Vaccines based on mutant CALR and JAK2 and uses thereof

Abstract

Vaccines, polypeptides, and polynucleotides based on mutant CALR and JAK2 sequences, vectors, host cells, and viruses, as well as methods of making and using them, are provided. The present disclosure also provides methods of inducing an immune response and treating, preventing, reducing the risk of, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F or CALR exon 9 mutation, or both a JAK2V617F and a CALR exon 9 mutation, comprising multiple administrations of any of the compositions comprising the polynucleotides, polypeptides, or vectors disclosed herein.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 62 / 936,841, filed November 18, 2019, and U.S. Provisional Patent Application No. 62 / 936,846, filed November 18, 2019, each of which is incorporated by reference in its entirety herein.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on November 6, 2020, is named JBI6177USNP1_SL.txt, and is 65,758 bytes in size.

[0003] FIELD OF THEINVENTION Mutant calreticulin (CALR) and Janus Kinase 2 (JAK2) sequences, vectors, host cells, viral-based vaccines, polypeptides and polynucleotides, and methods of making and using the same are provided. [Background technology]

[0004] Classical myeloproliferative neoplasms (MPNs), also called BCR-ABL-MPNs, are the most frequent of the myeloproliferative disorders. MPNs are characterized by the excessive production of fully functional terminally differentiated blood cells. Classical MPNs are classified into three entities, namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), in which disease-related complications such as venous and arterial thrombosis, bleeding, and transformation to acute myeloid leukemia (AML) occur frequently. All MPN entities arise from a single somatically mutated hematopoietic stem cell (HSC) that clonally expands and gives rise to virtually all myeloid cells, as well as B and NK cells. Clonal expansion of MPN HSCs is associated with mono- or multilineage hyperplasia.

[0005] PV is characterized by excess red blood cells and involvement of erythroid predominance, as well as variable hyperplasia of megakaryocytic / granulocytic lineages. ET is characterized by an increased platelet count with megakaryocytic hyperplasia, whereas PMF is a more heterogeneous disorder defined by the presence of myelofibrosis (specifically excess collagen fibers) and megakaryocytic hyperplasia. Myeloproliferation in PMF initially predominates in the bone marrow and then expands to extramedullary sites such as the spleen. Diagnosis at disease onset is often challenging, but the 2016 edition of the World Health Organization (WHO) diagnostic criteria for MPNs helps define both the molecular, clinical, and symptomatic presentation of MPNs. However, progression of ET and PV to secondary myelofibrosis (MF) is observed in many patients. Furthermore, the boundaries between these three disorders are not well established, especially between ET and PMF. Thus, transitional entities may emerge that represent conditions such as prefibrotic PMF (or early PMF), which at the time of diagnosis exhibit an ET phenotype and typical bone marrow histology, but have a higher likelihood of progressing to MF and a worse prognosis than true ET.

[0006] More than 50% of MPN patients harbor the JAK2V617F mutation. Furthermore, mutations in exon 9 of the calreticulin (CALR) gene are found in approximately 60% of patients with JAK2 wild-type essential thrombocythemia (ET) or primary myelofibrosis (PMF).

[0007] In 2005, a somatic G to T mutation at nucleotide 1849 in Exon14 of JAK2 was discovered. This mutation results in a valine to phenylalanine substitution at codon 617 (V617F) in the pseudokinase domain of JAK2. This mutation can be found in approximately 70% of myeloproliferative neoplasms (MPN), 95% of polycythemia vera (PV), and 50%-60% of ET and PMF. JAK2V617F often transitions from heterozygous to homozygous due to the occurrence of mitotic recombination that results in copy-neutral loss of heterozygosity along a variable-sized region of the short arm of chromosome 9 (9pLOH). JAK2V617F arises in multipotent hematopoietic progenitor cells, is present in all myeloid lineages, and can also be detected in lymphoid cells, mainly B cells and natural killer (NK) cells, and more rarely, in T cells later in the disease. JAK2V617F is primarily restricted to classical MPNs, except for refractory anemia with ringed sideroblasts and thrombocytopathy (RARST). However, JAK2V617F has been detected at very low levels (<1%) in normal populations, including newborns. It is one of the most frequent mutations found in age-associated clonal hematopoiesis (clonal hematopoiesis of undetermined potential). The presence of JAK2V617F mutations results in constitutive activation of signal transducer and activator of transcription (STAT) signaling, leading to increased cell proliferation, activation, and autocrine / paracrine release. JAK2V617F mutations have also been identified in patients with cardiovascular disease.

[0008] In 2013, frameshift mutations in exon 9 of the CALR gene were identified in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) who were negative for the V617F mutation in the JAK2 gene (JAK2V617F) and mutations in the thrombopoietin receptor (MPL) gene. More than 50 frameshift mutations were identified, with more than 85% harboring the same 44 amino acid mutation in the C-terminal tail. Mutations in the C-terminal tail result in the KDEL (SEQ ID NO:29) The CALR motif is removed, resulting in loss of endoplasmic reticulum (ER) retention and translocation to the cell surface membrane. Furthermore, mutant CALRs have a positively charged C-terminal tail that disrupts Ca2+ binding and limits essential functions. The two most frequent CALR mutations are type 1, also known as type 2, and type 3, with a 52-bp deletion (p.L367fs * 46), and a 5-bp insertion (p.K385fs), also known as type 2. * 47). There is a large difference in frequency between type 1 and type 2 mutations in ET and PMF. Type 1 and type 2 mutations in ET are closely distributed (55% vs. 35%), whereas type 1 is largely prevalent in PMF (75% vs. 15%). These results indicate that mutant CALR is an oncogenic driver and that CALRmut induces transformation via the MPL-JAK2-STAT signaling pathway.

