung homogenates were collected on day 6 post-infection for viral quantification by plaque assay. Lung viral loads are presented as logw PFU per gram of tissue. Each symbol represents an individual animal; horizontal lines denote group means. The mock-vaccinated group (FB / FB) exhibited high viral titers, whereas MVA / MVA and single-dose MVA-LD10 groups displayed significantly reduced viral burdens. The FB / MVA group showed intermediate control with high inter-animal variability. Differences between groups were analyzed by one-way ANOVA with Tukey’s multiple comparisons.

[0050] DETAILED DESCRIPTION OF THE INVENTION

[0051] Definitions

[0052] Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. As used in this specification and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise, e.g., "a peptide" or a “chimeric polypeptide” includes a plurality of peptides or chimeric polypeptides. Thus, for example, a reference to "a method" includes one or more methods, and / or steps of the type described herein, and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0053] The term “about” as used herein means + / - 10%.

[0054] “Coding sequence” or “encoding nucleic acid” or “nucleic acid sequence encoding” or the like, as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes an amino acid sequence, for example, a polyprotein, polypeptide, protein, peptide, or fragment thereof. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of human or mammal to which the nucleic acid is administered. The terms “percent identical,” “percent homologous,” or “percent similarity”, and the like, when used in the context of nucleic acid sequences refers to the residues in the two sequences being compared which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over the full-length of the sequence, or, or alternatively a fragment of at least about 50 to 2500 nucleotides. Similarly, the terms “percent identical,” “percent homologous,” or “percent similarity”, may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof. Suitably, a fragment is at least about 8 amino acids in length and may be up to about 7500 amino acids. Examples of suitable fragments are described herein. Generally, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence. Alignments can be performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Examples of such programs include, “Clustal Omega”, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 6.1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference. Multiple sequence alignment programs are also available for amino acid sequences, e.g., the “Clustal Omega”, “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

[0055] As used herein, the term “immune checkpoint inhibitor” refers to therapy targeting immune checkpoint proteins, key regulators of the immune system that when expressed can dampen the immune response to an immunologic stimulus. Immune checkpoint inhibitors block inhibitory checkpoints, restoring immune system function. Immune checkpoint inhibitors include those targeting immune checkpoint proteins such as PD-1, PD-1 Ligand- 1 (PD- 1), PD-1 Ligand-2 (PD- L2), CTLA-4, LAG-3, TIM-3, cluster of differentiation 73 (CD73), and V-domain Ig suppressor of T-cell activation (VISTA), B7-H3 / CD276, indoleamine 2,3-dioxygenase (IDO), killer immunoglobulin-like receptors (KIRs), carcinoembryonic antigen cell adhesion molecules (CEACAM) such as CEACAM-1, CEACAM-3, and CEACAM-5, sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and B and T lymphocyte attenuator (BTLA) protein. Immune checkpoint inhibitors are known in the art.

[0056] The term "inducing a protective immune response" means eliciting a humoral response (e g., the production of neutralizing antibodies) or a cellular response (e.g., the activation of T cells), or both a humoral and a cellular response, suitable to protect a subject from a orthopoxvirus infection, including but not limited to an mpox virus infection, smallpox virus infection, borealpox virus infection, vaccinia virus, or disease causing orthopoxvirus, and / or the effects of an orthopoxvirus infection, including but not limited to an mpox virus infection, smallpox virus infection, borealpox virus infection, vaccinia virus, or disease causing orthopoxvirus, in a subject to which the MVA-LD10 has been administered.

[0057] The term "modified vaccinia Ankara," "modified vaccinia ankara," "Modified Vaccinia Ankara," or "MVA" generally refers to a highly attenuated strain of vaccinia virus developed by Dr. Anton Mayr by serial passage on chick embryo fibroblast cells; or variants or derivatives thereof. MVA is reviewed in Mayr, A. et al. 1975 Infection 3:6-14. The genomic sequence of MVA and various variants is described, for example, at GenBank Accession Numbers AY603355, U94848, and DQ983238. In some embodiments, the MVA as provided herein can be derived synthetically, for example, through chemically synthesized plasmids and reconstituted to the full length genomic MVA sequence in a host cell, for example, as described in US2018 / 0251736, US2021 / 0230560, and WO2021 / 158565, each incorporated herein by reference.

[0058] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0059] Nucleic acids can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[0060] “Operably linked” as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0061] A “peptide,” “protein,” “polypeptide,” or “polyprotein” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0062] “Promoter” as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating, or enhancing the transcription of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0063] The term “prevent,” “preventing,” and “prevention” refers to the inhibition of the development or onset of a orthopoxvirus infection, including but not limited to an mpox virus infection, smallpox virus infection, borealpox virus infection, vaccinia virus, or disease causing orthopoxvirus, or the prevention of the recurrence, onset, or development of one or more symptoms of orthopoxvirus infection, including but not limited to an mpox virus infection, smallpox virus infection, borealpox virus infection, vaccinia virus, or disease causing orthopoxvirus, in a subject resulting from the administration of, for example, MVA-LD10.

[0064] The “host,” “patient,” or “subject” treated is typically a human patient, although it is to be understood the methods described herein are effective with respect to other animals, such as mammals. More particularly, the term patient can include animals used in assays such as those used in preclinical testing including but not limited to mice, rats, monkeys, dogs, pigs and rabbits, and the like, and animals susceptible to orthopoxvirus infections.

[0065] The term "therapeutically effective amount" refers to the amount of MVA and immune checkpoint inhibitor, for example MVA-LD10, which, when administered to a subject for treating or preventing an orthopoxvirus infection, including but not limited to an mpox virus infection, smallpox virus infection, borealpox virus infection, vaccinia virus, or disease causing orthopoxvirus, or inducing an immune response to such orthopoxvirus, is sufficient to affect such treatment of, prevention of, or immune induction against such orthopoxvirus, or results in the prevention of the development, recurrence, or onset of such orthopoxvirus infection, or a symptom thereof or symptom associated therewith, or an amount sufficient to induce a humoral and / or cellular immune response thereto.

[0066] For the recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Modified Vaccinia Ankara (MVA)-LDIO Vector

[0067] Modified vaccinia Ankara (MV A) in particular has been employed as a safe and potent viral vector vaccine against infectious diseases. MVA is a highly attenuated strain of vaccinia virus derived by extensive serial passages in chicken embryo fibroblasts (CEF) (Sutter G, Staib C. Vaccinia vectors as candidate vaccines: the development of modified vaccinia virus Ankara for antigen delivery. Current Drug Targets-Infectious Disorders. 2003;3:263-71). MVA is distinguished by its great attenuation, as demonstrated by diminished virulence and reduced ability to replicate in primate cells, while maintaining good immunogenicity. The MVA virus has been analyzed to determine alterations in the genome relative to the parental strain chorioallantois vaccinia virus Ankara (CVA) strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H. et al. 1991 J Gen Virol 72: 1031 -1038). The resulting MVA virus is host cell restricted to avian cells. Accordingly, MVA vaccines can be produced in large scale in chicken cell lines.

[0068] Modified vaccinia Ankara (MVA) has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr A, et al. Abstammung, eigenschafter und verwendung des attenuierten vaccinia-stammes. Infection 3: 6-14, 1975; Swiss Patent No. 568,392). The MVA virus is publicly available from American Type Culture Collection as ATCC No. VR-1508.

[0069] In particular embodiments, the MVA of the MVA-LD10 vector is the MVA available as ATCC VR-1566, a virus isolated by serial passage of CVA (Ankara) strain in chick embryo fibroblasts (CEF) in the laboratory of Professor Anton Mayr, then given to the National Institutes of Health, where it was plaque purified three times in CEF cells. VR-1566 was derived by limited further passage of stock received from the NIH in the SL-29 chicken embryo fibroblast cell line [ATCC CRL-1590],

[0070] In particular embodiments, the MVA is MVA-LD10. MVA-LD10 comprises the vaccinia virus strain modified vaccinia Ankara (MVA) comprising a polycistronic nucleic acid insert encoding engineered, multimeric LD10 peptides. In some alternative embodiments, the MVA is MVA-LD01. MVA-LD01 comprises the vaccinia virus strain modified vaccinia Ankara (MVA) comprising a polycistronic nucleic acid insert encoding engineered, multimeric LD01 peptides. In alternative embodiments, the MVA of the MVA-LD10 vector can be derived from an MVA having the genomic sequence as described in at GenBank Accession Numbers AY603355, U94848, and DQ983238. In some embodiments, the MVA of the MVA-LD10 can be derived synthetically, for example, through chemically synthesized plasmids and reconstituted to the full length genomic MVA sequence in a host cell, for example, as described in US2018 / 0251736, US2021 / 0230560, and WO2021 / 158565, each incorporated herein by reference.

[0071] The construction of the MVA-LD10 vector of the present invention can be prepared by methods known in the art, for example, as described in WO2022 / 169895 and US2024 / 0344082, incorporated herein by reference. For example, a DNA-construct which contains the heterologous polycistronic nucleic acid sequence described herein can be flanked by MVA DNA sequences adjacent to a predetermined insertion site (e.g. between two conserved essential MVA genes such as I8R / G1L (see, e.g., U.S. Pat. No. 9,133,478, incorporated herein by reference in its entirety); in restructured and modified deletion III (see, e.g., U.S. Pat. No. 9,133,480, incorporated herein by reference in its entirety); or at other non-essential sites within the MVA genome) is introduced into cells infected with MVA, to allow homologous recombination. Once the DNA-construct has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired MVA-LD10 vector in a manner known per se, preferably with the aid of a marker. The DNA-construct to be inserted can be linear or circular. A plasmid or polymerase chain reaction product is preferred. Such methods of making recombinant MVA vectors are described in, e g., U.S. Pat. No. 9,133,478, incorporated by reference herein. For the expression of the LD10 multimeric polypeptide, it is necessary for regulatory sequences, which are required for the transcription of the polycistronic nucleic acid sequence, to be present on the DNA. Such regulatory sequences (called promoters) are known to those skilled in the art, and include for example those described further below. The DNA-construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al. 1973 Virol 52:456-467; Wigler et al. 1979 Cell 16:777-785), by means of electroporation (Neumann et al. 1982 EMBO J. 1 :841-845), by microinjection (Graessmann et al. 1983 Meth Enzymol 101 :482-492), by means of liposomes (Straubinger et al. 1983 Meth Enzymol 101 :512-527), by means of spheroplasts (Schaffher 1980 PNAS USA 77:2163-2167) or by other methods known to those skilled in the art. In some embodiments, the MVA-LD10 vector as provided herein can be derived synthetically, for example, through chemically synthesized plasmids and reconstituted to the full length genomic MVA sequence in a host cell, for example, as described in US2018 / 0251736, US2021 / 0230560, and WO2021 / 158565, each incorporated herein by reference.

[0072] As described above, the LD10 polycistronic nucleic acid sequence can be inserted into any suitable site within the rMVA genomic sequence. In some embodiments, the polycistronic nucleic acid sequence is inserted into the MVA vector in a natural deletion site, a modified natural deletion site, or between essential or non-essential MVA genes. In some embodiments, the LD10 polycistronic nucleic acid sequence is inserted between two essential genes I8R / G1L.

[0073] The MVA-LD10 vector comprises a polycistronic nucleic acid sequence comprising tandem repeats of the LD10 immune checkpoint inhibitor capable of being processed into monomers and secreted from the cell to enhance the immunogenicity of the MVA vector as an mpox vaccine. By expressing localized, high quantities of multiple LD10 immune checkpoint inhibitor peptides capable of downregulating the PD-1 / PD-L1 pathways, immune modulating activities which typically hinder the development of sufficient antigenicity to induce immunity can be downregulated.

[0074] The general structure of the multimeric LD10 polypeptide of the MVA-LD10 vector is as follows: (M)(Secretion Signal Peptide-LDIO Immune Checkpoint Inhibitor Peptide-Cleavable Peptide)4(Secretion Signal Peptide-LDIO Immune Checkpoint Inhibitor Peptide), wherein M = methionine. The various components of the multimeric LD10 polypeptide are described further below and in Table 1.

[0075] The LD10 peptide comprises the amino acid sequence STGQISTLRVNITAPLSQ (SEQ ID NO: 1). LD10 has previously been described in, for example, U.S. Pat. Pub. No. 2018 / 0339044, incorporated herein by reference.

