CROSS-REFERENCE TO RELATED APPLICATION AND PRIORITY CLAIM

[0001] This application claims priority to U.S. Provisional Application Nos. 63/369,199, filed July 22, 2022; 63/370,922, filed August 9, 2022, and 63/380,718, filed October 24, 2022; the entire contents of all three provisional applications is hereby incorporated by reference as is fully set forth herein.

BACKGROUND

[0002] Mpox, formerly known as monkeypox, has been the center of a global health emergency that was declared in the summer of 2022. Mpox belongs to the same orthopoxvirus genus as smallpox and exists in two clades. The Central African clade has a high mortality rate of 10%, while infection with the West African clade results in about 1 % case-fatality rate ³ . Mpox is endemic in some Central African countries, where it causes more than 1 ,000 cases annually ^3-5 . The unprecedented mpox outbreak outside of its endemic boundaries had an epicenter in Europe and the U.S. caused over 30,000 cases and 40 deaths in the U.S. alone. In addition, the outbreak has disproportionally impacted members of the LGBTQ+ community and racial and ethnic minority groups.

[0003] The 2022 outbreak was caused by a Clade 2b.1 mpox virus (MPXV) that demonstrated unusually efficient human-to-human transmission and that appeared to have lower case fatality rate than the other circulating MPXV clades, 1 and 2a, which primarily affect Western and Central African countries ^{48 49} . Despite the low mortality in healthy individuals, mpox has been commonly associated with debilitating lesions and severe complications, especially in immunocompromised individuals ⁵⁰ .

[0004] Replication-competent vaccinia virus strains of different origin have been used around the world for orthopoxvirus vaccination campaigns. For example, during the smallpox vaccination campaign in the U.S., which ended in 1972 ⁶ , vaccinia strain Dryvax grown on calf skin formed the first-generation smallpox vaccine and was later substituted a second-generation vaccine, ACAM2000, which was plaque purified from Dryvax and produced using modern cell culture technology. While ACAM2000 was highly immunogenic, it was also associated with a high risk of myocarditis/pericarditis (1 in 175 naive adults), and this risk also extended to close contacts of vaccinated subjects, posing a threat, albeit low, for children, pregnant women, and immunocompromised individuals ⁶ . For this reason, use of ACAM2000 has been licensed with a medication guide and its use restricted to designated U.S. military personnel and laboratory researchers working with certain poxviruses.

[0005] Modified vaccinia Ankara (MVA) is a highly attenuated, propagationdefective (late block in life cycle) orthopoxvirus that was derived from its parental strain chorioallantois vaccinia virus Ankara by over 500 passages on chicken embryo fibroblasts ⁷ . MVA was developed as a third-generation smallpox vaccine and was administered at the end of the smallpox eradication campaign to more than 120,000 individuals, including children with immunodeficiencies and HIV infected individuals ⁷⁸ . Jynneos® is that FDA-approved third-generation vaccine based on modified vaccinia Ankara (MVA). Consequently, Jynneos® has been added to the Strategic National Stockpile (SNS) as a safer alternative to ACAM2000 that could be administered to the broader population. Due to the recent MPXV outbreak, Jynneos® is now offered as a prophylactic vaccine in at-risk subjects and for ring vaccinations in possible contacts of infected individuals. Given its robust safety and immunogenicity profile, MVA has also been extensively used as a viral vector for delivery of heterologous antigens and tested as a vaccine against infectious diseases and cancer ^{8 11} ' ¹³ .

[0006] Due to vaccine shortage during the recent mpox outbreak, Jynneos® received emergency use authorization (EUA) for intradermal (ID) administration, which allowed use of one-fifth of the dose normally used for the previously approved subcutaneous (SC) route of administration. While MVA demonstrated efficacy to protect against Mpox in animal models, recent real-world vaccine efficacy estimates ranged between 66% and 86% ⁵¹ ’ ⁵² , and preclinical data for the capacity of MVA to elicit MPXV cross-reactive immune responses is limited. Recent clinical data has shown low-to-absent MPXV cross-reactive neutralizing antibody (NAb) responses in Jynneos® vaccinated subjects born after the end of the smallpox vaccination campaign ⁴³⁵³ .

[0007] In summary, the 2022 Mpox outbreak has worried health officials about the availability and efficacy of a safe and effective smallpox/MPox vaccine ¹ . This disclosure provides methods and vaccine compositions to address the ongoing needs of preventing poxviral infection. SUMMARY

[0008] Provided herein, in some embodiments, are methods of vaccinating or protecting a subject against one or more diseases or one or more conditions caused by a coronavirus infection, a poxvirus infection, or both in a subject. Such methods include administering to a subject a composition comprising a synthetic MVA (sMVA) backbone and/or a recombinant synthetic MVA (rsMVA) vector comprising or capable of expressing one or more DNA sequences encoding the SARS-CoV-2 Spike (S) protein and the SARS-CoV-2 Nucleocapsid (N) protein or variants or mutants thereof.

[0009] Also provided according to some embodiments is the use of an sMVA backbone and/or an rsMVA vector that is capable of expressing one or more DNA sequences encoding the S protein and the N protein or variants or mutants thereof for preventing or reducing the severity of one or more diseases or one or more conditions caused by a coronavirus infection, a poxvirus infection, or both in a subject.

[0010] In other embodiments, methods of stimulating an immune response against a coronavirus and an orthopoxvirus in a subject are provided. Such methods may include administering to the subject a first immunogenic composition comprising a recombinant synthetic MVA (rsMVA) vector that includes one or more nucleotide sequences encoding the SARS-CoV-2 S protein and the SARS-CoV-2 N protein or variants or mutants thereof. In such embodiments, the rsMVA vector stimulates an immune response against the coronavirus and the orthopoxvirus.

[0011] Also provided according to some embodiments is the use of a recombinant synthetic MVA (rsMVA) vector to prevent or reduce the severity of a disease or condition caused by a coronavirus or an orthopoxvirus, wherein the rsMVA vector (i) includes one or more nucleotide sequences encoding the SARS-CoV-2 S protein and the SARS-CoV-2 N protein or variants or mutants thereof, and (ii) is capable of stimulating an immune response against both the coronavirus and the orthopoxvirus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] This application contains at least one drawing executed in color. Copies of this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fees. [0013] Figure 1 shows MVA-specific T cell response in COH04S1 vaccinees. Shown is the gating strategy used to identify MVA-specific T cells. Gating was performed on single cells>lymphocytes>live cells>GD3 ⁺ cells> CD8 ⁺ or GD4 ⁺ cells> CD107a ⁺ /IFNy ⁺ or CD69 ⁺ /IFNy ⁺ cells. CD69 ⁺ /IFNy ⁺ double positive cells were used to identify CD8 ⁺ and CD4 ⁺ T cell memory subsets. Naive cells were identified as CCR7 ⁺ /CD45RA ⁺ ; central memory (TCM) cells were identified as CCR7 CD45RA-; effector memory (TEM) cells were identified as CCR77CD45RA-; and terminally differentiated effector memory (TEM A) cells were identified as CCR77CD45RA7

[0014] Figure 2 shows MVA-specific binding IgG in COH04S1 vaccinees. MVA- specific binding IgG was measured by ELISA in subjects before vaccination, postprime vaccination, and at one month and five months post-booster vaccination with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Subjects who received placebo vaccination were used as negative controls. Shown are the absorbance readings (OD) at 450 nm using serial dilutions of the serum.

[0015] Figures 3A-3C show MVA-specific binding IgG in COH04S1 vaccinees. Figures 3A and 3B: Binding antibodies. MVA-specific IgG titers were measured by ELISA in subjects before vaccination, post-prime vaccination, and at one month and five months post-booster vaccination with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Subjects who received placebo vaccination were used as negative controls. Triangles pointing up in Figure 3B indicate time point of vaccination in DL1/DL1 , DL2/DL2, and DL3/DL3 groups. Triangles pointing down indicate time of DL1 vaccinations in DL1/placebo/DL1 group. Pound signs indicate subjects in DL1/DL1 and DL2/DL2 cohorts that were born before 1972. The asterisk indicates the DL3 subject born in 1986 with suspected orthopoxvirus pre-existing immunity. Kruskal-Wallis test followed by Dunn's multiple comparison test was used in A (*=p<0.05, **=p<0.01 ). Figure 3C: Seroconversion rate. Shown is the percentage of seroconverted volunteers with MVA-specific NAb titers MSfold above baseline at different time points post-vaccination with COH04S1 .

[0016] Figures 4A-4C show MPXV-specific IgG binding curves to MPXV NAb targets in COH04S1 vaccinees. Absorbance at 450nm of MPXV-specific binding IgG to H3 (Figure 4A), A35 (Figure 4B), and A29 (Figure 4C) were measured by ELISA in subjects at one month and five months post-booster vaccination with COH04S1 at dose-level (DL) 1 (DL1 /DL1 and DL1 /placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Subjects who received placebo vaccination were used as negative controls. Shown are the absorbance readings (OD) at 450 nm using serial dilutions of the serum.

[0017] Figures 5A-5C show MVA-specific neutralizing response in COH04S1 vaccinees. Figures 5A-5B: Neutralizing antibodies (NAb). MVA-specific NAb titers preventing 50% infection (NT50) were measured with a high-throughput neutralization assay. NAb were measured before vaccination, post-prime vaccination, and at one month and five months post-booster vaccinations with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Subjects who received placebo vaccination were used as negative controls. Triangles pointing up in B indicate time of vaccinations in DL1/DL1 , DL2/DL2, and DL3/DL3 groups. Triangles pointing down indicate time of DL1 vaccinations in DL1 /placebo/DL1 group. Pound signs indicate subjects in DL1/DL1 and DL2/DL2 cohorts that were born <1972. The asterisk indicates the DL3 subject born in 1986 with suspected orthopoxvirus preexisting immunity. Kruskal-Wallis test followed by Dunn’s multiple comparison test was used in A (*=p<0.05, **=p<0.01 ). Figure 50: Seroconversion rate. Shown is the percentage of seroconverted volunteers with MVA-specific NAb titers above baseline at different time points post-vaccination with COH04S1 .

[0018] Figure 6 shows MVA-specific NAb titers in COH04S1 vaccinees. MVA- specific NAb were measured using a high-throughput neutralization assay in serum samples of subjects before vaccination, post-prime vaccination, and at one month and five months post-booster vaccinations with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Subjects who received placebo vaccination were used as negative controls. Shown are percentages of neutralization using serial dilutions of serum. Dotted lines mark the 50% neutralization used to derive NT50 titers.

[0019] Figures 7A-7B show MVA-specific T cell response in COH04S1 vaccinees. Example of T cell analysis. Shown is an example of gating of double positive CD107a7CD69 ⁺ and IFNy ⁺ CD8 ⁺ (Figure 7A) or CD4 ⁺ (Figure 7B) T cells measured in unstimulated or MVA-stimulated PBMC samples of a volunteer (COH044, DL1 ) before and after vaccination with COH04S1 . [0020] Figures 8A-8B show MVA-specific T cell response in COH04S1 vaccinees. Figure 8A: IFNY ⁺ /CD1 07 ⁺ and IFNy ⁺ /CD69 ⁺ CD8 ⁺ and CD4 ⁺ T cell percentages were measured by cytofluorimetry in PBMC samples at baseline (0), at one-month post-prime (prime), and at one month post-boost (boost) month and six months (6mo) post-prime vaccination with COH04S1 at all dose levels. Activated T cell percentages at baseline and after vaccination were compared using two-tailed Wilcoxon signed-rank test. Figure 8B: Phenotypic analysis of antigen-specific T lymphocytes was performed using samples collected one month post-second dose. Shown are percentages of naive, central memory (TCM), effector memory (TEM), and terminally differentiated effector memory (TEMRA) T cells measured in IFNy ⁺ /CD69 ⁺ CD8 ⁺ and CD4 ⁺ T cell populations. 2-way ANOVA followed by Sidak’s multiple comparison test was used to compare groups. P values are indicated in the Figure. In Figures 8A-8B box plots extend from the 25th to the 75th percentiles, median values are shown as a line, whiskers extend from minimum to maximum values.