[0009] MPN patients suffer from a high symptom burden, life-threatening complications, and risk of progression to acute leukemia, while also having limited therapeutic options. Treatment for MPN patients best falls into the categories of observation, medical therapy, and allogeneic stem cell transplantation (allo-SCT). Medical therapy itself falls into the categories of cytoreductive agents, single agent JAK inhibitors, and the immunomodulator interferon (IFN)-α. The current standard of care, and the only approved therapeutic agent specifically for MPN patients, is the small molecule JAK1 / 2 inhibitor JAKAFI® (ruxolitinib). JAKAFI® (ruxolitinib) was the first targeted agent approved by the US Food and Drug Administration (FDA) for intermediate-to-high risk PMF, post-polycythemia vera myelofibrosis (PPV-MF), and post-essential thrombocythemia myelofibrosis (PET-MF), but has recently been approved for both PV and graft-versus-host disease (GVHD) patient populations. Efficacy was established in the COMFORT-I and COMFORT-II trials, which showed a significant reduction in spleen size as the primary endpoint. However, discontinuation due to diminished efficacy, disease progression, and treatment-related adverse events occurred in approximately 50% of patients at 3 years and 75% at 5 years. JAKAFI® (ruxolitinib) therapy has also been associated with an increased risk of aggressive B-cell lymphoma in patients with MF. Indeed, in a study of 107 MF patients who discontinued JAKAFI® (ruxolitinib) treatment, the median overall survival was only 14 months. Although there is a subset of patients who may derive a survival benefit from the use of JAKAFI® (ruxolitinib), the majority of MPN patients continue to experience disease progression. Summary of the Invention [Means for solving the problem]

[0010] The present disclosure provides polypeptides comprising at least two or more epitope sequences selected from the following: the Janus kinase 2 (JAK2) epitope of SEQ ID NO: 6 (FCGDENILV), the JAK2 epitope of SEQ ID NO: 5 (VLNYGVCFC), the JAK2 epitope of SEQ ID NO: 4 (KLSHKHLVLNYGVCFCGDENILVQEFVKFG), the calreticulin (CALR) epitope of SEQ ID NO: 1 (MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE), and the CALR epitope of SEQ ID NO: 2 (EEAEDNCRRMMRTK).

[0011] The present disclosure also provides a polypeptide comprising two or more repeats of SEQ ID NO: 6. In some embodiments, the polypeptide comprises an AAY, RR or DPP, HHAA (SEQ ID NO:56) , HHA, HHL, RKSYL (SEQ ID NO:57) , R.K.S.Y. (SEQ ID NO:58) , SSL, or REKR (SEQ ID NO:59) The present invention includes 2, 3, 4, 5, or more than 5 repeats of SEQ ID NO:6 separated by linker sequences such as:

[0012] The disclosure also provides a polypeptide of SEQ ID NO:28 (FCGDENILVAAYFCGDENILV), which comprises two polypeptides of SEQ ID NO:6 linked by the non-natural linker sequence AAY.

[0013] The present disclosure also provides polynucleotides encoding the polypeptides disclosed herein, and vectors comprising the polynucleotides encoding the polypeptides disclosed herein.

[0014] The present disclosure also provides methods of inducing an immune response and treating, preventing, reducing the risk of developing, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F or a CALR exon 9 mutation, or both a JAK2V617F and a CALR exon 9 mutation, comprising administering to a subject in need of a method of inducing an immune response and treating, preventing, reducing the risk of developing, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F or a CALR exon 9 mutation, or both a JAK2V617F and a CALR exon 9 mutation, any of the polynucleotides, polypeptides, or vectors disclosed herein.

[0015] The present disclosure also provides methods of inducing an immune response and treating, preventing, reducing the risk of developing, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F or a CALR exon 9 mutation, or both a JAK2V617F and a CALR exon 9 mutation, comprising multiple administration of any of the compositions comprising a polynucleotide, polypeptide, or vector disclosed herein.

[0016] The present disclosure also provides for the administration of an anti-CTLA-4 antibody, an anti-PD-1, or an anti-PD-L1 antibody in combination with any of the compositions comprising the polynucleotides, polypeptides, or vectors disclosed herein.

[0017] The disclosure also provides methods of inducing an immune response and treating, preventing, reducing the risk of, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F or a CALR exon 9 mutation, or both a JAK2V617F and a CALR exon 9 mutation, the methods comprising: The method includes administering to a subject in need thereof two or more doses of a vector comprising a polynucleotide encoding the JAK2 epitope of SEQ ID NO: 6 (FCGDENILV), the JAK2 epitope of SEQ ID NO: 5 (VLNYGVCFC), the CALR epitope of SEQ ID NO: 1 (MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE), and the CALR epitope of SEQ ID NO: 2 (EEAEDNCRRMMRTK), wherein the vector is Ad26, GAd20, MVA, or a self-replicating RNA.

[0018] In some embodiments, the method of inducing an immune response and treating, preventing, reducing the risk of developing, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F or a CALR exon 9 mutation, or both a JAK2V617F and a CALR exon 9 mutation, comprises administering two or more doses of a vector comprising a polynucleotide encoding a polypeptide comprising two or more repeats of SEQ ID NO:6 to a subject in need of a method of inducing an immune response and treating, preventing, reducing the risk of developing, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F or a CALR exon 9 mutation, or both a JAK2V617F and a CALR exon 9 mutation, wherein the vector is Ad26, GAd20, MVA, or a self-replicating RNA.