[0076] The LD10 inhibitor peptides expressed by the MVA are secreted from the cell. Secretion of the LD10 immune checkpoint inhibitor peptide can be accomplished by expressing a chimeric polypeptide that includes a secretion signal peptide fused to the N-terminus of the LD10 immune checkpoint inhibitor peptide. During the translation of the chimeric polypeptide comprising the secretion signal peptide and LD10 immune checkpoint inhibitor peptide, the signal peptide is recognized as it emerges from the ribosome; it is bound by the signal recognition particle (SRP) and translation is halted. This entire complex is transported to the external face of the Endoplasmic Reticulum (ER) where it binds to the SRP receptor, and the signal sequence is transferred to a translocon. While bound to the translocon, translation is reinitiated and the protein passes through the ER membrane and into the lumen. As it does this, the signal peptide is recognized by a signal peptidase and is cleaved to generate the immune checkpoint inhibitor peptide, which is trafficked through the Golgi network before being secreted from the cell via the classical secretory pathway. Accordingly, the LDlO-secretion signal chimeric protein includes the secretion signal derived from tissue plasminogen activator (tPA) amino acid sequence DAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGAR (SEQ ID NO: 2).

[0077] In addition to the secretion signal peptide on the N-terminus of each LD10 peptide, the polypeptide also includes a hybrid cleavable peptide sequence comprising the amino acid sequence RAKR (SEQ ID NO: 3) fused to a cis-acting hydrolase element (CHYSEL) peptide capable of inducing ribozyme skipping comprising amino acid sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 4), resulting in a hybrid cleavable peptide RAKR GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 5). By providing a self-cleaving peptide sequence and ribozyme skipping peptide fused to the C-terminus of the LD10 peptide, the multiple LD10 peptides can be cleaved into multiple monomers during or following translation. Suitable cleavage sequences are known in the art (see, e.g., Donnelly et al., Analysis of the aphthovirus 2A / 2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J. Gen. Virol. 82, 1013-1025 (2001), incorporated by reference in its entirety herein). As provided herein, the most C-terminus LD10 peptide of the multimeric polypeptide does not include a cleavable peptide.

[0078] Accordingly, the amino acid sequence of the multimeric form of MVA-LD10 is provided in SEQ ID NO: 6, which is encoded by the MVA optimized nucleic acid sequence of SEQ ID NO: 7.

[0079] Table 1 - Components of Multimeric LD10 Polypeptide

[0080] In some embodiments, the MVA-LD10 vector encodes five repeats of the LD10 peptide.

[0081] In some embodiments, the MVA-LD10 vector encodes four repeats of the LD10 peptide. In some embodiments, the MVA-LD10 vector encodes three repeats of the LD10 peptide. In some embodiments, the MVA-LD10 vector encodes two repeats of the LD10 peptide. In some embodiments, the MVA-LD10 vector encodes a single LD10 peptide.

[0082] In an alternative aspect, the MVA encodes programmed cell death protein 1 (PD1) inhibitory peptide LD01 (MVA-LD01). The LD01 peptide comprises the amino acid sequence CRRTSTGQISTLRVNITAPLSQ (SEQ ID NO: 8). LD01 has previously been described in, for example, U.S. Pat. Pub. No. 2018 / 0339044, incorporated herein by reference. The amino acid sequence of the multimeric form of MVA-LD01 is provided in SEQ ID NO: 9, which is encoded by the MVA optimized nucleic acid sequence of SEQ ID NO: 10. The various components of the multimeric LD01 polypeptide are described further below and in Table 2. Table 2 - Components of Multimeric LD01 Polypeptide

[0083] In some embodiments, the MVA-LD01 vector encodes five repeats of the LD01 peptide.

[0084] In some embodiments, the MVA-LD01 vector encodes four repeats of the LD01 peptide. In some embodiments, the MVA-LD01 vector encodes three repeats of the LD01 peptide. In some embodiments, the MVA-LD01 vector encodes two repeats of the LD01 peptide. In some embodiments, the MVA-LD01 vector encodes a single LD01 peptide.

[0085] Regulatory Sequences When the MVA vector encodes the immune checkpoint inhibitor, for example as provided in MVA-LD10, the immune checkpoint inhibitor polypeptide is expressed from a nucleic acid sequence inserted into a suitable location within the MVA genomic sequence. For the expression of the nucleic acid insert within the rMVA genomic backbone, it is necessary for regulatory sequences such as promoters, which are required for the transcription of the polycistronic nucleic acid encoding the polyprotein, to be located in the 5’ region of the nucleic acid insert adjacent to the transcription start site in order to initiate transcription. Wherein the nucleic acid insert is a polycistronic nucleic acid encoding multiple proteins / peptides as a single polyprotein, one or more promoters can be located 5’ to the transcriptional start site of the ORF encoding the N-terminus most polypeptide of the polyprotein.

[0086] Because MVA is a cytoplasmic virus, suitable promoters, in some embodiments, include those derived from naturally occurring poxviral promoters. Poxviral genes, promoters, and transcription factors are divided into early, intermediate, and late classes, depending on their expression timing during orthopoxvirus infections (see, e.g., Assarsson et al., Kinetic analysis of a complete orthopoxvirus transcriptome reveals an immediate-early class of genes. PNAS 2008;105(6):2140-2145; Yang Zet al., Genome-wide analysis of the 5' and 3' ends of vaccinia virus early mRNAs delineates regulatory sequences of annotated and anomalous transcripts. J Virol. 2011 ; 85( 12): 5897— 5909). MVA replication in most mammalian cells (non-permissive cells) ceases during the assembly of progeny virions after all stages of expression occur. This supports the utility of all promoter classes, including late promoters, for controlling transgene expression (Sancho et al., The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis. J Virol. 2002;76(16):8318-8334; Geiben-Lynn et al., Kinetics of recombinant adenovirus type 5, vaccinia virus, modified vaccinia ankara virus, and DNA antigen expression in vivo and the induction of memory T-lymphocyte responses. Clin Vaccine Immunol. 2008; 15(4):691—696). Some poxviral promoters have both early and late elements, allowing their open-reading frames (ORFs) or recombinant antigens to be expressed early in the virus infection and late after the viral genome replication, respectively (Broyles SS, Vaccinia virus transcription. J Gen Virol. 2003;84(Pt 9):2293— 2303). Poxviral promoters can be utilized cross-strain (see Prideaux et al., Comparative analysis of vaccinia virus promoter activity in fowlpox and vaccinia virus recombinants. Virus Res. 1990; 16(1):43— 57; Tripathy et al., Regulation of foreign gene in fowlpox virus by a vaccinia virus promoter. Avian Dis. 1990;34(l):218— 220).

[0087] Such MVA promoter sequences are known to those skilled in the art, and include for example the pl 1 promoter, which drives expression of the 1 Ik protein encoded by the F17R ORF (Wittek et al., Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome. J Virol. 1984;49(2):371- 378); the p7.5 promoter (Cochran et al., In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. J Virol. 1985;54(l):30- 37); the pHL promoter (Schmitt et al., Sequence and transcriptional analysis of the vaccinia virus Hindlll I fragment. J Virol. 1988;62(6): 1889- 1897); the pTK promoter (Weir and Moss, Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase. Virology. 1987; 158(l):206— 210); the pF7L promoter (Coupar et al., Effect of in vitro mutations in a vaccinia virus early promoter region monitored by herpes simplex virus thymidine kinase expression in recombinant vaccinia virus. J Gen Virol. 1987;68(Pt 9):2299-2309); the pH5 promoter (Perkus et al., Cloning and expression of foreign genes in vaccinia virus, using a host range selection system. J Virol. 1989;63(9):3829- 3836); the short synthetic promoter pSyn (Chakrabarti et al., Compact, synthetic, vaccinia virus early / late promoter for protein expression. Biotechniques. 1997;23(6): 1094-1097; Hammond et al., A synthetic vaccinia virus promoter with enhanced early and late activity. J Virol Methods. 1997;66(1): 135-1380); the pmH5 promoter (Wyatt et al., Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. Vaccine. 1996; 14(15): 1451—1458); the pHyb promoter (Sancho et al., The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis. J Virol. 2002;76(16):8318-8334); the LEO promoter (Wyatt et al., Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine. 2008;26(4):486-493); the pB8 promoter (Orubu et al., Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA. PLoS One. 2012;7(6):e40167); the pFl l promoter (Orubu et al., Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA. PLoS One. 2012;7(6):e40167). In some embodiments, the promoter is selected from one or more of pMH5, pl 1, pSyn, pHyb, or a combination thereof.

[0088] In some embodiments, the promoter is the pH5 promoter aaaaaatgaaaataaatacaaaggttcttgagggttgtgttaaattgaaagcgagaaataatcataa (SEQ ID NO: 11), or a nucleic acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the promoter is the pH5 promoter aaaaaatgaaaataaatacaaaggttcttgagggttgtgttaaattgaaagcgagaaataatcataaatt (SEQ ID NO: 12), or a nucleic acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0089] In some embodiments, the promoter is the modified pH5 promoter (pmH5) aaaaattgaaaataaatacaaaggttcttgagggttgtgttaaattgaaagcgagaaataatcataa (SEQ ID NO: 13), or a nucleic acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the promoter is the modified pH5 promoter (pmH5) aaaaattgaaaataaatacaaaggttcttgagggttgtgttaaattgaaagcgagaaataatcataaata (SEQ ID NO: 14), or a nucleic acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto. In some embodiments, the promoter is the modified pH5 promoter (pmH5) aaaaaatgaaaataaatacaaaggttcttgagggttgtgttaaattgaaagcgagaaataatcataaata (SEQ ID NO: 15), or a nucleic acid sequence at least 85%, 90%, 95%, 97%, or 99% identical thereto.

[0090] Additional vaccinia virus promoters that may be particularly suitable as promoters in the present invention include those derived from natural promoter sequences.

[0091] Table 3 - Vaccinia Virus Promoters Sequence Optimization

[0092] When the MVA vector encodes the immune checkpoint inhibitor, for example as provided in MVA-LD10, one or more nucleic acid sequences comprising the nucleic acid insert expressing the immune checkpoint inhibitor may be optimized for use in an MVA vector. Optimization includes codon optimization, which employs silent mutations to change selected codons from the native sequences into synonymous codons that are optimally expressed by the host-vector system. Other types of optimization include the use of silent mutations to interrupt homopolymer stretches or transcription terminator motifs. Each of these optimization strategies can improve the stability of the gene, improve the stability of the transcript, or improve the level of protein expression from the sequence. In exemplary embodiments, the number of homopolymer stretches in the heterologous DNA insert sequence will be reduced to stabilize the construct. A silent mutation may be provided for anything similar to a vaccinia termination signal.

[0093] In exemplary embodiments, the sequences are codon optimized for expression in MVA; sequences with runs of > 5 deoxyguanosines, > 5 deoxycytidines, > 5 deoxyadenosines, and > 5 deoxythymidines are interrupted by silent mutation to minimize loss of expression due to frame shift mutations.

[0094] In particular, the nucleic acid for insertion can be optimized by codon optimizing the original DNA sequence. For example, the “Invitrogen GeneArt Gene Software” can be used to codon optimize the DNA sequence. To fully optimize the gene sequence, homopolymer sequences (G / C or T / A rich areas) are interrupted via silent mutation(s). To the extent present in the nucleic acid insert sequence, the MVA transcription terminator (T5NT (UUUUUNU)) is interrupted via silent mutation(s). Further optimizations can include, for example, adding a Kozak sequence (GCCACC / ATG), adding a second stop codon, and adding a vaccinia virus transcription terminator, specifically “TTTTTAT”, or variations and / or combinations thereof.

[0095] MVA Vector and Immune Checkpoint Inhibitor Combination

[0096] In one aspect as provided herein, an MVA vector is administered in combination with an immune checkpoint inhibitor, for preventing, for reducing the effects of, or inducing a protective immune response against an orthopoxvirus, for example mpox virus, smallpox virus, or borealpox virus, in a subject), wherein the MVA and immune checkpoint inhibitor are administered only one time. In some embodiments, MVA-LD10 is administered. Numerous studies have explored the combination of MVA vaccines with immune checkpoint inhibitors for cancer treatment. For example, use of the Nous-209 genetic vaccine, which includes both a prime (GAd20-209-FSP) and boost (MVA-209-FSP) vaccination is being evaluated in combination with the PD-1 antibody pembrolizumab for patients with microsatellite instability-high (MSI-H) solid tumors. A Phase I-II bridge expansion cohort, aiming to assess safety, tolerability, and preliminary efficacy, and tumor response, determined that the combination of Nous-209 was safe and well tolerated, with encouraging clinical efficacy having reported 5 / 7 subjects achieving objective response (NCT04041310; Overman, MJ. et al. Results of phase I-II bridging study for Nous-209, a neoantigen cancer immunotherapy, in combination with pembrolizumab as first line treatment in patients with advanced dMMR / MSI-h colorectal cancer. Meeting Abstract: 2023 ASCO Annual Meeting I. 41(16_suppl):el4665-el4665(2023)). Other MVA and immune checkpoint inhibitor combinations for the treatment of cancer have been studied (Remy-Ziller, C. et al. Sequential administration of MVA-based vaccines and PD-1 / PD-L1- blocking antibodies confers measurable benefits on tumor growth and survival: Preclinical studies with MVA-PGal and MVA-MUC1 (TG4010) in a murine tumor model. Hum Vaccin Immunother. 14(1): 140-145(2018 Jan 2); Medina-Echeverz, J. et al. Synergistic cancer immunotherapy combines MVA-CD40L induced innate and adaptive immunity with tumor targeting antibodies. Nat Commun. 10(1): 5041(2019 Nov 6); Heery, C., et al. "Novel applications of MVA to improve outcomes in immunooncology." Annals of Oncology 30:i3(2019)). The use of MVA in combination with immune checkpoint inhibitors for the treatment of infectious orthopoxviruses, however, has not been explored.