[0021] Figures 9A-9D show MVA-specific T cell response in COH04S1 vaccinees. Shown are activated MVA-specific T cells after vaccination with COH04S1 . Percentages of MVA-specific IFNY ⁺ /CD69 ⁺ CD8 ⁺ (Figure 9A) and CD4 ⁺ (Figure 9B) T cells, and MVA-specific IFNY ⁺ /CD1 07 ⁺ CD8 ⁺ (Figure 9C) and CD4 ⁺ (Figure 9D) T cells were measured in PBMC samples by cytofluorimetry at baseline, one-month after the first vaccination, and one- and five-months post-booster vaccinations with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Subjects who received placebo vaccination were used as negative controls. Only two DL3 volunteer had available PBMC samples for the analysis.

[0022] Figures 10A-10D show MVA-specific T cell response in COH04S1 vaccinees. Shown are activated T cell memory subtypes after vaccination with COH04S1. Figures 10A-10B: Phenotypic analysis of antigen-specific T lymphocytes was performed using samples collected one month after the second dose of COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Shown are percentages of naive, central memory (TCM), effector memory (TEM), and terminally differentiated effector memory (TEMRA) T cells measured in IFNY+/CD69+ CD8+ (Figure 10A) and CD4+ (Figure 10B) T cell populations. Median values are indicated with lines. Figures 10C-10D: Percentages of activated CD8+ (Figure 10C) and CD4+ (Figure 10D) T cell memory subtypes in subjects vaccinated with COH04S1 at baseline, one-month after the first vaccination, and one- and five- months post-second dose. Bars represent mean values-SD.

[0023] Figure 11 shows the sequence of clinical vaccine candidate COH04S1 (SEQ ID NO: 1 ), confirming the sMVA-N/S vaccine structure.

[0024] Figures 12A-12D show MVA-specific humoral responses in sMVA- and COH04S1 -vaccinated NHP. NHP were vaccinated once with 5x10 ⁸ pfu (Figure 12A, 12C) or two-times vaccinated with 2.5x10 ⁸ pfu (Figure 12B, 12D) of sMVA (n=3) or COH04S1 (n=6). Mock-vaccinated NHP were used as controls (n=3). Figures 12A- 12B show MVA-specific IgG endpoint titers were measured by ELISA at baseline and one month after the first dose (A), and one month after the second dose (B). Figures 12C-12D show MVA-specific NAb titers. NAb specific for MVA were measured by microneutralization assay at baseline, one month after the first dose, and one month after the second dose (only in B). Dotted lines represent the lower limit of detection of the assay

[0025] Figure 13A-13D show MVA-specific humoral response in sMVA- and COH04S1 -vaccinated NHP. NHP were vaccinated once with 5x10 ⁸ pfu (Figures 13A, 13C) or two-times vaccinated with 2.5x10 ⁸ pfu (Figures 13B, 13D) of sMVA (n=3) or COH04S1 (n=6). Mock-vaccinated NHP were used as controls (n=3). Figures 13A- 13B show MVA-specific IgG endpoint titers were measured by ELISA at baseline, one month after the first dose (Figure 13A), and one month after the second dose (Figure 13B). Shown are absorbance values (OD) measured at 450 nm. Figures 13C-13D show MVA-specific NAb titers. NAb specific for MVA were measured by microneutralization assay at baseline, one month after the first dose, and one month after the second dose (only in Figure 13B). Dotted lines represent 50% neutralization used to derive NT50.

[0026] Figures 14A-14D show sMVA- and COH04S1 -induced binding antibodies to MPXV NAb targets. Figures 14A-14C: MPXV-specific IgG endpoint titers to MPXV H3 and A35 proteins were measured by ELISA in NHP vaccinated one (Figure 14A) and two times (Figure 14B) with sMVA or COH04S1 , and in healthy adults (Figure 14C) one month after-booster vaccination with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Mock- vaccinated NHP and subjects who received placebo vaccination were used as negative controls in Figures 14A-14C. Dotted lines represent lower limit of detection. Box plots extend from the 25th to the 75th percentiles, median values are shown as a line, whiskers extend from minimum to maximum values.

[0027] Figures 15A-15D show MPXV-specific binding IgG in NHP and healthy subjects vaccinated with sMVA and COH04S1. MPXV-specific IgG endpoint titers to MPXV H3 and A35 proteins were measured by ELISA in NHP vaccinated one and two times with sMVA or COH04S1 (Figures 15A-15B), and in healthy adults (Figure 15C) one month and five months after-booster vaccination with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Mock- vaccinated NHP and subjects who received placebo vaccination were used as negative controls. Shown are absorbance (OD) values at measured at 450 nm.

[0028] Figure 16 shows a correlative analysis of MVA- and MPXV-specific binding antibodies. MVA-specific endpoint titers measured in NHP’ and human serum were correlated to MPXV-specific endpoint titers measured in NHP’ and human serum using H3 and A35 proteins. Shown are Pearson’s correlation coefficients (r) and their two-tailed significance (p).

[0029] Figure 17 shows the sequence of the unmodified sMVA vector without inserted antigens assessed by PacBio sequencing indicating one confirmed nucleotide alteration at three base pairs downstream of open reading frame 021 of the published sequence by Antione and colleagues (U94848).

[0030] Figures 18A-18B show MVA-specific humoral and cellular responses in COH04S1 -vaccinated healthy adults. MVA-specific IgG endpoint titers (Figure 18A), and neutralizing antibodies (NAb) (Figure 18B) that were measured in subjects before vaccination, post-prime vaccination, and at one- and five-months post-booster vaccination with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Placebo controls were included. Box plots show 25th- 75th percentiles, lines indicate medians, whiskers go from minimum to maximum values. Two-way ANOVA followed by Tukey’s multiple comparison test was used in b- c after log transformation. Two-tailed Wilcoxon paired T test was used in d. P values <0.05 are shown. Dotted lines in b-c represent the lower limit of detection of the assay. Dotted lines in a-b represent the lower limit of detection of the assay.

[0031] Figures 19A-19C show MVA-specific humoral responses in COH04S1 - vaccinated healthy adults and sMVA- and COH04S1 -vaccinated NHP. Figure 19A shows the seroconversion rate. MVA-specific IgG endpoint titers and MVA-specific neutralizing antibodies (NAb) were measured in subjects before vaccination, postprime vaccination, and at one- and five-months post-booster vaccination with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Placebo controls were included. Shown is the percentage of seroconverted volunteers with a >2-fold increase in MVA-specific IgG titers and MVA NAb titers at different time points post-vaccination with COH04S1 compared to before vaccination. Figure 19B and 19C show MVA-specific humoral responses in COH04S1 - and sMVA-vaccinated NHP. NHP were two-times vaccinated with 2.5x108 pfu (DL3) of COH04S1 (n=6) or sMVA (n=3). Mock-vaccinated NHP were used as controls (n=3). MVA-specific IgG endpoint titers (Figure 19B) and MVA-specific NAb (Figure 190) were measured one month after the first dose (post-prime), and one month after the second dose (post-boost). Dotted lines represent lower limits of detection. Box plots show 25th-75th percentiles, lines indicate medians, whiskers go from minimum to maximum values.

[0032] Figure 20 shows the relative contribution of activated CD8 ⁺ and CD4 ⁺ T cell memory subtypes in subjects vaccinated with COH04S1 at baseline, one-month after the first vaccination, and one- and five-months post-second dose as related to Figures 8B, 18A, and 18B. Total indicates the average number of IFNy+/CD69+ CD8+ or CD4+ T cells/100 pl of blood.

[0033] Figures 21 shows the MPXV-specific humoral response in COH04S1 - vaccinated individuals. MPXV neutralization in serum diluted 1 :10 was measured in healthy adults one-month post-booster vaccination with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Placebo controls were included. Box plots show 25th-75th percentiles, lines indicate medians, whiskers go from minimum to maximum values. Kruskal-Wallis test followed by Dunn’s multiple comparison test was used. P values <0.05 are indicated.

[0034] Figures 22A-22B show MPXV-specific immunity in COH04S1 vaccinees and GOH04S1 and sMVA vaccinated NHP. Figure 22A shows MPXV neutralization. Percentage of MPXV neutralization was measured in serially diluted serum samples of subjects vaccinated with COH04S1 five-months post-booster vaccination at DL1 , DL2, and DL3. Placebo controls were included. Figure 22B shows MPXV neutralization. Percentage of MPXV neutralization after the second dose was measured in NHP serum diluted 1 :10. Box plots show 25th-75th percentiles, lines indicate medians, whiskers go from minimum to maximum values.

[0035] Figures 23A-23B show correlative analyses of orthopoxvirus-spec\ \c humoral responses in COH04S1 -vaccinated subjects. Spearman correlation analysis was performed between the indicated MVA-specific and MPXV-specific humoral responses in COH04S1 vaccinated subjects one-month after the second dose. In Figure 23A, the Spearman correlation coefficients were calculated and plotted as a matrix. In Figure 23B, two-tailed p values were calculated and indicated as: ns= not significant (p>0.05), *=0.05 < p <0.01 , **=0.01 < p < 0.001 , ***=0.001 < p < 0.0001 , ****=p < 0.0001.

[0036] Figure 24 shows the COH04S1 schedule and dosing in healthy adults for each of the 5 Groups. Large circles indicate timing of vaccination and dosage as indicated.

[0037] Figures 25A-25D show MPXV-specific humoral response in COH04S1 - and JynneosO-vaccinated individuals. Figures 25A and 25B show MPXV-specific IgG endpoint titers to MPXV virion proteins B6R, A35, M1 R, and H3 (A) and MPXV neutralizing antibody titers (PRNT50) (B) were measured in healthy adults (n=5/group) one-month post-booster vaccination with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Placebo controls were included (n=4). Figures 25C and 25D show MPXV-specific IgG endpoint titers to MVA and MPXV virion proteins B6R, A35, M1 R, and H3, (C) and MPXV-cross reactive neutralizing antibody titers (PRNT50) measured in the presence or absence of complement (D) were evaluated in healthy adults vaccinated with COH04S1 as described for a-b (n=20), and healthy adults vaccinated with two doses of Jynneos® (n=19). Red circles indicate volunteers born before 1973 who likely had smallpox vaccination during childhood. Box plots show 25 ^th -75 ^th percentiles, lines indicate medians, whiskers go from minimum to maximum values. Two-way ANOVA followed by Dunn’s Tukey’s multiple comparison test was used after log transformation. P values <0.05 are indicated. IMV= intracellular mature virions, EEV= extracellular enveloped virions.

[0038] Figures 26A-26D show sMVA and COH04S1 immunogenicity and protective efficacy against mpox in CAST/EiJ mice. Figure 26A Shows the study design. CAST/EiJ mice (n=9/group) were intramuscularly (IM) vaccinated two times with escalating doses of sMVA or COH04S1 ranging from 1 x10 ⁶ to 1 x10 ⁸ pfu. Serum samples were collected at baseline, day 28 and day 56 for immunological analysis. Mice were intranasally (IN) challenged with mpox at day 56 and weight was recorded for 5 days. At day 5, lungs were collected for viral load (VL) assessment. Figure 26B shows Neutralizing antibody (NAb) titers. NAb were evaluated at days 28 (post-prime) and 56 (post-boost) using a plaque reduction neutralization test (PRNT). 50% PRNT titers are shown. Dotted line indicates lower detection limit. Figure 26C shows body weight changes compared to baseline. Lines indicate mean values, error bars indicate SEM. Figure 26D shows lung VL. A 50% tissue culture infectious dose (TCID50) assay was used to evaluate lung VL at day 5 post-challenge. Dotted line indicates lower detection limit. In Figures 26B and 26D data are presented as box plots extending from 25th to 75th percentiles, with lines indicating medians, and whiskers going from minimum to maximum values. Two-way ANOVA followed by Tukey’s multiple comparison test was used following log transformation. *=0.05 < p <0.01 , **=0.01 < p < 0.001 , ***=0.001 < p < 0.0001 , ****=p < 0.0001 .