[0019] The present disclosure also provides a method for treating or preventing a myeloproliferative disorder in a subject, the method comprising administering to a subject in need of a method for treating or preventing a myeloproliferative disorder in a subject, two or more doses of a vector comprising a polynucleotide encoding the JAK2 epitope of SEQ ID NO: 6 (FCGDENILV), the JAK2 epitope of SEQ ID NO: 5 (VLNYGVCFC), the CALR epitope of SEQ ID NO: 1 (MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE), and the CALR epitope of SEQ ID NO: 2 (EEAEDNCRRMMRTK), wherein the vector is Ad26, GAd20, MVA, or a self-replicating RNA. [Brief description of the drawings]

[0020] [Figure 1] 1 shows JAK2V617F+HLAA02:01+MPN donor T cell responses against mutJAK2 class I epitopes 1 and 2. [Diagram 2] Flow cytometry intracellular cytokine staining of IFN-γ / TNF-α responses from both CD4+ and CD8+ T cells are shown. Normal donor dendritic cells were infected with adenovirus 26 vectors containing either an empty vector or a polynucleotide encoding the LS_CALR.JAK2 2X9mer of SEQ ID NO: 10. Autologous CD4 / CD8 T cells were added to the dendritic cells 24 hours after infection. Dendritic and T cells were co-cultured for 11 days and restimulated with a pool of peptide-specific mutCALR. [Diagram 3] Flow cytometry intracellular cytokine staining of IFN-γ / TNF-α responses from CD8+ T cells. Normal donor dendritic cells were infected with adenovirus 5 vectors containing either an empty vector or a polynucleotide encoding the LS_CALR / JAK2 2X9mer of SEQ ID NO: 10. Autologous CD8 T cells were added to the dendritic cells 24 hours after infection. Dendritic and T cells were co-cultured for 11 days and restimulated with 9mer peptides specific for either muJAK2 epitope 1 (SEQ ID NO: 5) or epitope 2 (SEQ ID NO: 6). [Figure 4] Flow cytometry intracellular cytokine staining of IFN-γ / TNF-α responses from CD8+ T cells. Normal donor dendritic cells were infected with adenovirus 5 vectors containing either an empty vector or a polynucleotide encoding LS_CALR.JAK2-30mer (SEQ ID NO:11). Autologous CD8 T cells were added to dendritic cells 24 hours after infection. Dendritic and T cells were co-cultured for 11 days and restimulated with 9mer peptides specific for either mutJAK2 epitope 1 (SEQ ID NO:5) or epitope 2 (SEQ ID NO:6). [Diagram 5] Figure 1 shows IFN-γ ELISpot responses in splenocytes from C57BL / 6 or Balb / c mice immunized IM with Ad26HEME001 (LS_CALR_JAK2-30mer, SEQ ID NO: 11) (n=8 / group) or Ad26.Empty (n=4) at a dose of 1010VP 2 weeks post-immunization. Splenocytes were stimulated overnight with a 15mer peptide pool spanning the HEME001 sequence. The number of IFN-γ SFU per 106 splenocytes was determined by ELISpot. The mean response per group is indicated by a horizontal line. The dotted line indicates the background of the assay, defined as the 95% percentile of SFU observed in unstimulated splenocytes (43 SFU / 106 cells). Statistical analysis was performed using the Wilcoxon Rank Sum test, with values ​​below 43 SFU / 106 cells set to this cutoff. VP, viral particles, LOD, limit of detection. [Figure 6]Figure 1 shows IFN-γ ELISpot responses in splenocytes from C57BL / 6 or Balb / c mice immunized IM with Ad26HEME002 (LS_CALR_JAK2-2x9mer, SEQ ID NO: 10) (n=8 / group) or Ad26.Empty (n=4) at a dose of 1010VP 2 weeks post-immunization. Splenocytes were stimulated overnight with a 15mer peptide pool spanning the HEME002 sequence. The number of IFN-γ SFU per 106 splenocytes was determined by ELISpot. The mean response per group is indicated by a horizontal line. The dotted line indicates the background of the assay, defined as the 95% percentile of SFU observed in unstimulated splenocytes (43 SFU / 106 cells). Statistical analysis was performed using the Wilcoxon Rank Sum test, with values ​​below 43 SFU / 106 cells set to this cutoff. VP, viral particles, LOD, limit of detection. [Figure 7] Figure 1 shows IFN-γ ELISpot responses in splenocytes from C57BL / 6 mice immunized IM with Ad26HEME002 (LS_CALR_JAK2-2x9mer, SEQ ID NO: 10), MVA-HCalJ-9.9 (TCE_CALR_JAK2-2x9mer, SEQ ID NO: 26), or Ad26.Empty (n=10 / group, except group 1 with 5 animals [MVA-HCalJ-9.9] and group 10 with 3 animals [Ad26.Empty]). At week 4, splenocytes were stimulated overnight with a peptide pool spanning the sequence of Ad26HEME002. The number of IFN-γ SFU per 106 splenocytes was determined by ELISpot. The geometric mean response per group is shown as the horizontal line. The dotted line on the graph indicates the background of the assay, defined as the 95% percentile of the SFU observed in unstimulated splenocytes. For difference tests comparing Ad26HEME002 prime alone with MVA-HCalJ-9.9 boost with Ad26HEME002 prime immunization, ANOVA was performed on log10 transformed ELISpot data. Values ​​below 52 SFU / 106 cells were set as cutoff. Bars correspond to the mean of each group. Gr: group. [Figure 8]Figure 1 shows IFN-γ ELISpot responses in splenocytes from C57BL / 6 mice immunized IM with Ad26HEME002 (LS_CALR_JAK2-2x9mer, SEQ ID NO: 10), MVA-HCalJ-9.9 (TCE_CALR_JAK2-2x9mer, SEQ ID NO: 26), or Ad26.Empty (n=10 / group, except group 1 [MVA-HCalJ-9.9] with 5 animals and group 4 [Ad26.Empty] with 3 animals). At week 4, splenocytes were stimulated overnight with a peptide pool spanning the CALRmut sequence. The number of IFN-γ SFU per 106 splenocytes was determined by ELISpot. The geometric mean response per group is shown as the horizontal line. The dotted line on the graph indicates the background of the assay, defined as the 95% percentile of the SFU observed in unstimulated splenocytes. For difference tests comparing Ad26HEME002 prime alone with MVA-HCalJ-9.9 boost with Ad26HEME002 prime immunization, ANOVA was performed on log10 transformed ELISpot data. Values ​​below 52 SFU / 106 cells were set as cutoff. Bars correspond to the mean of each group. Gr: group. [Figure 9]Figure 1 shows IFN-γ ELISpot responses in splenocytes from Balb / c mice immunized IM with Ad26HEME002 (LS_CALR_JAK2-2x9mer, SEQ ID NO: 10), MVA-HCalJ-9.9 (TCE_CALR_JAK2-2x9mer, SEQ ID