[0097] In some embodiments, the immune checkpoint inhibitor is an antibody, antibody fragment, antigen binding fragment, or peptide. In some embodiments, immune checkpoint inhibitor is encoded by the MVA vector. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor peptide LD10 that is encoded by an MVA vector (MVA-LD10). In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor peptide LD01 that is encoded by an MVA vector (MVA-LD01).

[0098] Immune Checkpoint Inhibitors

[0099] In some embodiments, the MVA administered does not encode an immune checkpoint inhibitor. Rather, the MVA is administered in combination with an immune checkpoint inhibitor. Suitable immune checkpoint inhibitors include PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, TIM-3 inhibitors, CD73 inhibitors, or an immune checkpoint inhibitor described herein, or a combination thereof.

[0100] PD-1 inhibitors

[0101] In one embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor that blocks the interaction of PD-1 and PD-L1 by binding to the PD-1 receptor, and in turn inhibits immune suppression. In one embodiment, the immune checkpoint inhibitor is a PD-1 immune checkpoint inhibitor selected from nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), cemiplimab (LIBTAYO®; Regeneron), dostarlimab (JEMPERLI®), pidilizumab (Medivation), AMP-224 (AstraZeneca / Medimmune), AMP-514 (AstraZeneca), sintilimab (IB 1308; Innovent / Eli Lilly) sasanlimab (PF-06801591; Pfizer), spartalizumab (PDR001; Novartis), retifanlimab (MGA012 / INCMGA00012); Incyte Corporation and MacroGenics), tislelizumab (BGB-A317; BeiGene), toripalimab (JS001), camrelizumab (SHR-1210; Jiangsu Hengrui Medicine Company and Incyte Corporation), CS1003 (Cstone Pharmaceuticals), zimberelimab (AB122; Arcus Biosciences) and JTX-4014 (Jounce Therapeutics).

[0102] PD-L1 inhibitors

[0103] In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor that blocks the interaction of PD-1 and PD-L1 by binding to the PD-L1 receptor, and in turn inhibits immune suppression. In some embodiments, the PD-L1 inhibitor is selected from atezolizumab (TECENTRIQ®, Genentech), durvalumab (IMFINZI®, AstraZeneca); avelumab (BAVENCIO®; Merck), envafolimab (KN035; Alphamab), BMS-936559 (Bristol-Myers Squibb), BMS-986189 (Bristol-Myers Squibb), lodapolimab (LY3300054; Eli Lilly), cosibelimab (CK-301; Checkpoint Therapeutics), sugemalimab (CS-1001; Cstone Pharmaceuticals), adebrelimab (SHR-1316; Jiangsu HengRui Medicine), CBT-502 (CBT Pharma), AUNP12 (Aurigene), CA-170 (Aurigene / Curis), or BGB-A333 (BeiGene). Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) Inhibitors

[0104] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor that blocks the interaction of costimulatory molecule CD28 with CD80 and CD86. CTLA-4 (CD 152) is a B7 / Cd28 family member that mediates immunosuppression by indirectly diminishing signaling through the co-stimulatory receptor CD28. CTLA-4 inhibits CD28-dependent proliferation and plays a crucial role in the activation of T cells. In some embodiments, the CTLA-4 inhibitor is selected from ipilimumab, tremelimumab, AGEN1884, or AGEN2041.

[0105] T cell immunoreceptor with immunoglobulin and HIM domain (TIGIT) Inhibitors

[0106] In some embodiments, the immune checkpoint inhibitor is a TIGIT inhibitor. TIGIT is upregulated by immune cells, including activated T cells, natural killer cells, and regulatory T cells. TIGIT binds to two ligands, CD155 (PVR) and CD112 (PVRL2, nectin-2) (Stanietsky et al., The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity. Proc Natl Acad Sci U S A 2009; 106: 17858-63). TIGIT (also called WUCAM, Vstm3, VSIG9) is a receptor of the Ig superfamily, which plays a critical role in limiting adaptive and innate immunity (Boles et al., A novel molecular interaction for the adhesion of follicular CD4 T cells to follicular DC. Eur J Immunol 2009; 39:695-703). TIGIT is expressed by activated CD8+ T and CD4+ T cells, natural killer (NK) cells, regulatory T cells (Tregs), and follicular T helper cells in humans (Joller et al., Cutting edge: TIGIT has T cell-intrinsic inhibitory functions. J Immunol 2011; 186: 1338— 42; Wu et al., Follicular regulatory T cells repress cytokine production by follicular helper T cells and optimize IgG responses in mice. Eur J Immunol 2016; 46: 1152-61).

[0107] In one embodiment, the immune checkpoint inhibitor is a TIGIT inhibitor that blocks the interaction of TIGIT and CD 155 by binding to the TIGIT receptor, and in turn inhibits immune suppression. TIGIT inhibitors include, but are not limited to, Vibostolimab (MK-7684; Merck), Etigilimab / OMP-313 M32 (OncoMed), Tiragolumab (MTIG7192A / RG-6058; Roche / Genentech), ociperlimab (BGB-A1217; Beigene), BMS-986207 (BMS), COM902 (Compugen), M6223 (Merck KGaA), domvanalimab (AB- 154; Arcus Biosciences), AZD2936 (AstraZeneca), JS006 (Shanghai Junshi Bioscience), IBI139 (Innovent Biologies), ASP-8374 (Astellas / Potenza), BAT6021 (Bio-Thera Solutions), TAB006 (Shanghai Junshi Bioscience), Domvanalimab (AB 154; Arcus Biosciences), EOS884448 (EOS-448; iTeos), SEA-TGT (Seattle Genetics), mAb- 7 (Stanwei Biotech); SHR-1708 (Hengrui Medicine), GS02 (Suzhou Zelgen / Qilu Pharma), RXT- 804 (Rxi Pharmaceuticals), NB6253 (Northern Biologies), ENUM009 (Enumreal Biomedical), CASC-674 (Cascadian Therapeutics), AJUD008 (AJUD Biopharma), AGEN1777 (Agenus, Bristol-Myers Squibb), HLX53 (Shanghai Henlius Biotech), BAT6005 (Bio-Thera Solutions), the anti-TIGIT / anti-PD-Ll bispecific antibody HLX301 (Shanghai Henlius Biotech), the anti- TIGIT / anti-PD-Ll antibody HB0036 (Shanghai Huaota Biopharmaceutical).

[0108] T-cell immunoglobulin and mucin domain 3 (TIM-3) inhibitors

[0109] In some embodiments, the immune checkpoint inhibitor is a TIM-3 inhibitor. TIM-3, encoded by Havcr2, is an immunoglobulin (Ig) and mucin domain-containing cell surface molecule that was originally discovered as a cell surface marker specific to interferon (IFN-y) producing CD4+ T helper 1 (Thl) and CD8+ T cytotoxic 1 (Tel) cell (Monney et al., Thl-specific cell surface protein TIM-3 regulates macrophage activation and severity of an autoimmune disease. Nature 2002; 415: 536-41). TIM-3 is coregulated and co-expressed along with other immune checkpoint receptors (PD-1, Lag-3, and TIGIT) on CD4+ and CD8+ T cells (Chihara et al., Induction and transcriptional regulation of the co-inhibitory gene module in T cells. Nature 2018; 558: 454-9; DeLong et al., 11-27 and TCR stimulation promote T cell expression of multiple inhibitory receptors. ImmunoHorizons 2019; 3: 13-25).

[0110] In one embodiment, the immune checkpoint inhibitor is a TIM-3 inhibitor that blocks the interaction of TIM-3 and galectin-9, phosphatidylserine (PtdSer), high-mobility group protein Bl (HMGB1), and / or CEACAM-1 by binding to the TIM-3 receptor, and in turn inhibits immune suppression. TIM-3 inhibitors include, but are not limited to, Cobolimab (TSR-022; Tesaro), RG7769 (Genentech), MAS825 (Novartis), sabatolimab (MBG453; Novartis), Sym023 (Symphogen), INCAGN2390 (Incyte), LY3321367 (Eli Lilly and Company), BMS-986258 (BMS), SHR-1702 (Jiangsu HengRui), AZD7789 (AstraZeneca); TQB2618 (Chia Tai Tianqing Pharmaceutical Group Co., Ltd.); and NB002 (Neologies Bioscience), BGBA425 (Beigene) and the TIM-3 and PD-1 bispecific RO7121661 (Roche).

[0111] Lymphocyte activation gene -3 (LAG-3) inhibitors In some embodiments, the immune checkpoint inhibitor is a LAG-3 inhibitor. LAG-3 (CD223) is a member of the immunoglobulin superfamily (IgSF) and exerts a wide variety of biologic impacts on T cell function (Triebel et al., LAG-3, a novel lymphocyte activation gene closely related to CD4. J Exp Med 1990; 171 : 1393-405). LAG-3 is expressed on cell membranes of natural killer cells (NK), B cells, tumor-infiltrating lymphocytes (TIL), a subset of T cells, and dendritic cells (DC) (Triebel et al., LAG-3, a novel lymphocyte activation gene closely related to CD4. J Exp Med 1990; 171 : 1393-405); Kisielow et al., Expression of lymphocyte activation gene 3 (LAG-3) on B cells is induced by T cells. Eur J Immunol 2005; 35: 2081-8; Grosso et al., LAG- 3 regulates CD8+ T cell accumulation and effector function in murine self- and tumor-tolerance systems. J Clin Invest 2007; 117: 3383-92; Workman et al., LAG-3 regulates plasmacytoid dendritic cell homeostasis. J Immunol 2009; 182: 1885-91; Andreae et al., Maturation and activation of dendritic cells induced by lymphocyte activation gene-3 (CD223). J Immunol 2002; 168: 3874-80). LAG-3 is one of the various immune-checkpoint receptors that are coordinately upregulated on both regulatory T cells (Tregs) and anergic T cells, and the simultaneous blockade of these receptors can result in an enhanced reversal of this anergic state relative to the blockade of one receptor alone (Grosso et al., Functionally distinct LAG-3 and PD-1 subsets on activated and chronically stimulated CD8 T cells. J Immunol 2009; 182: 6659-69).

[0112] In one embodiment, the immune checkpoint inhibitor is a LAG-3 inhibitor that blocks the interaction of LAG-3 with major histocompatibility complex 2 (MHC class II) by binding to the LAG-3 receptor, and in turn inhibits immune suppression. LAG-3 inhibitors include, but are not limited to, relatlimab (OPDUALAG®; BMS-986016; Bristol-Myers Squibb), GSK2831781 (GlaxoSmithKline), eftilagimod alpha (IMP321; Prima BioMed), leramilimab (LAG525; Novartis), favezelimab (MK-4280; Merck), fianlimab (REGN3767; Regeneron), TSR-033 (Tesaro / GSK), BI754111 (Boehringer Ingelheim), Sym022 (Symphogen, LBL-007 (Nanjing Leads Biolabs Co., Ltd), IBI110 (Innovent Biologies), IBI323 (Innovent Biologies), INCAGN02385 (Incyte Corporation), AVA021 (Avacta), MGD013 (Macrogenics), RO7247669 (Hoffman-LaRoche), EMB-02 (Shanghai Epimab Biotherapeutics), XmAb841 (Xencor), the dual PD-1 and LAG-3 inhibitor tebotelimab (MGD013; MacroGenics), CB213 (Crescendo Biologies), and SNA-03 (Microbio Group) and the dual PD-L1 and LAG-3 inhibitor FS118 (F-Star). Ecto-5 'nucleotidase; Cluster of differentiation 73 (NT5E; CD73) inhibitors

[0113] In some embodiments, the immune checkpoint inhibitor is a CD73 inhibitor. 5'- nucleotidase (5 '-NT), also known as ecto-5 '-nucleotidase or CD73 (cluster of differentiation 73), is an enzyme that in humans is encoded by the NT5E gene (Misumi Yet al., (August 1990). Primary structure of human placental 5 '-nucleotidase and identification of the glycolipid anchor in the mature form. European Journal of Biochemistry. 191 (3): 563-9). CD73 commonly serves to convert AMP to adenosine (Allard et al. Chapter Fifteen - Measurement of CD73 enzymatic activity using luminescence-based and colorimetric assays. Methods in Enzymology, Tumor Immunology and Immunotherapy - Molecular Methods, Academic Press, 629: 269-289). Together with CD39, CD73 plays a major role in promoting immunosuppression through the pathway degrading adenosine triphosphate (ATP) into adenosine. Adenosine is a well -described immunosuppressive agent which attenuates the effector functions of various immune cell populations, including T cells, and enhances the suppressive functions of T regs.