[0039] Figures 27A-27C show MPXV-specific immunity in COH04S1 vaccinees and COH04S1 and sMVA vaccinated NHP. Figure 27A shows MPXV-specific IgG. IgG endpoint titers to MPXV virion proteins B6R, A35, M1 R, and H3 were measured in healthy adult six months post-vaccination with COH04S1 at dose-level (DL) 1 (DL1/DL1 and DL1/placebo/DL1 ), DL2 (DL2/DL2), and DL3 (DL3/DL3). Placebo controls were included. P values <0.05 are indicated. Figure 27B shows MPXV- specific IgG in COH04S1 - and sMVA- vaccinated NHP. NHP were two-times vaccinated with 2.5x10 ⁸ pfu (DL3) of COH04S1 (n=6) or sMVA (n=3). Mock-vaccinated NHP were used as controls (n=3). MPXV-specific IgG endpoint titers to MPXV B6R, A35, M1 R, and H3 proteins were measured one month after the first dose (prime), and one month after the second dose (boost). Dotted line represents lower limit of detection. Figure 27C shows MPXV neutralization. Percentage of MPXV neutralization was measured at the indicated timepoints in serially diluted serum samples of subjects vaccinated with COH04S1 at DL1 , DL2, and DL3. Placebo controls were included. Box plots show 25th-75th percentiles, lines indicate medians, whiskers go from minimum to maximum values. Two-way ANOVA followed by Tukey’s multiple comparison test was used after log transformation. P values <0.05 are indicated. IMV= intracellular mature virions, EEV= extracellular enveloped virions.

DETAILED DESCRIPTION

[0040] Disclosed herein are methods of vaccinating or protecting a subject against one or more conditions or one or more infections caused by a coronavirus, a poxvirus, or both. In accordance with the embodiments described herein, the methods involve administering a synthetic MVA-based vaccine that is capable of protecting a subject against infection by at least two different viruses. Also disclosed herein is the use of a synthetic MVA-based vaccine to protect a subject against infection by at least two different viruses according to some embodiments. In certain embodiments, the synthetic MVA-based vaccine is capable of protecting a subject from infection from both a coronavirus and a poxvirus.

[0041 ] In some embodiments, the subject is at a risk of suffering from a condition or an infection caused by a coronavirus, a poxvirus, or both. In some embodiments, the subject is at a risk of suffering from a disease, condition, or infection caused by a coronavirus including, but not limited to, SARS-CoV-2 or any variants thereof. Nonlimiting examples of SARS-CoV-2 variants include, but are not limited to, VOC B.1 .1 .7 (alpha), VOC B.1 .351 (beta), VOC P.1 (gamma), VOC B.1 .617.2 (delta), VOC B.1 .621 , VOC Omicron BA.1 , VOC Omicron BA.2, VOC Omicron BA.4, VOC Omicron BA.5, Omicron BA.2.12.1 , Omicron BA.2.75, Omicron sublineage XBB (e.g. XBB.1.5, XBB.1.6, XBB.2.3), Omicron EG.5 or VOC C.1.2. In accordance with certain embodiments, a SARS-CoV-2 virus and variants thereof causes a disease or condition known as COVID-19. Thus, the compositions and methods and uses for such compositions can be used to protect the subject from developing COVID-19 in accordance with the embodiments described herein.

[0042] In some embodiments, the subject is at a risk of suffering from a condition or an infection caused by a poxvirus genus selected from orthopoxvirus, parapoxvirus, molluscipoxvirus, yatapoxvirus, capripoxvirus, suipoxvirus, leporipoxvirus, or avipoxvirus. In some embodiments, the orthopoxvirus is selected from smallpox virus, vaccinia virus, cowpox virus, monkeypox virus, rabbitpox virus, ectromelia virus, camelpox virus, horsepox virus, raccoonpox virus, skunkpox virus, taterapox virus, Uasin Gishu virus, or volepox virus. In some embodiments, the parapoxvirus is selected from bovine papular stomatitis virus, Orf virus, pseudocowpox virus, parapoxvirus of red deer, squirrel parapoxvirus, Camel contagious ecthyma (Ausdyk) virus, Chamois contagious ecthyma virus, parapoxvirus of reindeer virus, or Sealpox virus. In some embodiments, the molluscipoxvirus is Molluscum contagiosum. In some embodiments, the yatapoxvirus is selected from tanapox or Yaba monkey tumor virus. In some embodiments, the capripoxvirus selected from sheeppox virus, goatpox virus, or Lumpy skin disease virus. In some embodiments, the suipoxvirus is swinepox. In some embodiments, the leporipoxvirus selected from Myxoma virus, Shope fibroma virus, Squirrel fibroma virus, or Hare fibroma virus. In some embodiments, the avipoxvirus selected from Canarypox virus, Fowlpox virus, Juncopox virus, Mynahpox virus, Pigeonpox virus, Psittacinepox virus, Quailpox virus, Sparrowpox virus, Starlingpox virus, Turkeypox virus, Crowpox virus, Peacockpox virus, or Penguinpox virus.

[0043] In some embodiments, the condition or infection is caused by an orthopoxvirus. In some embodiments, the orthopoxvirus infection is caused by a smallpox virus (VARV), a vaccinia virus (VACV), a cowpox virus (CPXV), a monkeypox virus (MPXV), a rabbitpox virus, an ectromelia virus (ECTV), Raccoonpox virus (RCNV), Volepox virus (VPXV), and Skunkpox virus (SKPV), Abatino virus (OPVA), Ahkmeta virus (AKMV), Alaskapox virus, or camelpox virus. In some embodiments, the subject is at risk of suffering from a condition or an infection caused by a monkeypox virus (MPXV). In accordance with certain embodiments, the MPXV can cause a disease or condition known as mpox. Thus, the compositions and methods and uses for such compositions can be used to protect the subject from developing mpox in accordance with the embodiments described herein, poxvirus infection is caused by a smallpox virus (VARV), a vaccinia virus (VACV), a cowpox virus (CPXV), a monkeypox virus (MPXV), a rabbitpox virus, an ectromelia virus (ECTV), Raccoonpox virus (RCNV), Volepox virus (VPXV), and Skunkpox virus (SKPV), Abatino virus (OPVA), Ahkmeta virus (AKMV), Alaskapox virus, or camelpox virus.

[0044] The methods and uses disclosed herein include administering to the subject one or more compositions that include a synthetic MVA (sMVA) backbone, and/or any recombinant synthetic MVA (rsMVA) vector or reconstituted virus that expresses or is capable of expressing one or more heterologous nucleotide (e.g., DNA) sequences. In some embodiments, the methods and uses include administering a first composition that includes an rsMVA vector. In some embodiments, the rsMVA vector or reconstituted virus used in accordance with the methods and uses disclosed herein expresses or is capable of expressing one or more heterologous nucleotide (e.g., DNA) sequences encoding a SARS-CoV-2 Spike (S) protein and a SARS-CoV- 2 Nucleocapsid (N) protein; or variants or mutants of the S protein and N protein. In some embodiments the vector expresses additional SARS-CoV-2 antigens, including E, M, ORF1 a, ORF1 b, or variants, fragments, or peptides thereof. In one embodiment, the vector expressing the SARS-CoV-2 Spike RBD, either alone or in combination with other SARS-CoV-2 antigens as listed above.

[0045] In some embodiments, the rsMVA vectors used in accordance with the methods and uses disclosed herein include a synthetic MVA genome that serves as a viral backbone (referred to herein as the sMVA backbone or sMVA genome backbone) and one or more heterologous nucleotide sequences that encode (i) a SARS-CoV-2 S protein (or variant, mutant, and/or immunogenic fragment thereof) and (ii) a SARS- CoV-2 N protein (or variant, mutant, and/or immunogenic fragment thereof).

[0046] In certain embodiments, the recombinant synthetic MVA is the result of recombination of three plasmids followed by fowlpox infection of a permissive cell line as described in Chiuppesi et al., 2020 ¹⁵ (which is hereby incorporated by reference) without further modification with any exogenous gene sequence.

[0047] In some embodiments, the rsMVA vector comprises SEQ ID NO:1 (also referred to herein as COH04S1 ). A fully synthetic Modified Vaccinia Ankara (sMVA)- based vaccine platform is used to develop COH04S1 , a multi-antigenic poxvirus- vectored SARS-CoV-2 vaccine that co-expresses full-length spike (S) and nucleocapsid (N) antigens. Figure 1 1 shows the sequence of COH04S1 (SEQ ID NO:1 ).

[0048] According to some embodiments, the sMVA backbone or sMVA genome backbone used alone or as a backbone containing one or more heterologous nucleic acid (e.g., DNA) sequences as part of an rsMVA is SEQ ID NO:2 (Figure 17).

[0049] The one or more compositions disclosed herein may be given to a subject as a single, stand-alone dose. Thus, in some embodiments, the composition is administered to the subject as a single dose. In some embodiments, the composition is administered at a 1/2, 1/3 or 1/4 of a single dose. In other embodiments, the one or more compositions may be given as a multiple-dose regimen. For example, in some embodiments, a first composition is administered to the subject as a prime dose followed by a second composition as a booster dose. In some embodiments, the first composition is administered to the subject as a prime dose, followed by a second composition as a first booster dose and a third composition as a second booster dose. In some embodiments, one or more additional doses are administered to the subject after administration of the prime and booster doses.

[0050] In some embodiments the rsMVA is used as both the prime dose and the boost dose. In such embodiments, a first composition comprising the rsMVA is administered first, followed by a second composition comprising the rsMVA that is administered after the composition. In other embodiments, the rsMVA vector is administered in conjunction with an sMVA backbone vector or other poxvirus vaccine as a multiple-dose regimen. In such embodiments, a first composition comprising the rsMVA vector is administered first as a prime dose, followed by a second composition comprising the sMVA backbone vector (or other poxvirus vaccine) that is administered after the first composition as a boost dose. Alternatively, in some embodiments, the second composition comprising the sMVA backbone vector (or other poxvirus vaccine) is administered first as a prime dose, followed by the first composition comprising the rsMVA as a boost dose.

[0051] In some embodiments, this disclosure relates to methods and uses for a heterologous prime-boost regime. For example, the subject receives a prime dose of an sMVA vaccine (e.g., the sMVA backbone vector), or a monkeypox vaccine such as Jynneos®, Dryvax, ACAM2000 or ACAM3000, and followed by a boost dose of an sMVA-COVID vaccine such as COH04S1 . Alternatively, the subject receives a prime dose of an sMVA-COVID vaccine such as COH04S1 , and followed by a boost dose of an sMVA vaccine (e.g., the sMVA backbone), or a monkeypox vaccine such as Jynneos®, Dryvax, ACAM2000 or ACAM3000. In certain embodiments, a regimen incorporating MVA may be used as a priming vaccination followed by a boost dose (like those disclosed above) to protect against possible smallpox infection. In such case, the MVA prime may act to lessen the reactogenicity of the booster. In another embodiment, the heterologous prime-boost regimen may include sMVA or COH04S1 in combination with mRNA or adenoviral vectored vaccines expressing protective orthopoxvirus, smallpox, or mpox antigens involved in virus attachment, entry, or transmission, such as MPXV antigens M1 , A29, A35, and B6 or orthopoxvirus homologs thereof. In another embodiment, the heterologous prime-boost regimen may include sMVA or COH04S1 in combination with protein or virus-like particle vaccines composed of or containing orthopoxvirus, smallpox, or mpox antigens, such as MPXV antigens M1 , A29, A35, and B6 or orthopoxvirus homologs thereof. In another embodiment, the heterologous prime-boost regimen may include COH04S1 in combination with mRNA or adenoviral vectored vaccines expressing protective coronavirus or SARS-CoV-2, or protein or virus-like particle vaccines composed of coronavirus antigens.

[0052] According to some embodiments, the sMVA backbone and constructs discussed herein were disclosed in PCT Publication No. WO 2021/158565 entitled “ Poxvirus-based Vectors Produced by Natural or Synthetic DNA and Uses Thereof,” and the synthetic MVA-based coronavirus vaccines such as rsMVA expressing N protein and S protein (COH04S1 ) were disclosed in PCT Publication No. WO 2021/236550 entitled “Synthetic Modified Vaccinia Ankara (sMVA) Based Coronavirus Vaccines.” The relevant contents of the PCT publications are incorporated herein by reference. Other poxvirus vaccines that may be used in accordance with the multipledose regimen embodiments described herein include Jynneos®, Dryvax, ACAM2000 or ACAM3000.

[0053] In a multiple-dose regimen the first (or only) booster dose may be administered such that the interval between the prime dose and the booster is about 2 weeks, about 3 weeks, about 4 weeks, or about 30 days. Alternatively, in some embodiments, the booster administration may be delayed, such that the interval between the prime dose and the booster is greater than 30 days, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, or longer than 16 weeks. In some embodiments, the interval between the prime dose and the booster is about 90 days or longer than 90 days. In certain embodiments, the interval between the prime dose and the booster is about 8 weeks.