NO: 26), or Ad26.Empty (n=8 / group). At week 4, splenocytes were isolated and stimulated overnight with a peptide pool spanning the sequence of HEME002. The number of IFN-γ SFU per 106 splenocytes was determined by ELISpot. The average response per group is indicated by a horizontal line. Each individual data point was background subtracted using splenocytes not stimulated overnight with peptide to assess background SFU / 106 splenocytes. For difference tests comparing Ad26HEME002 prime alone with Ad26HEME002 prime immunization versus boost with MVA-HCalJ-9.9, unpaired t-tests with Welches correction (unequal SD) were performed to assess statistical significance. SFU: spot forming units; Ad empty = Ad26.Empty; MVA only: MVA-HCalJ-9.9 (TCE_CALR_JAK2-2x9mer; SEQ ID NO: 26); Ad: Ad26HEME002 (LS_CALR_JAK2-2x9mer; SEQ ID NO: 10). [Figure 10]Figure 1 shows the kinetics, over time, as measured by ELISpot, of induction of IFN-γ producing human HEME002 specific T cells in non-human primates immunized with Ad26HEME002, LS_CALR_JAK2-2x9mer; SEQ ID NO: 10 (Ad) and / or MVA-HCalJ-9.9 TCE_CALR_JAK2-2x9mer; SEQ ID NO: 26 (MVA) alone or in combination with YERVOY® (ipilimumab) (Ipi). Human HEME002 specific T cell responses per 106 PBMC were measured over time. Assay positivity was defined as background subtracted response SFU / 106 cells > 50 SFU / 106 cells and background subtracted response > 2x moderate response SFU / 106 cells. Values ​​not reaching the assay positivity criterion were adjusted to 50 SFU / 106 cells. Log10 transformed data is shown. The lower dotted line is 50 SFU / 106 cells and corresponds to LOD, the upper dotted line is 2,000 SFU / 106 cells and corresponds to ULOQ. Arrow points indicate time of immunization per group. Arrow bars are standard deviation. LOD: lower limit of detection, ULOQ: upper limit of quantification. Gr: group. Groups from the top of the graph: top line: Gr4; second line from the top: Gr3, third line from the top: Gr2, fourth line from the top: Gr1. [Figure 11]Figure 1 shows the induction of IFN-γ producing HEME002 specific T cells in non-human primates immunized with Ad26HEME002, LS_CALR_JAK2-2x9mer, SEQ ID NO: 10 (Ad) and / or MVA-HCalJ-9.9, TCE_CALR_JAK2-2x9mer; SEQ ID NO: 26 (MVA) alone or in combination with YERVOY® (ipilimumab) (Ipi) per group at week -2 of the experiment. IFNγ production was assessed by ELISpot. Values ​​shown were background subtracted to account for values ​​below 50 SFU / 106 cells (shown as open black circles). Animals showing high background responses >150 SFU / 106 cells are shown as grey marks. Horizontal bars indicate group means. Statistical analysis was performed on the full dataset using a Tobit model with likelihood ratio tests and Bonferroni adjustment, significant responses are p<0.05. The lower dotted line is 50 SFU / 106 cells, corresponding to the LOD, and the upper dotted line is 2,000 SFU / 106 cells, corresponding to the ULOQ. Log10 transformed data are shown. [Figure 12] Figure 2 shows the induction of IFN-γ producing HEME002 specific T cells in non-human primates immunized with Ad26HEME002, LS_CALR_JAK2-2x9mer, SEQ ID NO: 10 (Ad) and / or MVA-HCalJ-9.9, TCE_CALR_JAK2-2x9mer; SEQ ID NO: 26 (MVA) alone or in combination with YERVOY® (ipilimumab) (Ipi) per group during the second week of the experiment. IFNγ production was assessed by ELISpot. Values ​​shown were background subtracted to account for values ​​below 50 SFU / 106 cells (shown as open black circles). Animals showing high background responses >150 SFU / 106 cells are shown as grey marks. Horizontal bars indicate group means. Statistical analysis was performed on the full dataset using a Tobit model with likelihood ratio tests and Bonferroni adjustment, significant responses are p<0.05. The lower dotted line is 50 SFU / 106 cells, corresponding to the LOD, and the upper dotted line is 2,000 SFU / 106 cells, corresponding to the ULOQ. Log10 transformed data are shown. [Figure 13]Figure 1 shows the induction of IFN-γ producing HEME002 specific T cells in non-human primates immunized with Ad26HEME002, LS_CALR_JAK2-2x9mer, SEQ ID NO: 10 (Ad) and / or MVA-HCalJ-9.9, TCE_CALR_JAK2-2x9mer; SEQ ID NO: 26 (MVA) alone or in combination with YERVOY® (ipilimumab) (Ipi) per group at week 6 of the experiment. IFNγ production was assessed by ELISpot. Values ​​shown were background subtracted to account for values ​​below 50 SFU / 106 cells (shown as open black circles). Animals showing high background responses >150 SFU / 106 cells are shown as grey symbols. Horizontal bars indicate group means. Statistical analysis was performed on the full dataset using a Tobit model with likelihood ratio tests and Bonferroni adjustment, significant responses are p<0.05. The lower dotted line is 50 SFU / 106 cells, corresponding to the LOD, and the upper dotted line is 2,000 SFU / 106 cells, corresponding to the ULOQ. Log10 transformed data are shown. [Figure 14] Figure 1 shows the induction of IFN-γ producing HEME002 specific T cells in non-human primates immunized with Ad26HEME002, LS_CALR_JAK2-2x9mer, SEQ ID NO: 10 (Ad) and / or MVA-HCalJ-9.9, TCE_CALR_JAK2-2x9mer; SEQ ID NO: 26 (MVA) alone or in combination with YERVOY® (ipilimumab) (Ipi) per group at week 10 of the study. IFNγ production was assessed by ELISpot. Values ​​shown were background subtracted to account for values ​​below 50 SFU / 106 cells (shown as open black circles). Animals showing high background responses >150 SFU / 106 cells are shown as grey symbols. Horizontal bars indicate group means. Statistical analysis was performed on the full dataset using a Tobit model with likelihood ratio tests and Bonferroni adjustment, significant responses are p<0.05. The lower dotted line is 50 SFU / 106 cells, corresponding to the LOD, and the upper dotted line is 2,000 SFU / 106 cells, corresponding to the ULOQ. Log10 transformed data are shown. [Figure 15]Figure 1 shows the induction of IFN-γ producing HEME002 specific T cells in non-human primates immunized with Ad26HEME002, LS_CALR_JAK2-2x9mer, SEQ ID NO: 10 (Ad) and / or MVA-HCalJ-9.9, TCE_CALR_JAK2-2x9mer; SEQ ID NO: 26 (MVA) alone or in combination with YERVOY® (ipilimumab) (Ipi) per group at week 16 of the study. IFNγ production was assessed by ELISpot. Values ​​shown were background subtracted to account for values ​​below 50 SFU / 106 cells (shown as open black circles). Animals showing high background responses >150 SFU / 106 cells are shown as grey marks. Horizontal bars indicate group means. Statistical analysis was performed on the full dataset using a Tobit model with likelihood ratio tests and Bonferroni adjustment, significant responses are p<0.05. The lower dotted line is 50 SFU / 106 cells, corresponding to the LOD, and the upper dotted line is 2,000 SFU / 106 cells, corresponding to the ULOQ. Log10 transformed data are shown. [Figure 16] Figure 1 shows that the anti-CTLA4 antibody ipilimumab administered SC is equivalent to IV in priming antigen-specific T cell responses in NHPs. Ipilimumab was administered 3 mg / kg IV or SC at the time of vector immunization. IFNγ producing cells from 250K cynomolgus PBMCs were stimulated overnight with mutCALR-specific peptides and analyzed by Elipot. MutCALR peptide responses were calculated based on mutated CALR-specific responses minus background. Bars represent group means. Statistical analysis was performed on the full data set using a Tobit model with likelihood ratio tests and Bonferroni adjustment applied, with significant responses at p<0.05. [Figure 17]Figure 1 shows that incorporation of an anti-PD-1 antibody (nivolumab 10 mg / kg IV) into either one (group 6) or two (group 5) doses of Ad / Ad / MVA+ipilimumab (SC) starting at week 4 improves the magnitude of mutCALR-specific T cell responses compared to animals receiving Ad / Ad / MVA+ipilimumab (SC) alone. Ad26: Ad26HEME002, LS_CALR_JAK2-2x9mer, SEQ ID NO: 10; Ipi; ipilimumab; aPD1: nivolumab. [Figure 18A] FIG. 1 shows a schematic diagram of the Semliki Forest virus alphavirus genome, which is a positive-sense single-stranded RNA encoding the nonstructural polyproteins (nsP1-nsP4; replicase) at the 5′ end and the structural genes (capsid and glycoproteins) at the 3′ end. [Figure 18B] Schematic diagram of an exemplary self-replicating RNA (replicon) derived from an alphavirus replicon, in which a viral structural gene is replaced with a gene of interest under the transcriptional control of a subgenomic promoter (SGP). Conserved sequence elements (CSEs) at the 5' and 3' ends act as promoters for minus- and plus-strand RNA transcription. After the replicon is delivered into the cell, a nonstructural polyprotein precursor (nsP1234) is translated from the transcribed replicon in vitro. nsP1234 is initially autoproteolytically processed into fragments nsP123 and nsP4, which transcribe the minus-strand copy of the replicon. nsP123 is then fully processed into one protein, which combines with the (+)-strand replicase to transcribe a new plus-strand genomic copy and a (+)-strand subgenomic transcript encoding the gene of interest. The subgenomic RNA and the new genomic RNA are capped and polyadenylated. Inactive promoters are indicated by dotted arrows and active promoters by solid arrows. [Figure 19]Schematic diagram of an exemplary self-amplifying RNA from an alphavirus, containing the 5'-cap, nonstructural genes (NSP1-4), 26S subgenomic promoter (gray arrow), gene of interest (GOI), and 3'-polyadenylation tail. The diagram also shows the 2A ribosomal skipping element (P2A) and the overlapping first 193 nucleotides, excluding the start codon, downstream of the 5'-UTR of nsP1 and upstream of the DLP. [Figure 20] FIG. 1 is a schematic diagram of an exemplary lipid nanoparticle (LNP) encapsulating self-amplifying RNA with the molar ratio percentages of lipid components as indicated (Geall et al., PNAS, 2012, 109:14604-14609). [Figure 21A] Figure 1 shows the self-replicating RNA LS.CALR-JAK2-2x9mer priming an anti-CALR CD4 T cell response.Data show IFN production by splenocytes restimulated with overlapping CALR peptide libraries (open bars) or DMSO (grey bars) as measured by ELISpot. [Figure 21B] Self-replicating RNA LS.CALR-JAK2-2x9mer primes anti-CALR CD4 T cell responses. Data show IFN, TNFα and IL-2 production of splenocytes restimulated with overlapping CALR peptides measured by flow cytometry. Symbols represent individual mice (n=5), bars represent mean with SD. **p<0.01, Mann-Whitney test. [Figure 22A] Figure 1 shows lipid nanoparticle-formulated self-replicating RNA LS.CALR-JAK2-2x9mer priming anti-CALR CD4 T cell responses. Balb / c mice were immunized with the indicated doses of LNP-formulated self-replicating RNA LS.CALR-JAK2-2x9mer as well as 20 μg of the unformulated version and spleens were analyzed 14 days later. The figure shows IFN production by splenocytes restimulated with a duplicate CALR peptide library (DMSLO background subtracted) as measured by ELISpot. [Figure 22B]Figure 1 shows lipid nanoparticle-formulated self-replicating RNA LS.CALR-JAK2-2x9mer priming anti-CALR CD4 T cell responses. Data show IFN, TNFα and IL-2 production of splenocytes restimulated with overlapping CALR peptides measured by flow cytometry. Symbols represent individual mice (n=5) and bars represent mean with SD. *p<0.05, **p<0.01, Mann-Whitney test. [Figure 23A] Figure 1 shows a vaccine regimen that enhances anti-CALR CD4 T cell responses. Balb / c mice were primed and then boosted with various combinations of Ad26, self-replicating RNA molecules (replicons) or MVA as indicated in the figure, after which spleens were analyzed. The figure shows IFN production by splenocytes restimulated with a duplicate CALR peptide library (DMSO background subtracted) as measured by ELISpot. [Figure 23B] Figure 1 shows a vaccine regimen that enhances anti-CALR CD4 T cell responses. Data show IFN, TNFα and IL-2 production by splenocytes restimulated with overlapping CALR peptides as measured by flow cytometry. Symbols represent individual mice, bars represent mean with SD. *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] definition All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if fully set forth.