[0114] In one embodiment, the additional immune checkpoint inhibitor is a CD73 inhibitor that specifically binds to CD73 and blocks its extracellular 5'-nucleotidase activity. CD73 inhibitors include, but are not limited to, HLX23 (Shanghai Henlius Biotech), LY3475070 (Eli Lilly and Company), IPH5301 (Innate Pharma, AstraZeneca), AK119 (Akesobio Australia Pty Ltd.), PT199 (Phanes Therapeutics), mupadolimab (CPL006; Corvus Pharmaceuticals), Sym024 (Symphogen), oleclumab (MEDI9447; Astra Zeneca), IBI325 (Innovent Biologies), ORIC-533 (Oric Pharmaceuticals), JAB-BX102 (Jacobio Pharmaceuticals), TJ004309 (Tracon Pharmaceuticals), AB680 (Arcus Biosciences), NZV930 (Novartis), BMS-986179 (Bristol Myers Squibb), INCA00186 (Incyte Corporation), and the Anti-CD73-TGFP-Trap Bifunctional Antibody dalutrafusp alfa (Gilead Sciences).

[0115] Additional Immune Checkpoint Inhibitors

[0116] In some embodiments, the patient is administered an alternative immune checkpoint inhibitor. In some embodiments, the alternative immune checkpoint inhibitor is a B7-H3 / CD276 immune checkpoint inhibitor such as enoblituzumab (MGA217, Macrogenics) MGD009 (Macrogenics), 131I-8H9 / omburtamab (Y-mabs), and L8H9 / omburtamab (Y-mabs), an indoleamine 2,3 -dioxygenase (IDO) immune checkpoint inhibitor such as Indoximod and INCB024360, a killer immunoglobulin-like receptors (KTRs) immune checkpoint inhibitor such as Lirilumab (BMS-986015), a carcinoembryonic antigen cell adhesion molecule (CEACAM) inhibitor (e.g., CEACAM-1, -3 and / or -5). Exemplary anti-CEACAM-1 antibodies are described in WO 2010 / 125571, WO 2013 / 082366 and WO 2014 / 022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or a recombinant form thereof, as described in, e.g., US 2004 / 0047858, U.S. Pat. No. 7,132,255 and WO 99 / 052552. In other embodiments, the anti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng et al. PLoS One. 2010 September 2; 5(9). pii: el2529 (DOI: 10: 1371 / journal.pone.0021146), or cross-reacts with CEACAM-1 and CEACAM-5 as described in, e.g., WO 2013 / 054331 and US 2014 / 0271618.

[0117] In some embodiments, the alternative immune checkpoint inhibitor is an inhibitor directed to CD47, including, but not limited to, Hu5F9-G4 (Stanford University / Forty Seven), TI-061 (Arch Oncology), TTI-622 (Trillum Therapeutics), TTI-621 (Trillum Therapeutics), SRF231 (Surface Oncology), SHR-1603 (Hengrui), OSE-172 (Boehringer Ingelheim / OSE Immunotherapeutics), NI-1701 (Novimmune TG Therapeutics), IBI188 (Innovent Biologies); CC- 95251 (Celgene), CC-90002 (Celgene / Inibrx), AO-176 (Arch Oncology), ALX148 (ALX Oncology), IMM01 (ImmuneOnco Biopharma), IMM2504 (ImmuneOnco Biopharma), IMM2502 (ImmuneOnco Biopharma), IMM03 (ImmuneOnco Biopharma), IMC-002 (ImmuneOncia Therapeutics), IBI322 (Innovent Biologies), HMBD-004B (Hummingbird Bioscience), HMBD- 004A (Hummingbird Bioscience), HLX24 (Henlius), FSI-189 (Forty Seven), DSP107 (KAHR Medical), CTX-5861 (Compass Therapeutics), BAT6004 (Bio-Thera), AUR-105 (Aurigene), AUR-104 (Aurigene), ANTI-CD47 (Biocad), ABP-500 (Abpro), ABP-160 (Abpro), TJC4 (I- MAB Biopharma), TJC4-CK (I-MAB Biopharma), SY102 (Saiyuan), SL- 172154 (Shattuck Labs), PSTx-23 (Paradigm Shift Therapeutics), PDL1 / CD47BsAb (Hanmi Pharmaceuticals), NI-1801 (Novimmune), MBT-001 (Morphiex), LYN00301 (LynkCell), and BH-29xx (Beijing Hanmi).

[0118] In some embodiments, the alternative immune checkpoint inhibitor is an inhibitor directed to CD39, including, but not limited to TTX-030 (Tizona Therapeutics), IPH5201 (Innate Pharma / AstraZeneca), SRF-617 (Surface Oncology), ES002 (Elpisciences), 9-8B (Igenica), and an antisense oligonucleotide (Secarna).

[0119] In some embodiments, the alternative immune checkpoint inhibitor is an inhibitor directed to B and T lymphocyte attenuator molecule (BTLA), for example as described in Zhang et al., Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus, Clin Exp Immunol. 2011 Jan; 163(1): 77-87, and TAB004 / JS004 (Junshi Biosciences).

[0120] In some embodiments, the alternative immune checkpoint inhibitor is a sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) inhibitor, including, but not limited to, NC318 (an anti- Siglec-15 mAb).

[0121] Pharmaceutical Compositions

[0122] MVA including but not limited to a MVA-LD10 or MVA-LD01, for use as described herein are readily formulated and administered as a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable diluent, excipient, carrier, or adjuvant.

[0123] As used herein, the phrase "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as those suitable for parenteral administration, such as, for example, by intramuscular, intraarticular (in the joints), intravenous, intradermal, intraperitoneal, and subcutaneous routes. Examples of such formulations include aqueous and non-aqueous, isotonic sterile injection solutions, which contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. One exemplary pharmaceutically acceptable carrier is physiological saline. Carriers include excipients and diluents and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated. The carrier can be inert or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.

[0124] In some embodiments, the pharmaceutical composition comprises a MVA-LD10 vector formulated in 7.5% sucrose in PBS.

[0125] In some embodiments, the pharmaceutical composition comprises a MVA-LD10 vector formulated in 7% sucrose in PBS. In some embodiments, the pharmaceutical composition comprises a MVA-LD01 vector formulated in 7.5% sucrose in PBS.

[0126] In some embodiments, the pharmaceutical composition comprises a MVA-LD01 vector formulated in 7% sucrose in PBS.

[0127] Other physiologically acceptable diluents, excipients, carriers, or additional adjuvants and their formulations are known to those skilled in the art.

[0128] In some embodiments, additional adjuvants are used as further immune response enhancers. In various embodiments, the additional immune response enhancer is selected from the group consisting of alum-based adjuvants, oil based adjuvants, Specol, RIBI, TiterMax, Montanide ISA50 or Montanide ISA 720, GM-CSF, nonionic block copolymer-based adjuvants, dimethyl dioctadecyl ammoniumbromide (DDA) based adjuvants AS-1 , AS-2, Ribi Adjuvant system based adjuvants, QS21 , Quil A, SAF (Syntex adjuvant in its microfluidized form (SAF-m), dimethyldioctadecyl ammonium bromide (DDA), human complement based adjuvants m. vaccae, ISCOMS, MF-59, SBAS-2, SBAS-4, Enhanzyn®, RC-529, AGPs, MPL-SE, QS7, Escin; Digitonin; Gypsophila; and Chenopodium quinoa saponins.

[0129] In some embodiments, the pharmaceutical composition further comprises Army Liposome Formulation (ALF). ALF consists of liposomes containing saturated phospholipids, cholesterol, and monophosphoryl lipid A (MPLA) as an immunostimulant (see, e.g., Alving et al., Army Liposome Formulation (ALF) family of vaccine adjuvants. Expert Rev Vaccines. 2020 Mar;19(3):279-292, incorporated herein in its entirety).

[0130] The compositions utilized in the methods described herein can be administered by a route selected from, e.g., parenteral, intramuscular, intraarterial, intravascular, intravenous, intraperitoneal, subcutaneous, dermal, transdermal, ocular, inhalation, buccal, sublingual, perilingual, nasal, topical administration, and oral administration. The preferred method of administration can vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated). Formulations suitable for oral administration may consist of liquid solutions, such as an therapeutically effective amount of the composition dissolved in a diluent (e.g., water, saline, or PEG-400), capsules, sachets or tablets, each containing a predetermined amount of the vaccine. The pharmaceutical composition may also be an aerosol formulation for inhalation, e.g., to the bronchial passageways. Aerosol formulations may be mixed with pressurized, pharmaceutically acceptable propellants (e.g., dichlorodifluoromethane, propane, or nitrogen).

[0131] For the purposes of this invention, pharmaceutical compositions suitable for delivering a therapeutic or biologically active agent can include, e.g., tablets, gelcaps, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels, hydrogels, oral gels, pastes, eye drops, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. Any of these formulations can be prepared by well-known and accepted methods of art. See, for example, Remington: The Science and Practice of Pharmacy (21 st ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which is hereby incorporated by reference.

[0132] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an therapeutically effective amount of the vaccine dissolved in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the vaccine, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; (d) suitable emulsions; and (e) polysaccharide polymers such as chitins. The vaccine, alone or in combination with other suitable components, may also be made into aerosol formulations to be administered via inhalation, e.g., to the bronchial passageways. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0133] Suitable formulations for rectal administration include, for example, suppositories, which consist of the vaccine with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the vaccine with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons. The vaccines of the present invention may also be co-administered with cytokines to further enhance immunogenicity. The cytokines may be administered by methods known to those skilled in the art, e.g., as a nucleic acid molecule in plasmid form or as a protein or fusion protein.

[0134] In addition to the active compounds, the pharmaceutical formulations can contain other additives, such as pH-adjusting additives. In particular, useful pH-adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the formulations can contain antimicrobial preservatives. Useful antimicrobial preservatives include methylparaben, propylparaben, and benzyl alcohol. An antimicrobial preservative is typically employed when the formulations is placed in a vial designed for multi-dose use. The pharmaceutical formulations described herein can be lyophilized using techniques well known in the art.

[0135] When aqueous suspensions and / or elixirs are desired for oral administration, the compositions of the presently disclosed matter can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and / or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.

[0136] In yet another embodiment, the pharmaceutical composition is provided as an injectable, stable, sterile formulation comprising, for example, an MVA-LD10 vector as described herein, in a unit dosage form in a sealed container.

[0137] Classes of carriers include, but are not limited to binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidents, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others. Pharmaceutically acceptable carriers are carriers that do not cause any severe adverse reactions in the human body when dosed in the amount that would be used in the corresponding pharmaceutical composition. Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, powdered tragacanth, malt, gelatin; talc, and vegetable oils. Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the morphic form or pharmaceutical composition of the present invention.

[0138] Formulations suitable for administration to the lungs can be delivered by a wide range of passive breath driven and active power driven single / -multiple dose dry powder inhalers (DPI). The devices most commonly used for respiratory delivery include nebulizers, metered-dose inhalers, and dry powder inhalers. Several types of nebulizers are available, including jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. Selection of a suitable lung delivery device depends on parameters, such as nature of the drug and its formulation, the site of action, and pathophysiology of the lung. In certain embodiments, a pharmaceutical composition comprising MVA-LD10 as described herein, is administered as a pharmaceutical composition comprising one or more excipients from the Handbook of Pharmaceutical Excipients 9thEdition (or earlier).

[0139] In certain embodiments, a pharmaceutical composition comprising MVA-LD01 as described herein, is administered as a pharmaceutical composition comprising one or more excipients from the Handbook of Pharmaceutical Excipients 9th Edition (or earlier).

[0140] Additional-non-limiting examples of pharmaceutically acceptable excipients include vegetable oil, an animal oil, a fish oil or a mineral oil. For example an oil selected from the group consisting of medium chain fatty acid triglyceride, amaranth oil, apricot oil, apple oil, argan oil, artichokes oil, avocado oil, almond oil, acai berry extract, arachis oil, buffalo pumpkin oil, borage seed oil, borage oil, babassu oil, coconut oil, corn oil, cottonseed oil (cotton seed oil), cashew oil, carob oil, Coriander oil, camellia oil (Camellia oil), Cauliflower oil, cape chestnut oil, cassis oil, deer oil, evening primrose oil, grape syrup Oila oil (hibiscus oil), grape seed oil, gourd oil, hazelnut oil, hemp oil, kapok oil, krill oil, linseed oil, macadamia nut oil, Mongolia oil, moringa oil, malula oil, meadowfoam oil, mustard oil, niger seed oil, olive oil, okrao oil Hibiscus oil), palm oil, palm kernel oil, peanut oil, pecan oil, pine oil, pistachio oil, pumpkin oil, papaya oil, perilla oil (perilla oil), poppy seed oil, prune oil, saw palm oil, quinoa oil, rapeseed oil, rice germ oil, rice bran oil, rice oil, rarelman cheer oil, Safflower oil (safflower oil), soybean oil, sesame oil, sunflower oil, thistle oil, tomato oil, wheat germ oil, walnut oil, watermelon oil, docosahexaenoic acid (DHA), eicosapentaenoic acid (EP A), vitamin A oil, vitamin D oil, vitamin E oil, vitamin K oil, and derivatives thereof; and glycerophospholipids such as lecithin, and any combination thereof.

[0141] Methods of Use

[0142] The compositions of the invention can be used as vaccines for inducing an immune response to an orthopoxvirus, for example an mpox virus, smallpox virus, a borealpox virus, vaccinia virus, or other disease causing orthopoxvirus, In some embodiments, the orthopoxvirus is an mpox virus such as a Clade I and / or Clade II mpox virus.

[0143] In exemplary embodiments, the present invention provides a method of preventing a mpox virus infection to a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once. In some embodiments, the mpox virus is a Clade I mpox virus. In some embodiments, the mpox virus is a Clade II mpox virus.

[0144] In exemplary embodiments, the present invention provides a method of inhibiting an mpox virus infection in a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once. In some embodiments, the mpox virus is a Clade I mpox virus. In some embodiments, the mpox virus is a Clade II mpox virus. In some embodiments, the mpox virus is a Clade II b mpox virus.

[0145] In exemplary embodiments, the present invention provides a method of inducing an immune response to an mpox virus in a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once. In some embodiments, the mpox virus is a Clade I mpox virus. In some embodiments, the mpox virus is a Clade II mpox virus. The immune response may be a cellular immune response or a humoral immune response, or a combination thereof. In one embodiment, the immune response is a broadly neutralizing antibody response to mpox virus. In some embodiments, the immune response is a T- cell response to mpox virus.

[0146] In exemplary embodiments, the present invention provides a method of treating a mpox virus infection in a subject in need thereof (e.g., an exposed subject, such as a subject who has been recently exposed but is not yet symptomatic, or a subject who has been recently exposed and is only mildly symptomatic), said method comprising administering a pharmaceutical composition comprising MVA-LD10 vector to the subject in a therapeutically effective amount. The result of treatment is a subject that has an improved therapeutic profde. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once. In some embodiments, the mpox virus is a Clade T mpox virus. In some embodiments, the mpox virus is a Clade II mpox virus. In some embodiments, the mpox virus is a Clade lib mpox virus.

[0147] In an alternative aspect, provided herein is a method for preventing an mpox virus infection in a subject comprising administering to the subject a modified vaccinia Ankara (MVA) vector and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody, antibody fragment, antigen binding fragment, or peptide. In some embodiments, immune checkpoint inhibitor is encoded by the MVA vector.

[0148] In another aspect, provided herein is a method of reducing the effects of an mpox virus infection in a subject comprising administering to the subject an MVA vector and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody, antibody fragment, antigen binding fragment, or peptide. In some embodiments, immune checkpoint inhibitor is encoded by the MVA vector.

[0149] In another aspect, provided herein is a method of inducing a protective immune response against an mpox virus in a subject comprising administering to the subject an MVA vector and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody, antibody fragment, antigen binding fragment, or peptide. In some embodiments, immune checkpoint inhibitor is encoded by the MVA vector.

[0150] In some embodiments, the immune checkpoint inhibitor inhibits programmed-cell death protein 1 (PD-1), programed cell death ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In some embodiments, immune checkpoint inhibitor is encoded by the MVA vector. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is selected from nivolumab, pembrolizumab, cemiplimab, dostarlimab, pidilizumab, AMP -224, AMP-514, sintilimab, sasanlimab, spartalizumab, retifanlimab, tislelizumab, toripalimab, camrelizumab, CS1003, zimberelimab, or JTX-4014. In some embodiments, the immune checkpoint inhibitor is a PD-Ll inhibitor. In some embodiments, the PD-L1 inhibitor is selected from atezolizumab, durvalumab, avelumab, envafolimab, BMS- 936559, BMS-986189, lodapolimab, cosibelimab, sugemalimab, adebrelimab, CBT-502, AUNP12, CA-170, or BGB-A333. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is selected from ipilimumab, tremelimumab, AGEN1884, or AGEN2041. In one alternative aspect, provided herein is a method for inducing a protective immune response against an mpox virus in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising MVA-LD01. In some embodiments, the MVA- LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the induced protective immunity is to mpox Clade I. In some embodiments, the induced protective immunity is to mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen.

[0151] In another alternative aspect, provided herein is a method of reducing the effects of an mpox virus infection in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising MVA-LD01. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the mpox virus is mpox Clade I. In some embodiments, the mpox virus is mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen.

[0152] In another alternative aspect, provided herein is a method of preventing an mpox virus infection in a subject, such as a human, comprising administering to the subject a pharmaceutical composition comprising MVA-LD01. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the mpox virus is mpox Clade I. In some embodiments, the mpox virus is mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen.

[0153] In some embodiments, the subject is human. In some embodiments, the subject has not been previously exposed to an mpox virus. In some embodiments, the mpox virus is a Clade I mpox virus. In some embodiments, the mpox virus is a Clade II mpox virus. In some embodiments, the mpox virus is a Clade lib mpox virus. In some embodiments, the pharmaceutical composition is administered in a single dose only once. In some embodiments, the pharmaceutical composition is administered in a therapeutically effective amount in two doses during a prime boost regimen. In an alternative aspect, the compositions of the invention as described herein can be used as vaccines for inducing an immune response to a smallpox virus. In some embodiments, the subject has not been previously exposed to a smallpox virus.

[0154] In some embodiments, the present invention provides a method of preventing a smallpox virus infection to a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0155] In some embodiments, the present invention provides a method of inhibiting a smallpox virus infection in a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0156] In some embodiments, the present invention provides a method of inducing an immune response to a smallpox virus in a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in an therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0157] In some embodiments, the present invention provides a method of treating a smallpox virus infection in a subject in need thereof (e.g., an exposed subject, such as a subject who has been recently exposed but is not yet symptomatic, or a subject who has been recently exposed and is only mildly symptomatic), said method comprising administering a pharmaceutical composition comprising MVA-LD10 vector to the subject in a therapeutically effective amount. The result of treatment is a subject that has an improved therapeutic profile. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0158] In another alternative aspect, the compositions of the invention as described herein can be used as vaccines for inducing an immune response to a borealpox virus. Borealpox virus was formerly known as Alaskapox virus and was first documented in Alaska, United States in 2015 (Springer, Y.P. et al. Novel Orthoorthopoxvirus Infection in an Alaska Resident. Clin Infect Dis. 64(12): 1737-1741(2017 Jun 15)). In some embodiments, the subject has not been previously exposed to a borealpox virus.

[0159] In some embodiments, the present invention provides a method of preventing a borealpox virus infection to a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0160] In some embodiments, the present invention provides a method of inhibiting a borealpox virus infection in a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0161] In some embodiments, the present invention provides a method of inducing an immune response to a borealpox virus in a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0162] In some embodiments, the present invention provides a method of treating a borealpox virus infection in a subject in need thereof (e.g., an exposed subject, such as a subject who has been recently exposed but is not yet symptomatic, or a subject who has been recently exposed and is only mildly symptomatic), said method comprising administering a pharmaceutical composition comprising MVA-LD10 vector to the subject in a therapeutically effective amount. The result of treatment is a subject that has an improved therapeutic profile. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once. In an alternative aspect, the compositions of the invention as described herein can be used as vaccines for inducing an immune response to a vaccinia virus. In some embodiments, the subject has not been previously exposed to a smallpox virus.

[0163] In some embodiments, the present invention provides a method of preventing a vaccinia virus infection to a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0164] In some embodiments, the present invention provides a method of inhibiting a vaccinia virus infection in a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0165] In some embodiments, the present invention provides a method of inducing an immune response to a vaccinia virus in a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in an therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0166] In some embodiments, the present invention provides a method of treating a vaccinia virus infection in a subject in need thereof (e.g., an exposed subject, such as a subject who has been recently exposed but is not yet symptomatic, or a subject who has been recently exposed and is only mildly symptomatic), said method comprising administering a pharmaceutical composition comprising MVA-LD10 vector to the subject in a therapeutically effective amount. The result of treatment is a subject that has an improved therapeutic profile. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0167] In an alternative aspect, the compositions of the invention as described herein can be used as vaccines for inducing an immune response to a disease causing orthopoxvirus. Such disease causing orthopoxvirus may include, but are not limited to, Akhmeta virus, Camelpox virus, Cowpox virus, Ectromelia virus, Raccoonpox virus, Skunkpox virus, Taterapox virus, Volepox virus, smallpox virus, mpox virus, borealpox virus, buffalopox, and vaccinia virus. In some embodiments, the subject has not been previously exposed to a disease causing orthopoxvirus.

[0168] In some embodiments, the present invention provides a method of preventing a disease causing orthopoxvirus infection to a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0169] In some embodiments, the present invention provides a method of inhibiting a disease causing orthopoxvirus infection in a subject in need thereof (e g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in a therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0170] In some embodiments, the present invention provides a method of inducing an immune response to a disease causing orthopoxvirus in a subject in need thereof (e.g., an unexposed subject), said method comprising administering a pharmaceutical composition comprising an MVA-LD10 vector to the subject in an therapeutically effective amount. The result of the method is that the subject is partially or completely immunized against the virus. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once.

[0171] In some embodiments, the present invention provides a method of treating a disease causing orthopoxvirus infection in a subject in need thereof (e.g., an exposed subject, such as a subject who has been recently exposed but is not yet symptomatic, or a subject who has been recently exposed and is only mildly symptomatic), said method comprising administering a pharmaceutical composition comprising MVA-LD10 vector to the subject in a therapeutically effective amount. The result of treatment is a subject that has an improved therapeutic profile. In some embodiments, the pharmaceutical composition comprising an MVA-LD10 vector is administered only once. In another alternative aspect, provided herein is a method of vaccinating a subject against an mpox infection comprising administering a single dose of a pharmaceutical composition comprising an MVA-LD01 vector. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the mpox virus is mpox Clade I. In some embodiments, the mpox virus is mpox Clade II. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen.

[0172] In yet another alternative aspect, provided herein is a method of vaccinating a subject against a smallpox infection comprising administering a single dose of a pharmaceutical composition comprising an MVA-LD01 vector. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen.

[0173] In another alternative aspect, provided herein is a method of vaccinating a subject against a borealpox infection comprising administering a single dose of a pharmaceutical composition comprising an MVA-LD01 vector. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen.

[0174] In another alternative aspect, provided herein is a method of vaccinating a subject against a vaccinia infection comprising administering a single dose of a pharmaceutical composition comprising an MVA-LD01 vector. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen.

[0175] In another alternative aspect, provided herein is a method of vaccinating a subject against a disease causing orthopoxvirus infection comprising administering a single dose of a pharmaceutical composition comprising an MVA-LD01 vector. In some embodiments, the MVA- LD01 is administered in a therapeutically effective amount in a single dose. In some embodiments, the mpox virus is mpox Clade lib. In some embodiments, the MVA-LD01 is administered in a therapeutically effective amount in two doses during a prime boost regimen. A subject to be treated according to the methods described herein (e.g., a subject infected with mpox virus infection) may be one who has been diagnosed by a medical practitioner as having such a condition. Diagnosis may be performed by any suitable means. A subject in whom the development of an infection is being prevented may or may not have received such a diagnosis. One skilled in the art will understand that a subject to be treated according to the present invention may have been identified using standard tests or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., exposure to mpox, etc.).