[0054] In some embodiments, the multiple-dose regimen includes one or more additional booster doses (e.g., a second booster dose, a third booster dose, and so on). Said booster doses may be administered such that the interval between each booster dose is delayed as compared to the interval between the prime dose and the first booster. In certain embodiments, the interval between each booster dose is about 8 weeks, about 9 weeks, about 10 weeks, about 1 1 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, or longer than 16 weeks. In some embodiments, the interval between each booster dose is about 90 days or longer than 90 days. In some embodiments, the interval between each booster dose is about 90 days or longer than 90 days. In certain embodiments, the interval between each booster dose is between about 6 months to about 1 year. The interval between each booster may be on an annual or semi-annual schedule to account for additional variants that may arise each year.

[0055] In some embodiments, the prime dose (which may also refer to a dose given as a stand-alone dose) is between 1 .0 X 10 ⁶ PFU/dose and 5.0 X 10 ⁸ PFU/dose, for example, about 1.0 X 10 ⁶ PFU/dose, about 1.5 X 10 ⁶ PFU/dose, about 2.0 X 10 ⁶ PFU/dose, about 2.5 X 10 ⁶ PFU/dose, about 3.0 X 10 ⁶ PFU/dose, about 3.5 X 10 ⁶

PFU/dose, about 4.0 X 10 ⁶ PFU/dose, about 4.5 X 10 ⁶ PFU/dose, about 5.0 X 10 ⁶

PFU/dose, about 5.5 X 10 ⁶ PFU/dose, about 6.0 X 10 ⁶ PFU/dose, about 6.5 X 10 ⁶

PFU/dose, about 7.0 X 10 ⁶ PFU/dose, about 7.5 X 10 ⁶ PFU/dose, about 8.0 X 10 ⁶

PFU/dose, about 8.5 X 10 ⁶ PFU/dose, about 9.0 X 10 ⁶ PFU/dose, about 9.5 X 10 ⁶

PFU/dose, about 1.0 X 10 ⁷ PFU/dose, about 1 .5 X 10 ⁷ PFU/dose, about 2.0 X 10 ⁷

PFU/dose, about 2.5 X 10 ⁷ PFU/dose, about 3.0 X 10 ⁷ PFU/dose, about 3.5 X 10 ⁷

PFU/dose, about 4.0 X 10 ⁷ PFU/dose, about 4.5 X 10 ⁷ PFU/dose, about 5.0 X 10 ⁷

PFU/dose, about 5.5 X 10 ⁷ PFU/dose, about 6.0 X 10 ⁷ PFU/dose, about 6.5 X 10 ⁷

PFU/dose, about 7.0 X 10 ⁷ PFU/dose, about 7.5 X 10 ⁷ PFU/dose, about 8.0 X 10 ⁷

PFU/dose, about 8.5 X 10 ⁷ PFU/dose, about 9.0 X 10 ⁷ PFU/dose, about 9.5 X 10 ⁷

PFU/dose, about 1.0 X 10 ⁸ PFU/dose, about 1 .5 X 10 ⁸ PFU/dose, about 2.0 X 10 ⁸

PFU/dose, about 2.5 X 10 ⁸ PFU/dose, about 3.0 X 10 ⁸ PFU/dose, about 3.5 X 10 ⁸

PFU/dose, about 4.0 X 10 ⁸ PFU/dose, about 4.5 X 10 ⁸ PFU/dose, or about 5.0 X 10 ⁸ PFU/dose.

[0056] In some embodiments, the booster dose is in the same dosage as the prime dose. In some embodiments, the booster dose is a lower dosage than the prime dose. In some embodiments, the booster dose (e.g., the first booster dose or the second booster dose) is between 1.0 X 10 ⁶ PFU/dose and 5.0 X 10 ⁸ PFU/dose, for example, about 1.0 X 10 ⁶ PFU/dose, about 1.5 X 10 ⁶ PFU/dose, about 2.0 X 10 ⁶ PFU/dose, about 2.5 X 10 ⁶ PFU/dose, about 3.0 X 10 ⁶ PFU/dose, about 3.5 X 10 ⁶ PFU/dose, about 4.0 X 10 ⁶ PFU/dose, about 4.5 X 10 ⁶ PFU/dose, about 5.0 X 10 ⁶

PFU/dose, about 5.5 X 10 ⁶ PFU/dose, about 6.0 X 10 ⁶ PFU/dose, about 6.5 X 10 ⁶

PFU/dose, about 7.0 X 10 ⁶ PFU/dose, about 7.5 X 10 ⁶ PFU/dose, about 8.0 X 10 ⁶

PFU/dose, about 8.5 X 10 ⁶ PFU/dose, about 9.0 X 10 ⁶ PFU/dose, about 9.5 X 10 ⁶

PFU/dose, about 1.0 X 10 ⁷ PFU/dose, about 1 .5 X 10 ⁷ PFU/dose, about 2.0 X 10 ⁷

PFU/dose, about 2.5 X 10 ⁷ PFU/dose, about 3.0 X 10 ⁷ PFU/dose, about 3.5 X 10 ⁷

PFU/dose, about 4.0 X 10 ⁷ PFU/dose, about 4.5 X 10 ⁷ PFU/dose, about 5.0 X 10 ⁷

PFU/dose, about 5.5 X 10 ⁷ PFU/dose, about 6.0 X 10 ⁷ PFU/dose, about 6.5 X 10 ⁷

PFU/dose, about 7.0 X 10 ⁷ PFU/dose, about 7.5 X 10 ⁷ PFU/dose, about 8.0 X 10 ⁷

PFU/dose, about 8.5 X 10 ⁷ PFU/dose, about 9.0 X 10 ⁷ PFU/dose, about 9.5 X 10 ⁷

PFU/dose, about 1.0 X 10 ⁸ PFU/dose, about 1 .5 X 10 ⁸ PFU/dose, about 2.0 X 10 ⁸

PFU/dose, about 2.5 X 10 ⁸ PFU/dose, about 3.0 X 10 ⁸ PFU/dose, about 3.5 X 10 ⁸

PFU/dose, about 4.0 X 10 ⁸ PFU/dose, about 4.5 X 10 ⁸ PFU/dose, or about 5.0 X 10 ⁸ PFU/dose. The booster dose may be in a dosage the same as the prime dose or lower than the prime dose.

[0057] The composition may be administered to the subject in any suitable manner. In some embodiments, the composition is administered to the subject parenterally, e.g., by intramuscular injection. In some embodiments, the composition is administered to the subject by intranasal administration. In other embodiments, the composition is administered by subcutaneous, scarification, intradermal, intra-rectal, intra-li ngual , intravenous, or other known clinically acceptable routes of administration.

[0058] Given the urgency to vaccinate individuals at risk in both endemic and non-endemic countries to curtail further spread of the outbreak, there is a legitimate need for substantial doses of smallpox/MPXV vaccines which have become scarce. Recently, a fully synthetic MVA (sMVA) platform based on the genome sequence published by Antione ef al. was developed for reconstituting virus that is virtually identical to wild-type MVA in terms of replication properties, host cell tropism, and immunogenicity ¹⁵ . Using this platform, which allows rapid generation of sMVA recombinants encoding multiple transgenes, COH04S1 , a multiantigen sMVA-based COVID-19 vaccine encoding for Spike (S) and Nucleocapsid (N) antigens, was developed and disclosed in PCT Publication No. WO 2021 /236550, which is incorporated by reference herein.

[0059] COH04S1 and the sMVA backbone of the platform were extensively tested in animal models, including non-human primates (NHP), for safety and immunogenicity. Additionally, COH04S1 was found to be safe and to induce SARS- CoV-2-specific immune responses in healthy adults participating in a phase 1 , blinded, randomized, placebo-controlled clinical trial. As discussed in detail in the examples below, a retrospective analysis of vaccine-induced poxvirus immunity was performed in NHP vaccinated with COH04S1 and sMVA, and in a subgroup of volunteers enrolled in the phase 1 clinical trial that were vaccinated with COH04S1 at different doses. At all dose levels, vaccination with COH04S1 or sMVA (never together) resulted in robust MVA-specific binding and neutralizing antibody responses, which were sustained over six months post-vaccination. Importantly, both COH04S1 or sMVA vaccination induced elevated antibody titers binding MPXV-specific targets of neutralizing antibodies and MPXV-cross reactive neutralizing antibodies. Additionally, maximal levels of both CD8+ and CD4+ T cells specific for MVA were induced after one dose of COH04S1 and remained durable for at least six months. Given that both arms of the immune response are thought to be involved in protection against poxvirus infections, the results disclosed herein indicate that COH04S1 or other sMVA-based vaccines may represent valuable candidates for smallpox/monkeypox vaccination in addition to their use in protecting from SARS-CoV-2 and other infections.

[0060] COH04S1 has been extensively tested in animal models, including NHP, demonstrating robust immunogenicity and protective efficacy against SARS-CoV-2 and its variants through intramuscular and intranasal routes of vaccination ^15-17 ’ ⁵⁴ . Additionally, COH04S1 has been tested in a Phase I, randomized, placebo-controlled clinical trial in healthy adults, demonstrating safety and resulting in the induction of robust and durable humoral and cellular responses to both vaccine antigens. COH04S1 is clinically the most advanced MVA-based dual-antigen COVID-19 vaccine and it is currently, COH04S1 is being evaluated in three phase 2 clinical trials in healthy adults and immunocompromised patients (NCT04639466, NCT04977024, NCT05672355).

[0061] Following the recent MPXV outbreak numerous countries rushed to order Jynneos® for their nationals in need for a total of more than 3 million doses ⁴² . Additionally, the eventuality of a mass MPXV vaccination campaign in endemic African countries raises the issue of vaccine supply shortage and equitable distribution. Consequently, there is an urgency to replenish stockpiles around the world with safe and effective third generation smallpox/MPXV vaccines. The finding that both sMVA and COH04S1 induces robust and durable MVA- and MPXV-specific immunity represent the fundamental preliminary result for the basis that COH04S1 and sMVA- based vaccines may be tested and used as vaccines against smallpox/MPXV in addition to its use as a vaccine against SARS-Cov-2. This new use may confer dual protection against two of the most clinically relevant viruses of today.

[0062] Thus, according to some embodiments, the rsMVA vector, sMVA backbone vector, or other poxvirus vector used in accordance with the methods and uses described herein is part of a composition that is a vaccine composition or any immunogenic composition capable of providing full or partial protection against infection by a coronavirus and/or a poxvirus when administered to a subject or a population of cells. In certain embodiments, the rsMVA vector used in accordance with the methods and uses described herein is part of a composition that is a vaccine composition or any immunogenic composition capable of providing full or partial protection against infection by a coronavirus and a poxvirus when administered to a subject or a population of cells. In other embodiments, the sMVA backbone vector or other poxvirus vector used in accordance with the methods and uses described herein is part of a composition that is a vaccine composition or any immunogenic composition capable of providing full or partial protection against infection by a poxvirus when administered to a subject or a population of cells. In such embodiments, the sMVA backbone vector can be used as an alternative to other poxvirus vectors or in conjunction with other poxvirus vectors and/or the rsMVA vector as a prime and/or boost to enhance protection against any poxvirus protection provided by the rsMVA or other poxvirus vectors.

[0063] In certain embodiments, the rsMVA vector, sMVA backbone vector, or other poxvirus vector used in accordance with the methods and uses described herein is part of a composition that is a vaccine composition or any immunogenic composition capable of providing full or partial protection against infection by a coronavirus that causes COVID-19 and/or a poxvirus that causes mpox when administered to a subject or a population of cells. In certain embodiments, the rsMVA vector used in accordance with the methods and uses described herein is part of a composition that is a vaccine composition or any immunogenic composition capable of providing full or partial protection against infection by a coronavirus that causes COVID-19 and a poxvirus that causes mpox when administered to a subject or a population of cells. In other embodiments, the sMVA backbone vector or other poxvirus vector used in accordance with the methods and uses described herein is part of a composition that is a vaccine composition or any immunogenic composition capable of providing full or partial protection against infection by a poxvirus that causes mpox when administered to a subject or a population of cells. In such embodiments, the sMVA backbone vector can be used as an alternative to other poxvirus vectors or in conjunction with other poxvirus vectors and/or the rsMVA vector as a prime and/or boost to enhance protection against any poxvirus protection provided by the rsMVA or other poxvirus vectors.