[0022] It should be understood that the terms used herein are used only to describe particular embodiments and are not intended to be limiting. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0024] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0025] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on the limitations of how the value is measured or determined, i.e., the measurement system. Unless expressly stated otherwise in the Examples or elsewhere in the specification in the context of a particular assay, result, or embodiment, "about" means within one standard deviation or a range of up to 5%, whichever is greater, according to practice in the art.

[0026] "Adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response to the vaccine of the present disclosure.

[0027] "Alternative scaffold" refers to a single chain protein framework containing a structured core associated with highly conformationally tolerant variable domains that can be engineered and selected to bind specific antigens, as the variable domains tolerate polymorphisms introduced without compromising the integrity of the scaffold.

[0028] An "antigen presenting cell" (APC) refers to any cell that presents antigen on its surface in association with major histocompatibility complex molecules, either MHC class I or MHC class II molecules, or both.

[0029] "Antibody" refers to immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, antigen-binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified configuration of immunoglobulin molecules that contain an antigen-binding site of the required specificity.

[0030] "Antigen-binding fragment" refers to a portion of an immunoglobulin molecule that retains the antigen-binding properties of the original full-length antibody. Exemplary antigen-binding fragments are heavy chain complementarity determining regions (HCDRs) 1, 2 and / or 3, light chain complementarity determining regions (LCDRs) 1, 2 and / or 3, VH, VL, VH and VL, Fab, F(ab')2, Fd and Fv fragments, and domain antibodies (dAbs) consisting of either one VH domain or one VL domain. The VH and VL domains can be linked together via synthetic linkers to form various types of single chain antibody designs in which the VH / VL domains pair intramolecularly or intermolecularly when the VH and VL domains are expressed by separate chains to form monovalent antigen-binding sites such as single chain Fvs (scFvs) or diabodies. (These are described, for example, in WO 1998 / 44001, WO 1988 / 01649, WO 1994 / 13804 or WO 1992 / 01047).