[0176] Prophylactic treatment may be administered, for example, to a subject not yet exposed to or infected by an orthopoxvirus, but who is susceptible to, or otherwise at risk of exposure or infection with a orthopoxvirus.

[0177] Therapeutic treatment may be administered, for example, to a subject already exposed to or infected by a orthopoxvirus, who is not yet ill, or showing symptoms or infection, suffering from a disorder in order to improve or stabilize the subject's condition (e.g., a patient already infected with mpox virus). The result is an improved therapeutic profile. In some instances, as compared with an equivalent untreated control, treatment may ameliorate a symptom associated with mpox infection by, e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% as measured by any standard technique. In some instances, treating can result in the inhibition of viral replication, a decrease in viral titers or viral load, eradication or clearing of the virus. In other embodiments, treatment may result in amelioration of one or more symptoms of the infection, including any symptom identified above. According to this embodiment, confirmation of treatment can be assessed by detecting an improvement in or the absence of symptoms.

[0178] In other embodiments, treatment may result in reduction or elimination of the ability of the subject to transmit the infection to another, uninfected subject. Confirmation of treatment according to this embodiment is generally assessed using the same methods used to determine amelioration of the disorder, but the reduction in viral titer or viral load necessary to prevent transmission may differ from the reduction in viral titer or viral load necessary to ameliorate the disorder.

[0179] Dosage The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, immunogenic, and protective. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the immune system of the individual to synthesize antibodies, and, if needed, to produce a cell- mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be monitored on a patient-by-patient basis. However, suitable dosage ranges are readily determinable by one skilled in the art and generally range from about 5.0 x 106TCIDso / mL to about 5.0 x 109TCIDso / mL. The dosage may also depend, without limitation, on the route of administration, the patient's state of health and weight, and the nature of the formulation.

[0180] The pharmaceutical compositions of the invention are administered in such an amount as will be prophylactically and / or therapeutically effective, immunogenic, and / or protective against a pathogenic species of mpox virus. The dosage administered depends on the subject to be treated (e.g., the manner of administration and the age, body weight, capacity of the immune system, and general health of the subject being treated). The composition is administered in an amount to provide a sufficient level of expression that elicits an immune response without undue adverse physiological effects. In some embodiments, the MV A vector in the composition, e.g., MVA- LD 10 is administered at a dosage of, e.g., between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of the viral vector, preferably between about 1.0 x 105TCIDso / mL and about 1.0 x 1011TCIDso / mL, more preferably between about 1.0 x 106and about 1.0 x 1010TCIDso / mL, or most preferably between about 5.0 x 106and about 5.0 x 109TCIDso / mL. The composition may include, e.g., at least about 5.0 x 106TCIDso / mL of the viral vector (e.g., about 1.0 x 108TCIDso / mL of the viral vector). A physician or researcher can decide the appropriate amount and dosage regimen.

[0181] The composition may include, e.g., between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of the viral vector, preferably between about 1.0 x 105TCIDso / mL and about 1.0 x 1011TCIDso / mL, more preferably between about 1.0 x 106and about 1.0 x 1010TCIDso / mL, or most preferably between about 5.0 x 106and about 5.0 x 109TCIDso / mL. The composition may include, e.g., at least about 5.0 x 106TCIDso / mL of the viral vector (e.g., about 1.0 x 108TCIDso / mL of the viral vector). The method may include, e.g., administering the composition to the subject a single time. Alternative embodiments include the converted dosages from TCIDso / mL to PFU / mL by multiplying the TCIDso / mL by 0.7 (Poisson distribution):

[0182] TCIDso / mL x 0.7 = PFU / mL

[0183] To convert TCIDso / mL to PFU / mL, the Poisson distribution can be applied, wherein “P(o)” is the proportion of negative tubes and “m” is the mean number of infectious units per volume (PFU / mL), and P(o) = e(-m). For any titer expressed as TCIDso / mL, P(o) = 0.5. Thus, e(-m) = 0.5 and m = -ln(0.5), which is about 0.7. Therefore, one could multiply the TCIDso / mL titer by 0.7 to predict the mean number of PFU / mL. In a non-limiting example, a TCIDso titer of 1 x 105TCIDso / mL will produce approximately 0.7 x 105PFU / mL. When applying this calculation, the estimated mean will only be valid if the changes in the protocol required to visualize plaques do not alter viral expression as compared to conditions used to determine TCIDso / mL. For viruses normally propagated in chicken eggs, viral titer is calculated as the chicken embryo infectious dose (CEID). Viral cultures are serially diluted and are used to inoculate embryonated chicken eggs. Following incubation, allantoic fluid is harvested from each egg at all dilutions and virus titer is determined by the appearance of hemagglutination. A positive hemagglutination reaction indicates the virus is present at that dilution.

[0184] Alternative embodiments for suitable dosage ranges are readily determinable by one skilled in the art and generally range from about 3.50 x 106PFU / mL to about 3.50 x 109PFU / mL. The dosage may also depend, without limitation, on the route of administration, the patient's state of health and weight, and the nature of the formulation.

[0185] In alternative embodiments, the MV A vector in the composition, e.g., MVA-LD10 is administered at a dosage of, e.g., between about 7.0 x 103and about 7.0 x 1012PFU / mL of the viral vector, preferably between about 7.0 x 104PFU / mL and about 7.0 x 1010PFU / mL, more preferably between about 7.0 x 105and about 7.0 x 109PFU / mL, or most preferably between about 3.50 x 106and about 3.50 x 109PFU / mL. In alternative embodiments, the composition may include, e.g., at least about 3.50 x 106PFU / mL of the viral vector (e.g., about 7.0 x 107PFU / mL of the viral vector). A physician or researcher can decide the appropriate amount and dosage regimen.

[0186] In alternative embodiments, the composition may include, e.g., between about 7.0 x 103and about 7.0 x 1012PFU / mL of the viral vector, preferably between about 7.0 x 104PFU / mL and about 7.0 x I O10PFU / mL, more preferably between about 7.0 x 105and about 7.0 x 109PFU / mL, or most preferably between about 3.50 x 106and about 3.50 x 109PFU / mL. In alternative embodiments, the composition may include, e.g., at least about 3.50 x 106PFU / mL of the viral vector (e.g., about 7.0 x 107PFU / mL of the viral vector). The method may include, e.g., administering the composition to the subject a single time.

[0187] EXAMPLES

[0188] The claimed invention is further described by way of the following non-limiting examples. Further aspects and embodiments of the present invention will be apparent to those of ordinary skill in the art, in view of the above disclosure and following experimental exemplification, included by way of illustration and not limitation, and with reference to the attached figures.

[0189] Example 1. Construction of MVA-LD10

[0190] The construction of MVA-LD10 has been previously described in WO 2022 / 169895, incorporated herein in its entirety. MVA-LD10 was constructed to express an optimized nucleic acid sequence (SEQ ID NO: 7) encoding five repeats of LD10 (SEQ ID NO: 1) in polycistronic format and operably linked to the modified H5 early / late vaccinia promoter (SEQ ID NO: 13). A signal sequence (SEQ ID NO: 2) was added to the N-terminus of LD10 to route the peptides for secretion from the cell and a hybrid dual cleavage site (SEQ ID NO: 5) was added following the sequences to facilitate production of monomer peptides from the polycistronic design. The resultant LD10 insert encoded for the amino acid sequence of SEQ ID NO: 6. See LD10 insert illustration in FIG. 1. The starting material for recombinant virus production was parental MVA that had been harvested in 1974, before the appearance of Bovine Spongiform Encephalopathy / Transmissible Spongiform Encephalopathy (BSE / TSE) and plaque purified 3 times using certified reagents from sources free of B SE. A shuttle vector was used to insert the LD 10 sequences between two essential genes I8R / G1L of MVA by means of homologous recombination. The chosen insertion site has been identified as supporting high expression and insert stability. All inserted sequences were codon optimized for MVA.

[0191] Silent mutations were introduced to interrupt homo-polymer sequences (>4G / C and >4A / T), which reduce RNA polymerase errors that possibly lead to frameshift mutations. Vectors, Research Seed Virus (RSV), and Research Stocks (RS) were prepared in a dedicated room with full traceability and complete documentation of all steps using BSE / TSE-free raw materials, and therefore can be directly used for production of cGMP Master Seed Virus (MSV). For production of RSV for animal studies, a chicken embryo fibroblast cell line, DF-1 cells (ATCC, CRL-12203), were seeded into sterile tissue culture flasks and infected with MVA-LD10 at an MOI of 0.01.

[0192] Cells were recovered 3 days post-infection, disrupted by sonication, and bulk harvest material clarified by low-speed centrifugation. The clarified viral harvest was purified using sucrose cushion ultracentrifugation twice. The purified viruses were titrated by limiting dilution in DF1 cells, diluted to I MO8TCIDso / mL (7* 107PFU / mL) in sterile PBS + 7% sucrose, dispensed into sterile vials, and stored at -80°C.

[0193] To establish the expression of LD10 from the recombinant MVA-LD10 viral vector, DF1 cells were cultured and infected with either parental modified vaccinia Ankara (MV A), recombinant MVA-LD10, or left uninfected. Two days following infection, supernatant was harvested and dotted onto membrane along with 2.5 ng chemically synthesized LD10 peptide. The membrane was probed with a primary antibody specific for LD10 and a secondary

[0194] HRP antibody and visualized by chemiluminescence. Labeling of peptide and the MVA-LD10 sample confirmed LD10 expression in MVA-LDlO-infected cells (FIG. 2).

[0195] Example 2. Efficacy of MVA-LD10 in a lethal challenge model

[0196] Introduction

[0197] As a proof-of-concept, preclinical efficacy of a single prime dose of MVA-LD10 was evaluated in a lethal orthopoxvirus challenge model compared to single prime and double primeboost regiments of MVA.

[0198] Immunized mice were also evaluated for CD4+ T cell and CD8+ T cell functionality by re-stimulation of splenocytes with MVA and intracellular cytokine staining followed by flow cytometry to access the ability of MVA, MVA-LD10, and AC AM 2000 to induce cellular immunity.

[0199] Methods

[0200] C57B1 / 6 mice were immunized via the intramuscular (IM) route at a dose of IxlO7PFU on study days 0 and / or 28. On study day 150, animals were challenged intranasally (IN) with IxlO6PFU Vaccinia WR strain according to Table 4 below.

[0201] Table 4. Preclinical Study Design: Durability

[0202] On study days 0, 27, 55, 88, and 149 pre-challenge sera were collected for humoral immunity analysis. This study monitored mice daily for weight loss, clinical scoring, and survival. Fourteen days following challenge, on Day 164, all the surviving animals were euthanized and relevant tissues were harvested for immunogenicity. Relevant tissues were harvested for analysis of viral titers and immune profile from all animals that succumb to infection or reach the study endpoint.

[0203] Sample Collection and analysis

[0204] 1. Blood collection and serum processing were performed on days 0, 27, 55, 122, 153 (tail or cheek) and day 55 (terminal bleed) (n=4 / group).

[0205] 2. Lungs and spleens on days 150 (n=4 / group) were harvested. Lungs were fixed in 10% NBF for pathology. Spleens were incubated in media and analyzed by intracellular cytokine staining (ICS).

[0206] 3. Blood collection and serum processing were performed on days 3 -5 post infection. Viremia and known cytokines were analyzed.

[0207] 4. Lungs, Spleens and Liver from recovered animals were harvested on termination day, fixed, and assessed for pathology. If available, Formulation Buffer-treated animals before sacrificing will be harvested and assayed for pathology.

[0208] Stimulations and Intracellular Cytokine Staining (ICS) assay

[0209] Intracellular cytokine flow assay was used to detect the MVA specific cellular immune response induced by AC AM 2000, MVA, or MVA-LD10. Mice were euthanized on day 150 postimmunization and single cell suspension of spleens were prepared by homogenizing spleens and passing through 70 pm cell strainers and after RBC lysis, 1 x 106splenocytes per well were inoculated on 96-well plates and infected with MVA virus at a multiplicity of infection (MOI) of

[0210] 5. After 2 h at 37°C, brefeldin A and monensin were added to a concentration of 10 pg / ml, and incubation was continued for 6 h. At the end of stimulation, cells were stained with Live / Dead FITC dye (ThermoFisher; L23101) according to manufacturer recommendation and fixed and permeabilized with Cytofix / Cytoperm solution (Pharmingen, Inc.). Cells were then incubated with of CD3-APC Cy7, CD4-PE Cy7, CD8-Percp Cy5.5 and IFN-y -APC fluorochrome conjugated antibodies for 30 min at 4°C. Approximately 200,000 lymphocytes were acquired on the Attune NxT flow cytometer and analyzed for MVA-specific CD4 and CD8 T cells using FloJo software (Treestar Inc. San Carlos, CA). Results

[0211] Mice were administered a lethal, intranasal dose at 17 weeks (day 150) post -vaccination. At this late challenge timepoint, over 17 weeks following vaccination (day 150), MVA-LD10 demonstrated the highest degree of protection from disease as observed by minimal weight loss (FIG. 3A) and disease scores (FIG. 3B). As previously seen, a single dose of MVA-LD10 offered a higher degree of protection from death and disease following late challenge, while a single prime dose of MVA exhibited a reduced probability of survival in comparison (FIG. 3C).