[0064] Further, as discussed in the examples below, it was shown that administering rsMVA COH04S1 results in a significantly increased titer against the monkeypox antigen M1 R, a marker of the intracellular mature virus (IMV) of monkeypox. The IMV and the extracellular enveloped virus (EEV) of monkeypox are the two separate, main infectious forms of MPXV. Thus, rsMVA COH04S1 may cause more a robust or complete immunity or other protective effect against intracellular mature MPXV while maintaining immunity against extracellular enveloped MPXV. This may in turn confer more robust or complete protection against MPXV infection as compared to Jynneos® or other MVA-based vaccines. Accordingly, in some embodiments, the rsMVA vector, sMVA backbone vector used in accordance with the method and uses described herein is part of a composition that is a vaccine composition or any immunogenic composition capable of providing a full or partial protection against both virus forms that mediate poxvirus transmission and dissemination, including the intracellular mature virus (IMV) and the intracellular envelope virions (EEV). In certain embodiments, the full or partial protection provided by the rsMVA vector or sMVA backbone is more complete or robust as compared to other poxvirus vectors.

[0065] In accordance with the embodiments described herein, providing full or partial protection against infection by the coronavirus and/or poxvirus, as used herein, means preventing infection completely, but also means lowering or reducing the spread of infection, lowering or reducing the chance or risk of becoming infected, reducing the severity of infection, lowering or reducing the risk of developing or suffering a complication or consequence resulting from infection (e.g., moderate to severe symptoms, hospitalization, death), or otherwise alleviating symptoms and/or lowering the risk associated with developing a disease or condition caused by the coronavirus e.g., COVID-19) and/or poxvirus (e.g., mpox). As such, in certain embodiments, the rsMVA vector, sMVA backbone vector (or other poxvirus vector) used in accordance with the methods and uses described herein is part of a composition that is a vaccine composition or any immunogenic composition capable of providing full or partial protection from developing moderate or severe symptoms as a result of developing a condition or disease caused by a coronavirus (e.g., COVID- 19) and/or a condition or disease caused by a poxvirus (e.g., mpox). In other embodiments, the composition is a vaccine composition or any immunogenic composition capable of providing protection from hospitalization or death as a result of developing a condition or disease caused by a coronavirus (e.g., COVID-19) and/or a condition or disease caused by a poxvirus (e.g., mpox).

[0066] In accordance with the embodiments described herein, an immunogenic composition is a composition that includes one or more components (e.g., an antigen) to provoke an immune response when administered to a subject. For example an immunogenic composition may cause activation of one or more immune cells (e.g., T cells, B cells, natural killer (NK) cells, monocytes/macrophages, neutrophils, eosinophils, basophils, mast cells, or other immune cells) or immune system component (e.g., complement), that in turn act to kill infected cells, produce antibodies, release cytokines and cytotoxic molecules, among other actions. In some embodiments, the immunogenic composition provokes an immune response against both a coronavirus and/or a poxvirus. And in some embodiments, the immunogenic composition provokes a humoral immune response, e.g., an antibody-mediated immune response, against both a coronavirus and/or a poxvirus. In such embodiments, the immunogenic composition stimulates the production of antibodies specific to both the coronavirus and the poxvirus. Such antibodies may be binding or neutralizing antibodies.

[0067] In some embodiments, the immunogenic composition is a vaccine composition. A vaccine composition is a composition that provokes an immune response that provides a subject with full or partial immunity to a disease or condition. In certain embodiments, the full or partial immunity is a protective immunity, which limits pathogen replication before symptoms of the disease or condition develop or shortly thereafter causing only mild symptoms. In other embodiments, the full or partial immunity is a sterilizing immunity, which eliminates or neutralizes the pathogen before it replicates, thereby preventing the subject from developing the disease or condition. In some embodiments, the vaccine composition can provide protection from future infection for a certain amount of time or even lifetime immunity.

[0068] The following examples are intended to illustrate various embodiments of the invention. As such, the specific embodiments discussed are not to be constructed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.

EXAMPLES

Overview

[0069] Given the recent approval of MVA as a vaccine against smallpox and MPXV, the studies discussed below were performed to evaluate whether COH04S1 - or sMVA-vaccinated NHP and healthy volunteers ¹⁸ mount orthopoxviral-specific immunity. Here it was shown that COH04S1 -vaccinated healthy adults develop robust orthopoxvirus-specific humoral and cellular responses, including cross-reactive antibodies to MPXV-specific virion proteins and MPXV-specific neutralizing antibodies. Additionally, it was demonstrated that COH04S1 -induced humoral responses are as good or better than orthopoxvirus-specific responses measured in healthy adults vaccinated with FDA-approved Mpox vaccine Jynneos® based on wildtype MVA. Finally, it was shown that Mpox-susceptible CAST/EiJ mice vaccinated with COH04S1 or empty sMVA vector are protected from MPXV infection of the lungs. These results demonstrate the capacity of sMVA-based vaccines to elicit cross- reactive and protective orthopox-specific immunity against MPXV. These results suggest that COH04S1 and sMVA represent unique vaccine candidates to control the unforeseen global MPXV outbreak.

[0070] As demonstrated herein, sMVA and multiantigen sMVA-based SARS- CoV-2 vaccine COH04S1 induces robust orthopoxviral-specific immunity in NHP and in healthy adults vaccinated during a Phase I clinical trial aimed at testing safety and immunogenicity of COH04S1 as a COVID-19 vaccine ¹⁸ . These findings show that sMVA- and COH04S1 -vaccinated NHP and COH04S1 -vaccinated volunteers at all tested dose levels develop robust MVA-specific humoral and cellular responses that remain detectable for up to six months post vaccination. Importantly, vaccination of NHP and healthy subjects with sMVA and COH04S1 induced elevated levels of binding antibodies targeting MPXV proteins which are known target of NAb, suggesting that sMVA and COH04S1 represent unique vaccine candidates that can be used as MPXV single-agent or MPXV/COVID-19 multi-agent vaccines to control both the COVID-19 and MPXV outbreaks.

[0071] MVA-specific binding and neutralizing antibodies developed in all volunteers after two vaccine doses resulting in 100% seroconversion. Consistent with a dose-escalation trial using a wild-type MVA ²³ (ACAM3000), post-prime antibody titers were affected by the vaccine dose, with lower post-prime antibody titers in DL1 than in DL2/DL3 vaccinated subjects. In contrast, following the second dose, COH04S1 was similarly immunogenic at all DL tested. Increased timing between vaccinations with DL1 did not affect the peak antibody response; however, it seemed to marginally increase the magnitude of the long-term response compared to volunteers vaccinated in a shorter interval, although a larger number of volunteers would be needed to bolster this conclusion. No differences across DL were observed in magnitude and phenotype of MVA-specific activated T cells indicating that the lowest dose was sufficient to induce robust and durable cellular responses. This result is concordant with maximal induction of SARS-CoV-2 S- and N-specific T cells by COH04S1 at all DL tested ¹⁸ .

[0072] As previously observed by others, early post-vaccine T cell response to poxvirus antigens was largely comprised of CD8 ⁺ T cells ^{25 26} . Interestingly, COH04S1 induced higher MVA-specific CD8 ⁺ than CD4 ⁺ T cells, which is opposite to the previously observed SARS-CoV-2 S- and N-specific CD8 ⁺ and CD4 ⁺ T-cells induced by COH04S1 ¹⁸ , indicating that different antigens can preferentially activate different T cell subtypes even during concomitant antigen stimulation. While it has been shown that TEM are the predominant CD8 ⁺ subtype and TCM the predominant CD4 ⁺ T cell subtypes in SARS-CoV-2 infected or vaccinated individuals ¹⁸ ’ ^{27 28} , MVA-specific TEM cells were the predominant subtype in both CD8 ⁺ and CD4 ⁺ T cell populations of COH04S1 vaccinees. Interestingly, TEM cells, but not TCM cells, have been demonstrated to protect against peripheral infection with vaccinia virus ²⁹ , highlighting an important protective role of TEM cells in orthopoxviral infections. Comprehensive studies of long-term immunity to vaccinia have shown that TEM cells decay with time after antigen encounter while TCM cells have a greater capacity to persist in vivo ^3Q . Whether the phenotype observed at six months post-vaccination in COH04S1 - vaccinated subjects is maintained long-term or whether TCM cells may overtake TEM cells as the main MVA-specific T cell subpopulation can only be clarified in long-term studies.

[0073] Smallpox and vaccinia immunity has been shown to be stable for decades after infection or vaccination and to decline only slowly over time ^{31 32} . Of the two volunteers born before 1972, the year of the end of the smallpox vaccination campaign, only one had an indication of low levels pre-existing poxviral immunity. However, both volunteers responded to two COH04S1 doses with higher-than- average long-lasting MVA- and MPXV-specific humoral responses, suggesting that vaccination with COH04S1 successfully recalled low-to-undetectable vaccinia immunity acquired 50 years or more before, which resulted in more robust and durable responses than in naive subjects. Interestingly, one DL3 subject born in 1986, and therefore not subjected to smallpox vaccination during childhood, showed low-level poxviral pre-existing humoral immunity at baseline. The same subject had a drastic increase in MVA-specific humoral response post-prime vaccination, and binding and NAb at five months post-boost were exceptionally high. It is plausible that this subject was recently inoculated with vaccinia due to work exposure risk and that a combination of high COH04S1 dose with short time since smallpox vaccination contributed to the elevated MVA-specific responses. Importantly, this subject and the two volunteers born before 1972 developed robust SARS-CoV-2 S- and N-specific humoral and cellular responses ¹⁸ , suggesting that vector-specific pre-existing immunity did not prevent induction of robust immunity to the SARS-CoV-2 antigens of COH04S1 .

[0074] Definition of correlates of protection against smallpox and MPXV is complicated by the contrasting findings emerged in the past decades. Orthopoxvirusspecific NAb but not CD8 ⁺ T cells correlated with an attenuated Dryvax skin lesion or “take” at the inoculation site in one study ³³ , and NAb were necessary and sufficient to protect monkeys against MPXV in another study ³⁴ . On the other hand, a study in mice demonstrated an important protective role of T cell immunity in the absence of an antibody response ³⁵ , and patients with defects in their T cell responses are those at risk to develop severe progressive vaccinia disease when vaccinated ³⁶ . Consequently, it seems likely that a complex interplay of immunological factors contributes to the establishment of immunity to orthopoxviruses and these immunological correlates may vary based on species and viral strain. Therefore, it is encouraging that COH04S1 - vaccination induced a comprehensive MVA-specific immunological response in vaccinated volunteers.

[0075] Intramuscular doses of both ACAM3000 (1x10 ⁷ pfu) and Jynneos® (1x10 ⁸ pfu) have been shown to result in a significantly attenuated response to a Dryvax “take” in human challenge studies. ⁹ ’ ³³ ’ ³⁷ Considering that COH04S1 induced comparable humoral and cellular responses post-boost at all dose levels tested, it is possible that COH04S1 would confer similar protection to wild-type MVA vaccines against challenge starting from the lowest dose tested.

[0076] The demonstrated results of seroconversion and cellular responses in all subjects post-boost independent of vaccine dose indicates that vaccination with COH04S1 induces robust MVA-specific immunity. Because of biosafety limitations, vaccinia or MPXV viruses were not used for assessment of vaccine immunogenicity. However, magnitude of MVA- and vaccinia-specific humoral immunity have been shown to be equivalent and vaccinia and MPXV NAb targets to be >94% conserved, indicating high degree of antibody cross-recognition between orthopoxviruses ^{2023 33} . Additionally, a comparison of binding and NAb titers induced by COH04S1 with titers measured in the WHO “International Standard for Anti-Smallpox Serum” 63/024 ⁴⁰ was not possible since the product is currently not available in the NIBSC repository. Finally, to measure NAb, a high throughput neutralization assay was utilized based on the use of purified MVA intracellular mature virions, which are the main viral form liberated by cell lysis. Vaccine-mediated neutralization of extracellular enveloped virions has not been evaluated although this viral form is believed to be responsible for vaccinia virus inter-host dissemination ⁴¹ .