[0031] The transitional phrases "comprising," "consisting essentially of," and "consisting" are intended to connote their generally accepted meanings in patent language, i.e., (i) "comprising" is synonymous with "comprising," "containing," or "characterized by," is inclusive or open-ended, and does not exclude other unrecited elements or method steps, (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim, and (iii) "consisting essentially of" limits the scope of the claim to the materials or steps specified, and those that do not materially affect the "basic and novel characteristic(s)" of the claimed invention. An embodiment described with respect to the phrase "comprising" (or its equivalents) also provides as an embodiment what is described independently with respect to "consisting of" and "consisting essentially of."

[0032] "CALR" refers to human calreticulin. The human CALR protein comprises an amino acid sequence as set forth, for example, in UniProt Accession No. P27797.

[0033] "Downstream loop" or "DLP motif" refers to a polynucleotide sequence that contains at least one RNA stem loop, which, when placed downstream of the start codon of an open reading frame (ORF), increases translation of the ORF compared to an otherwise identical construct that does not contain the DLP motif.

[0034] "Enhancing" or "inducing," in reference to an immune response, refers to increasing the scale and / or efficiency of an immune response or extending the duration of an immune response. These terms are used interchangeably with "boosting."

[0035] As used herein, "epitope sequence" refers to a portion of a polypeptide or amino acid sequence, e.g., a portion of a primary, secondary, tertiary, or quaternary structure of a polypeptide or amino acid sequence, that is recognized by the immune system, e.g., antibodies, B cells (e.g., B lymphocytes), and / or T cells.

[0036] An "expression vector" refers to a vector that can be utilized in a biological system or reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0037] "Facilitating element" refers to any polynucleotide or polypeptide element operably linked to a polynucleotide or polypeptide, including promoters, enhancers, polyadenylation signals, stop codons, protein tags such as histidine tags, and the like.

[0038] "Heterologous" refers to two or more polynucleotides or two or more polypeptides that are not found in the same relationship to each other in nature.

[0039] An "immunogenic fragment" refers to a polypeptide that is recognized by cytotoxic T lymphocytes, helper T lymphocytes, or B cells when the fragment is in a complex with an MHC class I or MHC class II molecule.

[0040] "In frame" refers to the reading frame of the codons in a first polynucleotide being the same as the reading frame of the codons in a second polynucleotide that are joined together to form a heterologous polynucleotide. An in-frame polynucleotide encodes a polypeptide that is encoded by both the first and second polynucleotides.

[0041] "Immunogenic" refers to a polypeptide that contains one or more immunogenic fragments.

[0042] "Immune response" refers to any response to an immunogenic polypeptide or polynucleotide or fragment by the immune system of a vertebrate subject. Exemplary immune responses include local and systemic cellular and humoral immunity, such as CTL responses, including antigen-specific induction of CD8+ cytotoxic T lymphocytes (CTLs), helper T cell responses, including T cell proliferative responses and cytokine release, and B cell responses, including antibody responses.

[0043] "In combination with" means that two or more therapeutic agents are administered to a subject together in a mixture, simultaneously as single agents, or sequentially as single agents in any order.

[0044] "Isolated" refers to a homogenous population of molecules that have been substantially separated and / or purified away from other components of the system in which they are produced, such as in a recombinant cell (e.g., a synthetic polynucleotide or polypeptide), as well as to a protein that has been subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free of other cellular material and / or chemicals, and includes molecules that have been isolated to greater degrees of purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0045] "JAK2" refers to human Janus kinase 2. The human JAK2 protein comprises the amino acid sequence as set forth, for example, in UniPort Accession No. O060674.

[0046] "Mutant CALR" refers to CALR with one or more exon 9 mutations.

[0047] "Mutant JAK2" refers to JAK2 having a V617F mutation.

[0048] "Non-naturally occurring" refers to a molecule that does not exist in nature.

[0049] "Operably linked" sequences include both expression control sequences adjacent to the nucleic acid sequence they regulate, and regulatory sequences at a distance to act in trans or control the nucleic acid sequence being regulated.

[0050] "Philadelphia chromosome" or "Ph" refers to a well-known chromosomal translocation between chromosomes 9 and 22 that results in an oncogenic BCR-ABL gene fusion with constitutively active tyrosine kinase activity. This translocation fuses a portion of the BCR gene on chromosome 22q11 with a portion of the ABL gene on chromosome 9q34, which is designated t(9;22)(q34;q11) under the International System for Human Cytogenetic Nomenclature (ISCN). Depending on the exact location of the fusion, the molecular weight of the resulting fusion protein can range from 185 to 210 kDa. The "Philadelphia chromosome" refers to all BCR-ABL fusion proteins formed by the (9;22)(q34;q11) translocation.

[0051] "Polynucleotide" refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Examples of polynucleotides include RNA and DNA.

[0052] A "polypeptide" or "protein" refers to a molecule comprising at least two amino acid residues linked by a peptide bond to form the polypeptide.

[0053] "Preventing," "preventing," "prevention," or "prophylaxis" of a disease or disorder means preventing the disorder from occurring in a subject.

[0054] "Prime-boost" or "prime-boost regimen" refers to a method of treating a subject that includes priming a T cell response with a first vaccine, followed by boosting the immune response with a second vaccine. The first and second vaccines are typically different. These prime-boost immunizations elicit a larger and broader immune response than can be achieved by priming and boosting with the same vaccine. The priming step initiates memory cells, and the boosting step expands the memory response. The boosting can be performed one or more times.

[0055] "Recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules that are prepared, expressed, produced, or isolated by recombinant means.