[0212] Analysis of post-vaccination, pre-challenge cellular immunity revealed an increase of CD4+ T cells (FIG. 4A) and significant increase of functional antigen-specific CD8+ T cells (FIG. 4B) 17 weeks following vaccination from single dose MVA-LD10 as compared to one dose of MVA. This indicates a strong, durable T cell memory response formed from a single-dose of MVA-LD10.

[0213] Together, these data demonstrate that MVA-LD10 offers superior protection from orthopoxvirus challenge than one or two doses of MVA, and disease protection correlated with cellular and not humoral immunity.

[0214] Example 3. Efficacy of MVA and MVA encoding LD10 vaccine in a lethal challenge model Introduction

[0215] Having demonstrated a durable response of prime MVA-LD10 in response to a standard lethal IxlO6PFU Vaccinia WR strain intranasal dose challenge at Day 150, the same prime MVA- LD10 regimen was tested at an earlier time point with an increased challenge dose. The preclinical efficacy of MVA-LD10 was therefore evaluated in a orthopoxvirus challenge model with an increased vaccinia dose of IxlO7PFU Vaccinia WR strain at substantially earlier challenge points relative to Example 2 (Day 56, Day 90) to analyze the ability of a prime MVA-LD10 regimen to reduce viral load in sera of mice shortly following immunization as compared to prime or primeboost parental MVA regimens. Methods

[0216] C57B1 / 6 mice (n=16 / group) were immunized via the intramuscular (IM) route at a dose of IxlO7PFU on study days 0 and / or 28. On study days 55 and 90, animals (n=6 / group) were challenged intranasally (IN) with IxlO7PFU Vaccinia WR strain according to Table 5 below.

[0217] Table 5. Study Design

[0218] Pre-challenge sera were collected for humoral immunity analysis. This study monitored mice daily for weight loss, clinical scoring, and survival. Fourteen days following challenge, at either Day 70 or Day 104, all the surviving animals were euthanized and relevant tissues were harvested for immunogenicity. Relevant tissues (TBD) were harvested for analysis of viral titers and immune profde from all animals that succumb to infection or reach the study endpoint.

[0219] Sample Collection and analysis

[0220] 1. Blood collection and serum processing were performed. Live neutralizing assays of Vaccinia and Monkey pox strains were performed.

[0221] 2. Lungs and spleens were harvested. Lungs were fixed in 10% NBF for pathology. Spleens were incubated in media and analyzed by intracellular cytokine staining (ICS).

[0222] 3. Blood collection and serum processing were performed on days 3 -5 post infection. Viremia and known cytokines were analyzed.

[0223] 4. Lungs, Spleens and Liver from recovered animals were harvested on termination day, fixed, and assessed for pathology. If available, Formulation Buffer-treated animals before sacrificing will be harvested and assayed for pathology.

[0224] Viremia analysis PrimeTime Gene Expression 2x Mastermix (IDT) was utilized in 40 pl qPCR reactions. Here, 16.25 pl of DNA extracted from blood was utilized to determine the relative concentration of viral PFUs to cell concentration via delta CT analysis between E3L and B-Actin. Primers and probes were utilized at 100 nmol concentration. B-Actin utilized 0.3 pl primer and 0.15 pl probe while A12L and E3L viral genes were detected utilizing 0.6 pl and 0.3 pl probe this was used as a manner of primer limiting of B-Actin. Standard curves were generated from DNA extracted from 20 pl of 1.7xl09PFU / mL Viral sample utilizing the HotShot Lysis Method utilizing 100 pl oflysis solution and 100 pl of neutralization solution. This was then diluted down at a 1 : 10 to form the base concentration utilized for standard curve setup. Mouse DNA was extracted from 4x106L929 cells also utilizing the Hot Shot Cell Lysis method as described above. Viral and Mouse DNA were added together at a 1:10 ratio to form the base DNA utilized for the standard curve. A seven step 2-fold dilution was utilized to more accurately detect the amount of virus and mouse DNA range that was observed in our samples. A total of 11 pl of DNA from each dilution was utilized and supplemented with molecular grade water to represent the 16.25 pl of DNA used in reactions. qPCR reactions were completed in the Quant Studio 6 Pro where the reaction was first activated at 95°C for 3 minutes, then completed 40 cycles of 95°C Denaturing 50°C annealing and then 60°C elongation. Mouse DNA alone was utilized to set the zero-point of detection for A12L and E3L to account for off-target effects and Viral DNA was utilized to set the zero-point of detection for B-Actin.

[0225] Results

[0226] Mice were administered a lethal, intranasal dose at 4 weeks (day 55) and 9 weeks (day 90) post-vaccination.

[0227] The challenge timepoint of 4 weeks (day 55) post-vaccination revealed that body weight was stable across MVA-LD10 and MVA treatment groups (FIG. 5 A). All MVA treatment groups exhibited low clinical scores following the day 55 challenge (FIG. 5B), whereas the control mice had clinical scores of greater than 4 as soon as 4 days post-challenge (FIG. 5C). Unexpectedly, one dose of MVA-LD10 offered complete protection from death similar to two doses of MVA, whereas one dose was not fully protective (FIG. 5D). Evaluation of viremia 3 days following the Day 56 challenge revealed a significant reduction in viral load in sera of mice immunized with MVA-LD10 as compared to MVA (FIG. 6).

[0228] The extended challenge timepoint of day 90 post-vaccination revealed that body weight was also stable across MVA-LD10 and MVA treatment groups (FIG. 7A). Following this day 90 lethal challenge, a single dose of MVA-LD10 surprisingly provided the lowest clinical scores as compared to single and double dose of MVA vaccination (FIG. 7B). For example, subjects administered control and MVA prime regimen had clinical scores of 5 or greater as soon as 5 days post-challenge (FIG. 7C), whereas MVA-LD10 had a clinical score of approximately 1 at the same time point. In agreement with this, MVA-LD10 surprisingly offered a higher degree of protection from death and disease than two doses of MVA, as no mice died following challenge compared to the MVA single-dose group where 4 of 6 mice succumbed to challenge (FIG. 7D).

[0229] In another study, the mock-vaccinated group (FB / FB) exhibited high viral titers, whereas MVA / MVA and single-dose MVA-LD10 groups displayed significantly reduced viral burdens (FIG. 7E). Furthermore, no difference was observed between the MVA / MVA and single-dose MVA-LD10 groups in a PFU Assay following VACV challenge, indicating comparable suppression of viral replication at the site of infection (FIG. 7F).

[0230] Together, these data demonstrate that vaccination with a single dose of MVA-LD10 strikingly offered superior protection from weight loss and disease compared to one or two doses of MVA at lethal challenge timepoints at Day 55 and Day 90.

[0231] Example 4. Assessing Vaccine Humoral Immunity

[0232] Terminal whole blood will be collected by cardiac puncture and sera will be separated at the study endpoint. Animal sera will be evaluated by ELISA to determine the presence of IgG antibody against MVA, vaccinia virus, and / or MP XV antigens A29L, B6R, H3L, HL, Ml, A35R. Sera reactivity will be evaluated to each MPXV antigen individually. IgG subtypes will also be measured by ELISA with a view toward identifying Thl / Th2 -balanced or Thl-oriented response.

[0233] Serial dilutions of mouse sera will also be used in a live virus mpox clade I neutralization assay to determine 50% plaque reduction neutralization titers of each vaccine group as performed previously for preliminary studies. Finally, evaluation of antibody-dependent effector mechanisms as described previously will be conducted. Briefly, sera from the vaccine groups will be incubated with mpox antigen coated fluorescent beads and incubated with either mouse neutrophils or monocytes. Cells will then be analyzed by flow cytometry to quantify phagocytic intake of fluorescent bead / antibody complexes. Sera will also be used to assess NKL antibody mediated activity against MPXV by adding sera to plates coated with dominant mpox virus antigens. Followed by the addition of freshly isolated mouse NK cells which will then be assessed by flow cytometry for expression of three activation markers: CD107a, ITNy, and MIP-ip.

[0234] Example 5. Assessing Vaccine Cellular Immunity

[0235] Following vaccination of 6-8 weeks of BALB / c mice with MVA or MVA-LD10, the Thl or Th2 profde of CD4+ and CD8+ T cells will be evaluated in splenocytes by ex vivo stimulation with dominant mpox antigens by intracellular cytokine staining (ICS). The study will further extend to characterize mpox-specific long-persistent memory T cell response 4 weeks after the final immunization. CD4+ and CD8+ T cells isolated from lymph nodes (LN) and spleens will undergo surface staining of IL-7Ra (CD127)+ and CD62L+ which will define subpopulations of naive and memory T cells. For example, follicular helper T (Tfh) cells have been implicated as being essential for germinal center (GC) responses. Tfh cells will also be measured from vaccinated or control splenocytes by surface staining of CD4+ICOS+CXCR5+ or CD4+PD- lhiCXCR5+ cells, the latter likely indicating a “GC-like” phenotype.

[0236] Additionally, regulatory T cells will be measured by staining of CD4+CD25+FoxP3+ to determine if blocking PD1 with LD10 from the MVA-LD10 vaccination, alters the regulatory T cell population.

[0237] Example 6. Determining Vaccine Efficacy According to Morbidity and Mortality Against Lethal Clade I MPOX in a CAST / Ei-mpox Mouse Model

[0238] Five groups of 12 female CAST Ei / J mice will be immunized with either 1) Formulation buffer, 2) Two doses of MVA, 3) One dose of MVA, 4) One dose of MVA-LD10, or 5) MVA plus anti-PD-Ll antibody. Following 4 weeks after the final immunization, CAST Ei / J mice will be challenged with mpox clade I intranasally (IN) with a challenge dose determined during the model testing phase. See Table 6 below. Table 6. Mouse study groups for Clade T Mpox challenge

[0239] Animals will be observed for changes in body temperature, visually inspected clinical symptoms such as ruffled fur, hunched posture, respiratory distress and lethargy. Clinical score will be generated by combining all these parameters. Animals will be monitored for weight loss by measuring body weights daily post challenge and finally mortality will be reported as a Kaplan- Meier survival analysis.

[0240] Example 7. Determining Vaccine Efficacy According to Viremia and Disease Pathology Against Lethal Clade I MPOX in a CAST / Ei-mpox Mouse Model

[0241] Seven days following challenge with mpox clade I, study day 63, blood will be collected from all groups and inactivated by addition of ATL / Proteinase K lysis buffer and incubating at 56°C for 1 hour. Whole blood will be evaluated for viremia by quantitative PCR (qPCR) through evaluation of genome copies using primers specific for the orthopoxvirus polymerase as described previously.

[0242] Sera will also be separated and evaluated for a panel of proinfl ammatory cytokines including using a predefined mouse Luminex cytokine panel.

[0243] Seven days post-challenge 4 animals will be euthanized, and terminal blood will be collected by cardiac puncture. Additionally, lungs, liver, spleen, ovaries, and brain will be harvested. Tissues will be split in half for assessing the viremia and the other half for immunopathology to inform viral load, dissemination and histopathology. Viremia analyses will be done by grounding half of each tissue, isolating DNA, and virus titers determined by qPCR as performed at day 3 post-challenge above. The other half of the tissue will be fixed in 10% neutral buffered formalin and subsequently embedded in paraffin wax. Formalin fixed paraffin embedded (FFPE) blocks will be sectioned and evaluated for gross pathology and histopathology by H&E staining, and T cell infiltration by CD3 staining and viral burden with anti-vaccinia antibodies that cross react to mpox virus. Serum collected from terminal bleed will also be assessed for viremia by qPCR and a panel of cytokines by Luminex as described for day 3 above. Fourteen days following challenge, all surviving animals will be euthanized, tissues will be collected, and same sample analysis will be performed as described from day 63 harvest. The primary endpoint of this study is disease-free survival. Efficacy analysis, however, will also include viral load and histopathology. With these analyses, efficacy of one-dose MVA-LD10 will be compared to a one- dose or two-dose MVA regimen.