Materials and Methods

[0077] Given the approval of MVA as a vaccine against smallpox/MPXV, studies disclosed in the examples below were evaluated to determine whether COH04S1 - vaccinated healthy volunteers and COH04S1 - or sMVA-vaccinated NHP developed orthopoxvirus- and MPXV-specific immunity. The following materials and methods are used in accordance with the examples below unless otherwise indicated:

[0078] Non-human primates: In life portion of NHP studies were conducted at Bioqual Inc. (Rockville, MD). The studies were conducted in compliance with local, state, and federal regulations and were approved by Bioqual and City of Hope Institutional Animal Care and Use Committees (IACUC). African green monkeys (Chlorocebus aethiops) were randomized by weight and sex to vaccine and control groups. NHP were intramuscularly vaccinated twice four weeks apart with 2.5x10 ⁸ pfu of COH04S1 (n = 6) or sMVA (n = 3) diluted in PBS. Mock-vaccinated NHP immunized twice with PBS only were used as additional controls (n = 3). The evaluation of SARS- CoV-2 immunity following NHP-vaccination with COH04S1 has been previously described ¹⁶ .

[0079] Human subjects: COH04S1 immunogenicity was investigated at City of Hope (COH) as part of a clinical protocol (IRB#20447) approved by an external Institutional Review Board (Advarra IRB). This open-label and randomized, placebo controlled, phase 1 clinical study is registered (NCT04639466). Among others, exclusion criteria included aged 8 or >55, previous SARS-CoV-2 infection, BMI<18 or >35, underlying health conditions, and poxvirus vaccination within a six-months period. All subjects gave informed consent at enrollment. Out of the 51 subjects who received one or two doses of COH04S1 , 5 subjects were selected from each dose group, for a total of 20 subjects, based on a 2 dose regimen and availability of frozen PBMCs samples. Subjects were not required to provide their smallpox vaccination status, and poxvirus serostatus at enrollment was not evaluated. However, an exclusion criterion was any poxv/ri/s-vaccination within six months of enrollment in the trial. Subjects received were vaccinated intramuscularly (IM) with two doses of COH04S1 at days 0 and 28 with 1 x10 ⁷ pfu (DL1 ), 1 x10 ⁸ pfu (DL2), and 2.5x10 ⁸ pfu (DL3). An additional five subjects received DL1 at day 0, placebo at day 28, and another DL1 at day 56. Four volunteers who received placebo at days 0 and 28 were included in the study. Two subjects, one in DL1/DL1 group and one in DL2/DL2 group, were born before 1972 and therefore may have had been previously vaccinated against smallpox. A summary of study subjects, vaccination schedule and age at enrollment is presented in Tables 1 and 2 below. COH04S1 -induced SARS-CoV-2 immunity in this population has been previously described ^{18 19} . [0080] Table 1. COH04S1 -Vaccinated Volunteer Characteristics Median (Range); ² n (%); ³ self-reported. DL= dose level. N/A=not reported

[0081] Table 2. Individual characteristics of subjects vaccinated with COH04S1

[0082] Plasma samples of JynneosO-vaccinated healthy volunteers were collected as part of a registered observational trial. 19 volunteers vaccinated with two doses of Jynneos® were enrolled. Median time from full vaccination to sample collection was 98 days. Vaccination was via homologous (SC or ID), or heterologous (SC/ID or ID/SC) route. Volunteers’ characteristics are presented in Table 3 below.

[0083] Table 3. Jynneos® vaccinated volunteers characteristics

[0084] CAST/EiJ mice. In life portion of the CAST/EiJ mouse study was conducted at Bioqual Inc. (Rockville, MD). The mouse study was conducted in compliance with local, state, and federal regulations and was approved by Bioqual and City of Hope IACUC. Male CAST/EiJ mice, 6-8 weeks of age, were randomly assigned to groups (9-10 mice/group) and vaccinated twice in 4 weeks interval with either 1 x10 ⁶ , 1 x10 ⁷ , or 1 x10 ⁸ pfu/ml of sMVA or COH04S1 vaccines via intramuscular route. Postprime and post-boost blood samples were collected for the assessment of mpox- specific NAb. One-month post-boost, mice were intranasally challenged with 6.03 x 10 ⁶ pfu/ml of mpox USA/MA001/2022 (expanded in house from NR-58622, BEI Resources) and weight was recorded daily for 5 days. At day 5 post-challenge lungs were collected for viral load assessment. See Figure 26A

[0085] MVA and MPXV IgG Endpoint ELISA: MVA-specific binding antibodies were evaluated by ELISA. ELISA plates (3361 , Corning) were coated overnight at 4°C with 1 pg/mL of MVA expressing Venus fluorescent marker (MVA-Venus) ¹³ , or with 1 pg/mL of B6R (40902-V08H), A35 (40886-V08H), M1 R (40904-V07H), or H3L (40893- V08H1) MPXV antigens (SinoBiological) in PBS pH 7.4. Plates were washed (0.1% Tween-20/PBS), then blocked with 250 pl/well of assay buffer (0.5% casein/154mM NaCI/10mM Tris-HCI/0.1 % Tween-20 [pH 7.6]/8% Normal goat serum [NGS]; 4% NGS for NHP samples ELISA) for 2 hours 37°C. After washing, serially diluted heat- inactivated serum in blocking buffer was added to the plates in duplicate wells. Plates were wrapped in foil and incubated 2 hours at 37°C after which plates were washed and 1 :3,000 dilution of anti-human IgG HRP secondary antibody (BioRad 204005), or 1 :10,000 anti-monkey lgG(H+L) HRP secondary antibody (Thermo Fisher PA1 -84631 ) in assay buffer was added for 1 hour at room temperature. Plates were washed and developed with 1 Step TMB-Ultra (Thermo Fisher 34029). The reaction was stopped with 1 M H2SO4 and 450nm absorbance was immediately quantified on FilterMax F3 (Molecular Devices). Endpoint titers were calculated as the highest dilution to have an absorbance >0.1 nm for all the assays with the exception of B6R (>0.3 nm), and M1 R (>0.2 nm). Seroconversion was defined as a two or more times increase in baseline titer ⁹ .

[0086] MVA neutralization assay. ARPE-19 cells were seeded in 96-well plates (1 .5 x 10 ⁴ cells/well). The following day, 2-fold serial dilutions of serum starting from 1 :10 were incubated for 2 hours with MVA-Venus (multiplicity of infection [MOI]=2). The serum-virus mixture was added to the cells in duplicate wells and incubated for 24 hours. After the 24 -hour incubation period, the cells were imaged using Leica DMi8 inverted microscope. Pictures from each well were processed using Image-Pro Premier (v9.2; Media Cybernetics) and fluorescent cells corresponding to infection events were counted. The neutralization titer for each dilution was calculated as follows: NT = [1 -(fluorescent cells with immune sera/fluorescent cells without immune sera)] x 100. The titers that gave 50% neutralization (NT50) were calculated by determining the linear slope of the graph plotting NT versus serum dilution by using the next higher and lower NT using Office Excel (v2019). Seroconversion was defined as an increase of two or more times the baseline titer ⁹ .

[0087] Quantification of vaccine-induced MVA specific T cells: Peripheral blood mononuclear cells (PBMC) were isolated from fresh blood using Ficoll and counted using Luna-FL cell counter (Logos Biosystems). Frozen PBMCs were thawed, counted and 1 X10 ⁶ PBMCs were stimulated with MVA-Venus (MOI=1 ) for 24 hours in a total volume of 200 pl of RPMI media with 5% of human serum in a 96 wells plate. Unstimulated cells and PHA (20 pg/ml) were used as negative and positive controls, respectively. Anti-CD107a-APC, Golgi Plug (Brefeldin A) and Golgi Stop (Monesin) were added 4 hours before staining. Cells were washed with PBS and stained 15 minutes at room temperature with Live and dead near IR, anti-CD3-FITC, anti-CD4- BV421 , anti-CD8-BV605, anti-CD69-PE, anti-CCR7-PE/Dazzle 594 and anti-CD45- PerCP. After washing, cells were permeabilized with Fix/Perm (BD) for 20 minutes at 4°C. Cells were washed with Perm/Wash (BD) and intracellular stained with anti-IFNy- PECy7 for 30 minutes at 4°C, washed and resuspend in FACS buffer until acquisition. Cells were acquired in Attune NxT cytometer (Thermofisher) and data was analyzed with Flow Jo X software following the gating strategy described in Figure 1 . Only one out of five DL3 volunteers (COH027) had available PBMCs samples for the analysis.

[0088] MPXV Plaque Reduction Neutralization Test (PRNT). The day before the assay, 2.5 x 10 ⁵ Vero E6 cells (ATCC, CRL-1586) were seeded in each well of a 24-well plate using plate-seeding media

(DMEM/Glutamax/10%FBS/Penicillin/Streptomycin). The following day, four-fold serum dilutions were prepared in 96-well plates using infection media (DMEM/Glutamax/2%FBS) and equal volumes of diluted MPXV virus stock (MA- 104/US-2003, West African clade) were added to each well. Plates were incubated for 15-18 hours at 2-8°C. After this time the serum/MPXV mix was added to Vero E6 cells and incubated 37°C. After 1 hour of gentle rocking, 0.5% methylcellulose overlay medium (0.5% methylcellulose/DMEM/Glutamax/ 10%FBS/Peniciliin/Streptomycin) was added to each well and plates were further incubated for 48 hours. Once the infection step was complete, the overlay medium was removed, and a 0.4% Crystal Violet stain solution was added to each well. After removing of the staining solution, plates were scanned with a flatbed scanner and manual counting of plaques performed. Number of plaques in virus control wells were used to calculate the percentage of neutralization in samples’ wells.

[0089] Statistical analysis: Statistical analysis was performed using GraphPad Prism 8.3.0. Differences in humoral responses across groups were compared using two-way ANOVA followed by Tukey’s multiple comparison test following log transformation. T cell percentages at different time-points were compared using two- sided Wilcoxon rank test. Differences in T cell subsets were evaluated using 2-way ANOVA followed by Sidak’s multiple comparison test. Pearson correlation coefficients and their p values were calculated for the correlative analysis. Example 1 . Induction of orthopoxyiral immunity in non-human primates

[0090] Orthopoxviral-specific humoral responses were retrospectively evaluated in NHP vaccinated with one or two doses of sMVA or COVID-19 vaccine candidate COH04S1 . NHP vaccinated with one dose received 5x10 ⁸ plaque forming units [pfu], while NHP vaccinated with two doses received 2.5x10 ⁸ pfu/dose. Saline mock- vaccinated NHP were used as controls. MVA-specific IgG were evaluated in NHP serum by ELISA. No orthopoxviral-specific pre-existing immunity was observed in pre- immune samples. One-month after the first dose all NHP vaccinated with sMVA and COH04S1 independently of the dose used developed MVA-specific IgG (Figure 12). NHP vaccinated with 5x10 ⁸ pfu tended to have higher post-prime MVA-specific IgG endpoint titers than NHP vaccinated with 2.5x10 ⁸ pfu. In NHP vaccinated with a second 2.5 x 10 ⁸ pfu dose, MVA-specific IgG were boosted in both sMVA- and COH04S1 -vaccinated animals to endpoint titers approaching 10 ⁴ . No differences in titers were observed between sMVA- and COH04S1 -vaccinated NHP (Figures 12A-B and 13). MVA-specific NAb responses were measured on epithelial cells using a high- throughput microneutralization assay. One month after the first dose NHP vaccinated with sMVA and COH04S1 at 2.5x10 ⁸ pfu developed MVA-specific NAb titers ranging from 1 :30 to 1 :750. Vaccination with 5x10 ⁸ pfu resulted in higher post-prime NT50 (range 240- 2,840). In NHP vaccinated with a second 2.5x10 ⁸ pfu dose, NAb were boosted in both sMVA- and COH04S1 -vaccinated animals to NT50 values ranging from 680 to 3,690. Similar NT50 titers were measured between sMVA- and COH04S1 - vaccinated NHP (Figures 12C-D and 13). These results showed that both sMVA, and sMVA-based vaccine COH04S1 promote robust induction of MVA-specific humoral responses after one dose and that a second dose can increase the magnitude of the antibody response.

Example 2. Study design to evaluate orthopoxyiral immunity in COHQ4S1 vaccinees

[0091] Orthopoxviral-specific responses were retrospectively evaluated for up to six months after vaccination with sMVA COVID-19 vaccine candidate COH04S1 in a subgroup of 20 volunteers enrolled in a phase 1 clinical trial aimed at testing safety and immunogenicity of COH04S1 at different dose levels (DL) (NCT04639466) ^{18 19} . Subjects were prime-boost vaccinated with low-dose (DL1 , 1x10 ⁷ pfu), medium-dose (DL2, 1 x10 ⁸ pfu), or high-dose (DL3, 2.5x10 ⁸ pfu) of vaccine. Of the 20 subjects vaccinated with COH04S1 , 15 (5 subjects/group) received two DL1 , DL2, or DL3 vaccinations 28 days apart, and 5 received two DL1 vaccinations 56 days apart with a placebo dose at day 28 (DL1/placebo/DL1 ). Four placebo-vaccinated subjects enrolled in the same trial were included as controls (Figure 24). Subjects were not required to provide their smallpox vaccination status, and poxvirus serostatus at enrollment was not evaluated. However, an exclusion criterion was any poxvirus- vaccination in the six months before enrolling in the trial. Summary of study subjects, vaccination schedule and age at enrollment is presented in Tables 1 -2.

Example s. Orthopoxyiral-specific binding antibodies induced in CQH04S1 - vaccinated subjects

[0092] MVA-specific IgG binding antibodies in serum of COH04S1 vaccinated subjects were measured against whole MVA virions by ELISA. Low binding (O.D.<0.4 nm) at low serum dilution (1 :150) was measured at baseline in most subjects (Figure 2). In contrast to all placebo control volunteers, all subjects vaccinated with COH04S1 showed an increase in MVA-specific IgG titers post-vaccination regardless of the dose vaccination regimen (Figure 3), demonstrating potent vaccine-elicited orthopoxviral- specific humoral immunity. Elevated MVA-specific IgG titers were measured following prime vaccination at all dose levels, although MVA IgG titers in DL2 and DL3 subjects tended to be higher than those in DL1 subjects, indicating a dose dependent response (Figures 3A-3B). While DL1 cohorts had a seroconversion rate of 30-60% following prime vaccination, DL2 and DL3 subjects showed 100% seroconversion after the first dose (Figure 3C). MVA-specific IgG titers further increased in all vaccine cohorts following the second dose resulting in similar responses in DL1 and DL2/3 subjects and 100% seroconversion in all vaccine cohorts. MVA-specific IgG titers slowly declined over five-months post vaccination in all vaccine cohorts, but they remained at elevated levels over baseline in all subjects independent of the dose immunization regimen, except for one subject in the DL1 cohort. Notably, two subjects in the DL1 and DL3 cohorts, one subject born in 1971 and one subject born in 1986, had high IgG endpoint titers of 4,050 and 1 ,350 before vaccination, possibly indicating preexisting orthopoxviral immunity (Figure 2). These two subjects showed particularly elevated MVA-IgG titers after only one vaccine dose and their MVA IgG titers remained stable over six months post vaccination. These results overall demonstrate that COH04S1 -vaccinated subjects develop potent orthopoxviral-specific IgG antibody responses.

Example 4. MPXV-sDecific antibodies to neutralizing antibody targets induced in CQH04S1 -vaccinees

[0093] Binding antibodies to MPXV major targets of NAb were evaluated by ELISA. H3 is the MPXV homologue of vaccinia H3L with whom it shares 94% sequence. Antibodies against H3L are likely a key contributor to protection against poxvirus infection and disease ^{20 21} . A29 and A35 are the more conserved MPXV homologues of vaccinia A27L and A33R proteins which are associated with intracellular mature virions (IMV) and intracellular enveloped virions (EEV), respectively ²³²² , the two forms of vaccinia infectious virus particles. One-month after COH04S1 second dose, binding antibodies specific for MPVX neutralizing antibody (NAb) targets H3, A29, and A35 were elevated in all vaccine groups. Similarly, at 5 months after the booster vaccination, all subjects vaccinated with COH04S1 had sustained binding antibody titers to MPXV antigens. These results demonstrate that MVA-based vaccine COH04S1 induces MPXV-specific binding antibodies against antigens involved in the protection from MPXV infection. See Figure 4A-4C.

Example s. Orthopoxyiral-specific neutralizing antibodies induced in COHQ4S1 - vaccinated subjects

[0094] Similar to the observed MVA IgG responses in DL1 -DL3 subjects, COH04S1 -vaccinated subjects in all vaccine cohorts showed a strong increase in MVA-specific NAb titers, consistent with potent vaccine-induced orthopoxviral-specific immunity (Figure 5). Following prime vaccination, only a minor proportion of the DL1 subjects showed elevated MVA-specific NAb titers, whereas all DL2 and DL3 subjects showed an increase in MVA-specific NAb titers, confirming a dose-dependent vaccine effect after one dose (Figures 5A-5B). While the DL1 cohorts showed a seroconversion rate of 0-60% after the first vaccination, DL2 and DL3 cohorts showed 100% seroconversion for MVA-specific NAb after the first dose (Figure 5C). All subjects of the different vaccine cohorts developed robust MVA-specific NAb titers at one month after the booster dose with NT50 titers ranging from 73 to >2,560 and a median NT50 of 303, resulting in 100% seroconversion in all vaccine cohorts. DL2 and DL3 subjects tended to have slightly higher NAb titers than DL1 vaccinated volunteers. Similar to the MVA IgG titers, MVA-specific NAb titers declined in all vaccine cohorts over five-months post-second vaccination, but they remained above baseline in most subjects, resulting in a median NT50 titer of 65 and comparable titers across vaccine groups. Only two volunteers in the DL2 and DL3 vaccine cohorts had undetectable MVA-specific NAb titers. Interestingly, the same two subjects with high baseline MVA binding IgG titers had pre-vaccination NAb titers approaching the NT50 detection limit of 20, suggesting pre-existing orthopoxvirus-specific NAb responses in these vaccinees (Figure 6). Placebo-vaccinated volunteers had consistently undetectable MVA-specific NAb throughout the observation period (Figure 5). These results demonstrate that subjects vaccinated with COH04S1 at different dose levels develop robust and durable orthopoxvirus-specific NAb responses.

Example 6. Orthopoxyiral-specific cellular immunity induced in COHQ4S1 - vaccinated subjects

[0095] Orthopoxvirus-specific T cells were evaluated after the first and the second dose by assessing co-expression of IFNy with CD107a or CD69 activation markers on MVA-stimulated T cells using flow cytometry (Figures 1 and 7). CD107a marks cells capable of cytotoxic effector functions while CD69 is an early T cell activation marker that is transiently upregulated by activated T cells. Low levels of activated CD8+ and CD4+ T cells secreting IFNy upon MVA stimulation were measured at baseline (Figures 8 and 9). COH04S1 vaccinees showed a significant increase in CD107+ and CD69+ IFNy-secreting CD8+ and CD4+ T cells to maximal levels at one month after the first dose. After the second dose CD107+ and CD69+ IFNy-secreting CD8+ and CD4+ T cells levels remained stable and significantly elevated levels of activated T cells were measured over five months post-boost (Figures 8A and 9). Similar levels of MVA-specific T cells were observed independently of dose level (Figure 9). In contrast, placebo subjects showed no or only low percentage of MVA-specific T cells (Figure 9).

[0096] Phenotypic analysis of activated MVA-specific T cell subsets revealed that both CD8+ and CD4+ T cell populations in COH04S1 vaccinees were mostly comprised of T effector memory (TEM) cells (Figure 8B). Terminally differentiated TEM cells (TEMRA) comprised about 20% of the CD8+ T cell population and were significantly higher than TEMRA cells in the CD4+ population. Low percentages of naive and central memory (TCM) T cells were measured in both CD8+ and CD4+ T cell populations. Comparable percentages of activated naTve/TcM/TEM/TEMRA T cells were measured across the different DL vaccine cohorts (Figures 10A-10B). Finally, a similar phenotype distribution with predominance of TEM/EMRA over TCM was observed in the CD8+ and CD4+ T cell populations at one month and five months post booster vaccination (Figures 10C-10D). These results demonstrate that at all tested dose levels vaccination with COH04S1 induces robust and durable orthopoxvirus-specific cellular responses with a predominant effector memory phenotype and that one COH04S1 dose is sufficient to obtain maximal induction of MVA-specific activated T cells.

[0097] Binding antibodies to known MPXV major targets of NAb were evaluated by ELISA. H3 is the MPXV homologue of vaccinia H3L with which it shares 94% sequence, and it is the least conserved of NAb target between the two members of the orthopoxvirus family ²⁰ . H3 is associated with intracellular mature virions (IMV) and antibodies against H3L are likely a key contributor to protection against poxvirus infection and disease ^{20 21} . A35 is the more conserved MPXV homologue of vaccinia A33R protein which is associated with intracellular enveloped virions (EEV) ²²²³ , the form of vaccinia infectious virus particle that is more resistant to neutralizing antibodies ²⁴ . After one sMVA or COH04S1 dose, NHP developed H3- and A35- specific IgG, with higher levels of H3- than A35-specific IgG (Figures 14A-B, 15-16). One-month after the second dose, increased MPXV-specific IgG titers were measured in both sMVA- and COH04S1 -vaccinated NHP, indicating comparable induction of MPXV-specific antibodies by the two vaccines. In healthy subjects vaccinated with two doses of COH04S1 , H3- and A35-specific IgG were elevated one month after the second COH04S1 dose (Figures 14C, 15-16). H3-specific IgG endpoint titers tended to be higher in subjects vaccinated with the higher DL, while A35-specific IgG levels were more evenly distributed across DL. At 5 months after the booster vaccination, sustained binding antibody titers to MPXV antigens H3 and A35 were measured in COH04S1 -vaccinees independent of the DL (Figures 14D, 15-16). These results demonstrate that MVA-based vaccines sMVA and COH04S1 induce MPXV-specific binding antibodies against antigens involved in the protection from MPXV infection.

Example 7. Synthetic modified vaccinia Ankara confers potent monkeypox immunity

[0098] In accordance with and by way of update to the examples above, MVA- specific binding antibodies and neutralizing antibodies (NAb) were measured in COH04S1 vaccinated subjects regardless of vaccine dose (Figures 18A-18B). In contrast to all placebo control volunteers, all subjects vaccinated with COH04S1 showed an increase in MVA-specific IgG titers following vaccination (Figure 18A). Consistent with a prior dose-escalation trial of wild-type MVA ²³ , MVA IgG titers in DL2 and DL3 subjects following prime vaccination tended to be higher than those in DL1 subjects, indicating a dose dependent response. MVA-specific IgG titers further increased in all vaccine cohorts following the second dose, resulting in similar responses and 100% seroconversion in all vaccine cohorts (Figures 19A-19C). MVA- specific IgG titers slowly declined over five-months post-vaccination in all vaccine cohorts, although they remained at elevated levels in all subjects independent of dose immunization regimen.

[0099] Following prime vaccination, only a minor proportion of DL1 subjects showed elevated MVA-specific NAb titers, whereas all DL2 and DL3 subjects showed an increase in MVA-specific NAb titers, confirming a dose-dependent vaccine effect (Figures 18B, 19A-190). All subjects developed robust MVA-specific NAb titers at one month after the booster dose, resulting in 100% seroconversion in all vaccine cohorts. Similar to the MVA-specific IgG titers, MVA-specific NAb titers declined over five- months post-second vaccination, but they remained above baseline in most subjects. In addition, comparable MVA-specific binding antibodies and NAb titers were measured in NHP prime-boost vaccinated with either COH04S1 or sMVA (Figures 19A-19C).