[0244] Example 8. NHP Study Design

[0245] Introduction

[0246] A preclinical efficacy study of MVA-LD10 will be evaluated in a nonhuman primate (NHP) challenge model compared to double prime-boost regiments of MVA.

[0247] Methods

[0248] NHPs will be immunized by IM injection at a dose of 0.5 mL IxlO8PFU on study days 0 and / or 28. On study day 56, animals will be intratracheally challenged with 5xl07PFU of clade I MPXV according to Table 7 below.

[0249] Table 7. NHP Study Design

[0250] Pre-challenge sera will be collected for analysis humoral immunity. This study will be monitored mice daily for weight loss, clinical scoring, and survival. Following challenge, surviving animals will be euthanized and relevant tissues will be harvested for immunogenicity. Relevant tissues will be harvested for analysis of viral titers and immune profile from all animals that succumb to infection or reach the study endpoint.

[0251] Sample Collection and analysis

[0252] 1. Blood collection and serum processing will be performed. 2. Lungs and spleens will be harvested. Lungs will be fixed in 10% NBF for pathology.

[0253] Spleens will be incubated in media and analyzed by intracellular cytokine staining (ICS).

[0254] 3. Blood collection and serum processing will be performed on days 3-5 post infection. Viremia and known cytokines will be analyzed.

[0255] 4. Lungs, Spleens and Liver from recovered animals will be harvested on termination day, fixed, and assessed for pathology. If available, Formulation Buffer-treated animals before sacrificing will be harvested and assayed for pathology.

Claims

CLAIMSWe Claim:

1. A method for preventing an mpox infection in a subject comprising administering to the subject a pharmaceutical composition comprising a modified vaccinia Ankara (MV A) vector encoding an amino acid sequence comprising SEQ ID NO: 6 (MVA- LD10 vector).

2. A method of reducing the effects of an mpox infection in a subject comprising administering to the subject a pharmaceutical composition comprising MVA-LD10 vector.

3. A method of inducing a protective immune response against an orthopoxvirus in a subject comprising administering to the subject a pharmaceutical composition comprising MVA-LD10 vector.

4. The method of claims 1-3, wherein the subject is human.

5. The method of claims 1-4, wherein the subject has not been previously exposed to an mpox virus.

6. The method of claims 1-5, wherein the mpox virus is a Clade I mpox virus.

7. The method of claims 1-5, wherein the mpox virus is a Clade II mpox virus.

8. The method of claims 1-5, wherein the mpox virus is a Clade lib mpox virus.

9. The method of claims 1-8, wherein the pharmaceutical composition is administered in a single dose only once.

10. The method of claims 1-9, wherein the pharmaceutical composition comprises between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of MVA-LD10 vector.

11. The method of claims 1 -9, wherein the pharmaceutical composition comprises between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of MVA-LD10 vector.

12. The method of claims 1 -9, wherein the pharmaceutical composition comprises between about 1.0 x 105TCIDso / mL and about 1.0 x 1011TCIDso / mL of MVA-LD10 vector.

13. The method of claims 1-9, wherein the pharmaceutical composition comprises between about 1.0 x 106and about 1.0 x 1010TCIDso / mL of MVA-LD10 vector.

14. The method of claims 1 -9, wherein the pharmaceutical composition comprises between about 5.0 x 106and about 5.0 x 109TCIDso / mL of MV A-LD 10 vector.

15. The method of claims 1 -9, wherein the pharmaceutical composition comprises at least about 5.0 x 106TCIDso / mL of MVA-LD10 vector.

16. The method of claims 1-9, wherein the pharmaceutical composition comprises about 1.0 x 108TCIDso / mL of MVA-LD10 vector.

17. A method of vaccinating a subject against an mpox infection comprising administering a single dose of a pharmaceutical composition comprising an MVA-LD10 vector.

18. The method of claim 17, wherein the subject has not been previously exposed to an mpox virus.

19. The method of claims 17-18, wherein the mpox virus is a Clade I mpox virus.

20. The method of claims 17-18, wherein the mpox virus is a Clade II mpox virus.

21. The method of claims 17-18, wherein the mpox virus is a Clade lib mpox virus.

22. A method of vaccinating a subject against a smallpox infection comprising administering a single dose of a pharmaceutical composition comprising an MVA- LDIO vector.

23. A method of vaccinating a subject against a borealpox infection comprising administering a single dose of a pharmaceutical composition comprising an MVA- LD10 vector.

24. A method of vaccinating a subject against a vaccinia infection comprising administering a single dose of a pharmaceutical composition comprising an MVA- LD10 vector.

25. A method of vaccinating a subject against a disease causing orthopoxvirus comprising administering a single dose of a pharmaceutical composition comprising an MVA- LD 10 vector.

26. The method of claim 25, wherein the disease causing orthopoxvirus is selected from an Akhmeta virus, Camelpox virus, Cowpox virus, Ectromelia virus, Raccoonpox virus, Skunkpox virus, Taterapox virus, Volepox virus, smallpox virus, mpox virus, borealpox virus, buffalopox, or vaccinia virus.

27. The method of claims 22-26, wherein the subject is human.

28. The method of claims 22-27, wherein the pharmaceutical composition is administered in a single dose only once.

29. The method of claims 22-28, wherein the pharmaceutical composition comprises between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of MVA-LD10 vector.

30. The method of claims 22-28, wherein the pharmaceutical composition comprises between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of MVA-LD10 vector.

31. The method of claims 22-28, wherein the pharmaceutical composition comprises between about 1.0 x 105TCIDso / mL and about 1.0 x 1011TCIDso / mL of MVA-LD10 vector.

32. The method of claims 22-28, wherein the pharmaceutical composition comprises between about 1.0 x 106and about 1.0 x 1010TCIDso / mL of MVA-LD10 vector.

33. The method of claims 22-28, wherein the pharmaceutical composition comprises between about 5.0 x 106and about 5.0 x 109TCIDso / mL of MV A-LD 10 vector.

34. The method of claims 22-28, wherein the pharmaceutical composition comprises at least about 5.0 x 106TCIDso / mL of MV A-LD 10 vector.

35. The method of claims 22-28, wherein the pharmaceutical composition comprises about 1.0 x 108TCIDso / mL of MVA-LD10 vector.

36. A method for preventing an mpox infection in a subject comprising administering to the subject a pharmaceutical composition comprising a modified vaccinia Ankara (MV A) vector encoding an amino acid sequence comprising SEQ ID NO: 10 (MVA- LD01 vector).

37. A method of reducing the effects of an mpox infection in a subject comprising administering to the subject a pharmaceutical composition comprising MVA-LD01 vector.

38. A method of inducing a protective immune response against an orthopoxvirus in a subject comprising administering to the subject a pharmaceutical composition comprising MV A-LD01 vector.

39. The method of claims 36-38, wherein the subject is human.

40. The method of claims 36-39, wherein the subject has not been previously exposed to an mpox virus.

41. The method of claims 36-40, wherein the mpox virus is a Clade I mpox virus.

42. The method of claims 36-40, wherein the mpox virus is a Clade II mpox virus.

43. The method of claims 36-40, wherein the mpox virus is a Clade Tib mpox virus.

44. The method of claims 36-43, wherein the pharmaceutical composition is administered in a single dose only once.

45. The method of claims 36-44, wherein the pharmaceutical composition comprises between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of MVA-LD01 vector.

46. The method of claims 36-44, wherein the pharmaceutical composition comprises between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of MVA-LD01 vector.

47. The method of claims 36-44, wherein the pharmaceutical composition comprises between about 1.0 x 105TCIDso / mL and about 1.0 x 1011TCIDso / mL of MVA-LD01 vector.

48. The method of claims 36-44, wherein the pharmaceutical composition comprises between about 1.0 x 106and about 1.0 x 1010TCIDso / mL of MVA-LD01 vector.

49. The method of claims 36-44, wherein the pharmaceutical composition comprises between about 5.0 x 106and about 5.0 x 109TCIDso / mL of MV A-LD01 vector.

50. The method of claims 36-44, wherein the pharmaceutical composition comprises at least about 5.0 x 106TCIDso / mL of MVA-LD01 vector.

51. The method of claims 36-44, wherein the pharmaceutical composition comprises about 1.0 x 108TCIDso / mL of MVA-LD01 vector.

52. A method of vaccinating a subject against an mpox infection comprising administering a single dose of a pharmaceutical composition comprising an MVA-LD01 vector.

53. The method of claim 52, wherein the subject has not been previously exposed to an mpox virus.

54. The method of claims 52-53, wherein the mpox virus is a Clade I mpox virus.

55. The method of claims 52-53, wherein the mpox virus is a Clade II mpox virus.

56. The method of claims 52-53, wherein the mpox virus is a Clade lib mpox virus.

57. A method of vaccinating a subject against a smallpox infection comprising administering a single dose of a pharmaceutical composition comprising an MVA- LD01 vector.

58. A method of vaccinating a subject against a borealpox infection comprising administering a single dose of a pharmaceutical composition comprising an MVA- LD01 vector.

59. A method of vaccinating a subject against a vaccinia infection comprising administering a single dose of a pharmaceutical composition comprising an MVA- LD01 vector.

60. A method of vaccinating a subject against a disease causing orthopoxvirus comprising administering a single dose of a pharmaceutical composition comprising an MVA- LD01 vector.

61. The method of claim 25, wherein the disease causing orthopoxvirus is selected from an Akhmeta virus, Camelpox virus, Cowpox virus, Ectromelia virus, Raccoonpox virus, Skunkpox virus, Taterapox virus, Volepox virus, smallpox virus, mpox virus, borealpox virus, buffalopox, or vaccinia virus.

62. The method of claims 57-61, wherein the subject is human.

63. The method of claims 57-62, wherein the pharmaceutical composition is administered in a single dose only once.

64. The method of claims 57-63, wherein the pharmaceutical composition comprises between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of MVA-LD01 vector.

65. The method of claims 57-63, wherein the pharmaceutical composition comprises between about 1.0 x 104and about 9.9 x 1012TCIDso / mL of MVA-LD01 vector.

66. The method of claims 57-63, wherein the pharmaceutical composition comprises between about 1.0 x 105TCIDso / mL and about 1.0 x 1011TCIDso / mL of MVA-LD01 vector.

67. The method of claims 57-63, wherein the pharmaceutical composition comprises between about 1.0 x 106and about 1.0 x 1010TCIDso / mL of MVA-LD01 vector.

68. The method of claims 57-63, wherein the pharmaceutical composition comprises between about 5.0 x 106and about 5.0 x 109TCIDso / mL of MV A-LD01 vector.

69. The method of claims 57-63, wherein the pharmaceutical composition comprises at least about 5.0 x 106TCIDso / mL of MVA-LD01 vector.

70. The method of claims 57-63, wherein the pharmaceutical composition comprises about 1.0 x lO8TCID5o / mL of MVA-LD01 vector.

71. The method of claims 1-70, wherein the MVA vector is administered as a pharmaceutical composition comprising 7.5% sucrose in PBS.

72. A method for preventing an mpox virus infection in a subj ect comprising administering to the subject a modified vaccinia Ankara (MVA) vector and an immune checkpoint inhibitor.

73. A method of reducing the effects of an mpox virus infection in a subject comprising administering to the subject an MVA vector and an immune checkpoint inhibitor.

74. A method of inducing a protective immune response against an mpox virus in a subject comprising administering to the subject an MVA vector and an immune checkpoint inhibitor.

75. The method of claims 72-74, wherein the immune checkpoint inhibitor is an antibody, antibody fragment, antigen binding fragment, or peptide.

76. The method of claims 72-75, wherein the immune checkpoint inhibitor inhibits programmed-cell death protein 1 (PD-1), programed cell death ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

77. The method of claims 72-76, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

78. The method of claim 77 wherein the PD-1 inhibitor is selected from nivolumab, pembrolizumab, cemiplimab, dostarlimab, pidilizumab, AMP -224, AMP-514, sintilimab, sasanlimab, spartalizumab, retifanlimab, tislelizumab, toripalimab, camrelizumab, CS1003, zimberelimab, or JTX-4014.

79. The method of claims 72-76, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor.

80. The method of claim 79, wherein the PD-L1 inhibitor is selected from atezolizumab, durvalumab, avelumab, envafolimab, BMS-936559, BMS-986189, lodapolimab, cosibelimab, sugemalimab, adebrelimab, CBT-502, AUNP12, CA-170, orBGB-A333.

81. The method of claims 72-76, wherein the immune checkpoint inhibitor is a CTLA-4 inhibitor.

82. The method of claim 81, wherein the CTLA-4 inhibitor is selected from ipilimumab, tremelimumab, AGEN1884, or AGEN2041.

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