[0100] Next, in accordance with Example 6, Orthopoxvirus-specific T cells in COH04S1 -vaccinated subjects were evaluated by assessing co-expression of IFNy with CD107a or CD69 activation markers on MVA-stimulated T cells (Figures 1 , 8A- 8B, 9A-9D, 20). COH04S1 vaccinees showed a significant increase in activated IFNy- secreting CD8 ⁺ and CD4 ⁺ T cells from baseline to maximal levels at one month after the first dose. After the second COH04S1 dose MVA-specific T cell levels remained stable and significantly elevated levels of activated T cells were measured over five months post-boost. Concordant with a dose-independent induction of SARS-CoV-2 antigen-specific T cells by COH04S1 ¹⁸ , MVA-specific T cells levels were comparable across dose groups. As previously observed by others, post-vaccine T cell response to orthopoxvirus antigens was largely comprised of CD8 ⁺ T cells ²⁴ . Phenotypic analysis revealed that at all time-points post-vaccination CD8 ⁺ and CD4 ⁺ MVA-specific T cells in COH04S1 vaccinees were predominantly T effector memory (TEM) cells, cell type that has been associated with protection against peripheral infection with VACV ²⁹ .

[0101] Next, by way of update to the examples above, it was addressed whether COH04S1 stimulated MPXV-specific immune responses to known protective antibody targets of intracellular mature virus (IMV) and extracellular enveloped virus (EEV), the two major virus forms mediating poxvirus transmission and dissemination ^{21 22} . In previous studies, single IMV and EEV antigens were effective in partially protecting mice against lethal vaccinia challenge ^{21 44} , whereas a combination of four vaccinia IMV and EEV antigens was completely protective in mice and elicited MPXV-specific humoral responses in NHP ²² . The selected antigens have 93.8% to 98.4% similarity with their VACV homologues ²⁰ . In healthy human subjects vaccinated with COH04S1 , elevated IgG specific for MPXV EEV proteins B6R and A35 (homologous of VACV B5R and A33R) and IMV proteins M1 R and H3 (homologous of VACV L1 R and H3L) were measured at one month after the second dose (Figure 25A). Except for A35- specific IgG, B6R, M1 R and H3-specific IgG titers tended to be higher in subjects vaccinated at higher DL. At five months post-booster vaccination, MPXV-specific antibodies appeared to decline, although elevated antibody titers to MPXV antigens were consistently measured in a proportion of COH04S1 vaccinees regardless of the DL used (Figures 27A). Elevated IgG titers against all IMV- and EEV-specific MPXV proteins were also measured in NHP after one or two doses of COH04S1 or sMVA (Figures 27B).

[0102] NAb preventing MPVX infection were measured in COH04S1 -vaccinated subjects using a clade 2 virus in a plaque reduction assay (Figures 27C; also see Figures 21 , 22A). After two vaccine doses, elevated MPXV NAb responses were measured in all vaccine cohorts regardless of dose level. At a dilution of 1 :10 most COH04S1 -vaccinated subjects’ serum samples neutralized at least 40% of the MPXV inoculum. In addition, MVA- and MPXV-specific humoral responses induced by COH04S1 correlated strongly (Figures 23A-23B), indicating cross-reactivity of vaccine-induced orthopoxviral-specific responses.

[0103] The studies discussed herein demonstrate that heathy adults vaccinated with multiantigen sMVA-vectored COVID-19 vaccine COH04S1 at different dose levels develop robust orthopoxviral-specific humoral and cellular immune responses, including antibodies to major MPXV virion proteins that neutralize MPXV infection in vitro. In addition, NHP vaccinated with either COH04S1 or sMVA develop similar and robust orthopoxviral- and MPXV-specific humoral responses, indicating comparable capacity of sMVA with and without inserted antigens to elicit orthopoxvirus/MP M- specific immunity. Considering that both humoral and cellular immunity have been shown to play a protective role against orthopoxviruses ⁴⁵ , it is encouraging that COH04S1 and sMVA vaccination induced robust and comprehensive orthopoxvirus- specific immunological responses.

[0104] In a cohort of 24 volunteers only two subjects (one in DL1/DL1 and one in DL2/DL2 groups) were older than 48 years, and therefore may have undergone routine smallpox vaccination before the smallpox vaccination campaign was ended in 1972. Additionally, vaccination against smallpox for work-related risk is only given to members of the army or laboratory personnel who directly handle replication competent VACV. Therefore, the MPXV cross-reactive immunity observed in most of the volunteers is likely to be due to a primary response to MVA and not to a recall response to smallpox vaccination.

[0105] While the protective efficacy of previously tested MVA vaccines against MPXV has been reported in NHP ^{38 39} , the vaccine immunogenicity analysis was primarily carried out using a different platform (VACV/MVA), and there is no data for the capacity of any vaccine to elicit MPXV-specific NAb responses in humans. Additionally, neither Dryvax, nor a combination of MVA and Dryvax were able to protect NHP with AIDS from a lethal MPXV challenge ³⁴ , questioning whether MVA vaccines would be protective against MPXV in the immunocompromised population. In contrast, the finding that COH04S1 -vaccinated healthy adults and NHP vaccinated either with COH04S1 or sMVA develop robust MPXV-specific binding antibodies to multiple virion IMV and EEV proteins suggests that sMVA has the capacity to elicit humoral responses that interfere with MPXV virus transmission and dissemination. In addition, all COH04S1 vaccine cohorts regardless of vaccine dose developed detectable MPXV-specific NAb, highlighting the potential of sMVA to stimulate cross- reactive antibodies that are considered essential for the protection against MPXV infection.

[0106] MVA has been extensively used as a viral vector for delivery of heterologous antigens and tested as a vaccine against infectious diseases and cancer ⁷ , which includes MVA-based HIV vaccine candidates that are being tested in clinical studies ⁷ . However, the benefits of these additional heterologous antigens in vaccine formulations such as MVA-based HIV vaccines are debatable because studies have shown that those additional antigen inserts cause issues that may be a barrier to widespread use ^{46 47} , which may negatively impact regulatory approval and public acceptance. In contrast, the safety and immunogenicity of COH04S1 observed in the recent phase 1 clinical trial, in addition to the strong protective capacity of COH04S1 measured in preclinical animal models ^{16 17} , suggest that COH04S1 represents a unique vaccine candidate to simultaneously protect against SARS-CoV- 2 and MPXV, which would be a desirable attribute especially in low-resource settings. Thus, in some embodiments, a dose of 1 x10 ⁸ pfu of an sMVA-based vaccine given as a prime and boost format (2-4-week interval between doses) would be the ideal clinical regimen to augment the limited availability of the Jynneos® vaccine, especially in limited resource countries where MPXV cases continue to climb and the need for vaccination is greatest.

Example 8. Evaluation of MPXV-specific neutralizing antibodies in COHQ4S1 - and Jynneos®-vaccinated healthy adults using complement

[0107] MPVX-specific NAb were measured in COH04S1 -vaccinated subjects using a clade 2b virus by plaque reduction assay (PRNT) with and without the addition of exogenous complement (Figure 25B and 27A-27C). Compared to baseline, after two vaccine doses, elevated cross-reactive MPXV-specific NAb responses were measured in all vaccine cohorts regardless of dose level (Figures 27A-27C). Postboost PRNT50 titers were measured in 9/20 volunteers. After the addition of complement, a significant increase of MPXV PRNT50 titers was observed, with most volunteers showing PRNT50 titers above baseline (Figure 25B). In addition, MVA- and MPXV-specific humoral responses induced by COH04S1 correlated strongly (Figure 23A-23B), indicating cross-reactivity of vaccine-induced orthopoxviral-specific responses.

Example 9. COHQ4S1 -elicited immune responses as compared to those induced by Jynneos®

[0108] To compare the stimulation of MPXV cross-reactive humoral responses between COH04S1 and FDA-approved Jynneos®, immune plasma was analyzed from a cohort of volunteers vaccinated through different routes with Jynneos® during the recent mpox health emergency (Table 3). Individuals were vaccinated two times with the standard subcutaneous dose (1 x10 ⁸ pfu), or with the low-dose-sparing regimen (2x10 ⁷ pfu) via intradermal route that has been recently given EUA. Additionally, some individuals were prime-boost vaccinated with a combination of the two doses/immunization routes. As shown in Figure 25C, M1 R-specific IgG was significantly elevated in COH04S1 vaccinees compared to volunteers who received Jynneos®, and comparable titers of other MVA-specific and MPXV-cross reactive IgG titers were measured between the two vaccine cohorts. In addition, comparable MPXV-specific NAb responses were measured in COH04S1 - and Jynneos® vaccinees when titers were evaluated both in the presence and absence of exogenous complement (Figure 25D). A minority of individuals in each cohort (2/20 in the COH04S1 cohort and 5/19 in the Jynneos® cohort) were born before 1973 and likely received smallpox vaccination during childhood. These subjects with VACV preexisting immunity were more likely to have elevated MPXV-cross reactive binding and neutralizing antibodies.

[0109] The Vaccinia homolog of M1 R is well known to elicit an immune response that contributes to protection. And, the enhanced MPXV M1 R -specific titers in COH04S1 vaccinated individuals compared to Jynneos vaccinated individuals may translate into enhanced protection by COH04S1 compared to Jynneos. As such, these results indicate that the sMVA-based COH04S1 vaccine elicits MPXV-cross reactive humoral responses that may be more robust and/or complete as compared to those induced by the FDA-approved Mpox vaccine Jynneos® due to an enhanced M1 R- specific IgG response against IMV form of MPXV.

[0110] The differing results in COH04S1 as compared to Jynneos® are indeed surprising, given that the MVA backbones are substantially similar. While the difference is unlikely to be explained by an S- or N-specific cross reactive immune response, the expression of S and/or N antigens may potentially enhance the immune responses to the M1 R homolog in MVA, potentially through a stabilization effect or enhanced expression, or alternatively through enhanced IMV production, thereby augmenting the induction of cross-reactive immune responses to MPXV IMV or the M1 R virion protein. This may be similar to an intrinsic adjuvant effect. Alternatively, since COH04S1 and Jynneos were administered through different immunization routes (intramuscular for COH04S1 versus intradermal or subcutaneous for Jynneos), the differential IMV M1 R specific immune responses in COH04S1 and Jynneos vaccinated individuals may also be explained through the use of different immunization routes, which may result in different antigen expression and processing in different target cells and potentially improve the induction IMV or M1 R-specific immune responses.

Example 10. COHQ4S1 and empty sMVA vector protect against Mpox in CAST/EiJ mice

[0111] To evaluate the capacity of COH04S1 and sMVA to confer protection against Mpox, vaccine-efficacy was assessed against MPXV lung infection utilizing the CAST/EiJ mouse model. CAST/EiJ mice (n=9-10/group) were vaccinated twice by intramuscular route in 4 weeks interval with escalating doses of either COH04S1 or sMVA and challenged intranasally 4 weeks later with a MPXV clade 2b isolate derived from a MPXV-infected individual (Figure 26A). As shown in Figure 26B, MPXV-specific cross-reactive NAb were detectable in a small number of animals post-prime vaccination. MPXV-specific NAb titers significantly increased after the second vaccination in the high-dose sMVA and COH04S1 vaccine groups, while intermediate- and low-dose sMVA and CHO04S1 groups showed low-to-absent MPXV-specific NAb titers after the booster vaccination.

Minimal weight gain/loss was observed after MPXV intranasal challenge, with a significant increase in body weight compared to controls only observed at days 4 or 5 post-challenge in mice vaccinated with low-dose sMVA or intermediate- and high-dose COH04S1 (Figure 26C). When MPXV viral load (VL) was measured in lung tissue of mice five days post-challenge, all sMVA- and COH04S1 -vaccinated animals showed markedly reduced MPXV VL compared to controls, independent of the used vaccine dose (Figure 26D). Lung VL in controls ranged from 10 ⁷ to 2x10 ⁹ TCID50/g, consistent with productive lung infection by MPXV in unvaccinated animals. Mice vaccinated with low-dose sMVA and COH04S1 had lung VL that ranged from below the detection limit (2.5x10 ³ TCID50/g) to 8x10 ⁶ TCID50/g. Mice vaccinated with sMVA and COH04S1 intermediate-dose had low-to-undetectable lung VL in most cases. Only 2/8 mice vaccinated with high-dose of sMVA and COH04S1 showed titers above the detection limit, indicating complete or potent viral control in these animals. Lung VL in sMVA and COH04S1 low-dose vaccine groups were significantly higher than in mice vaccinated with intermediate- and/or high-dose, suggesting improved protection through higher vaccine doses. Taken together, these results show that unmodified sMVA without inserted antigens and multiantigen sMVA-based COVID-19 vaccine COH04S1 expressing SARS-CoV-2 spike and nucleocapsid antigens confer protection against lung infection caused by MPXV, even when administered at very low dose.

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