[0056] "Refractory" refers to a disease that does not respond to treatment. A refractory disease can be resistant to treatment before or at the start of treatment, or a refractory disease can become resistant during treatment.

[0057] "Relapse" refers to the return of a disease or signs and symptoms of a disease after a period of improvement following prior treatment with a therapeutic agent.

[0058] "Replicon" refers to a viral nucleic acid that can direct the production of copies of itself and includes RNA and DNA. For example, a double-stranded DNA version of the Arterivirus genome can be used to generate the single-stranded RNA transcript that constitutes the Arterivirus replicon. Generally, a viral replicon contains the complete genome of the virus.

[0059] "RNA replicon" (or "self-replicating RNA", or "self-replicating RNA" or "srRNA") refers to an RNA molecule that contains all of the genetic information necessary to direct its own amplification or self-replication in a permissive cell. To direct its own replication, the RNA molecule 1) encodes a polymerase, replicase, or other protein that can interact with a protein, nucleic acid, or ribonucleoprotein derived from the virus or the host cell to catalyze the RNA amplification process, and 2) contains cis-acting RNA sequences necessary for the replication and transcription of the RNA encoding the replicon. Self-replicating RNA molecules are typically derived from the genome of a positive-stranded RNA virus and can be used as the basis for the introduction of foreign sequences into a host cell by replacing viral sequences encoding structural or nonstructural genes, or by inserting foreign sequences 5' or 3' of sequences encoding structural or nonstructural genes. Foreign sequences may be introduced into a subgenomic region of an alphavirus. Self-replicating RNA molecules may be packaged into recombinant viral particles, such as recombinant alphavirus particles, or delivered to the host using lipid nanoparticles (LNPs). The self-replicating RNA may be at least 1 kb, or at least 2 kb, or at least 3 kb, or at least 4 kb, or at least 5 kb, or at least 6 kb, or at least 7 kb, or at least 8 kb, or at least 10 kb, or at least 12 kb, or at least 15 kb, or at least 17 kb, or at least 19 kb, or at least 20 kb in size, and may be 100 bp to 8 kb, or 500 bp to 8 kb, or 500 bp to 7 kb, or 1 to 7 kb, or 1 to 8 kb, or 2 to 15 kb, or 2 to 20 kb, or 5 to 15 kb, or 5 to 20 kb, or 7 to 15 kb, or 7 to 18 kb, or 7 to 20 kb in size. Self-replicating RNA is described, for example, in WO 2017 / 180770, WO 2018 / 075235, and WO 2019143949(A2).

[0060] "Specifically bind", "specific binding", "specifically binding" or "bind" refers to a proteinaceous molecule binding to an antigen or an epitope within the antigen with an affinity higher than its affinity for other antigens. Typically, a proteinaceous molecule binds to an antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of about 1×10-7 M or less, e.g., about 5×10-8 M or less, about 1×10-8 M or less, about 1×10-9 M or less, about 1×10-10 M or less, about 1×10-11 M or less, or about 1×10-12 M or less, and typically the KD is at least 100-fold less than the KD for binding to non-specific antigens (e.g., BSA, casein). In the context of the molecules described herein, "specific binding" refers to binding of a proteinaceous molecule to a CALR / JAK2 mutant polypeptide, a CALR mutant polypeptide or a JAK2 mutant polypeptide alone, or in complex with HLA, without detectable binding to wild-type CALR or JAK2 alone, or in complex with HLA.

[0061] "Subgenomic RNA" refers to an RNA molecule that is smaller in length or size than the genomic RNA from which it is derived. Viral subgenomic RNA may be transcribed from an internal promoter whose sequence is present in the genomic RNA or its complement. Transcription of the subgenomic RNA may be mediated by a virus-encoded polymerase(s) associated with a host cell-encoded protein, a ribonucleoprotein(s), or a combination thereof. Many RNA viruses generate subgenomic mRNAs (sgRNAs) for expression of their 3'-proximal genes. Viral subgenomic RNA may be transcribed from an internal promoter whose sequence is present in the genomic RNA or its complement. Transcription of the subgenomic RNA may be mediated by a virus-encoded polymerase(s) associated with a host cell-encoded protein, a ribonucleoprotein(s), or a combination thereof.

[0062] "Subgenomic replicon" refers to a viral nucleic acid that contains less than the full complement of genes and other mechanisms of the viral genome, but is still capable of directing the production of copies of itself. For example, the subgenomic replicon of an adenovirus may contain most of the genes for the nonstructural proteins of the virus, but lacks most of the genes that code for the structural proteins. The subgenomic replicon can direct the expression of all of the viral genes necessary for the replication of the viral subgenome (replication of the subgenomic replicon) without producing viral particles.

[0063] A "subject" includes any human or non-human animal. A "non-human animal" includes any vertebrate, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" may be used interchangeably herein.

[0064] "Treating," "treating," or "treatment" of a disease or disorder, such as cancer, hereinafter refers to achieving one or more of the following: reducing the severity and / or duration of the disorder, inhibiting the worsening of symptoms characteristic of the disorder being treated, limiting or preventing the recurrence of the disorder in a subject who previously had the disorder, or limiting or preventing the recurrence of symptoms in a subject who was previously symptomatic for the disorder.

[0065] "Therapeutically effective amount" refers to an amount effective to obtain a desired therapeutic result at a required dosage and duration. The therapeutically effective amount may vary depending on factors such as the individual's condition, age, sex, and weight, and the ability of a therapeutic agent or combination of therapeutic agents to elicit a desired response in an individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, an improvement in the subject's health status.

[0066] "Vaccine" refers to a composition comprising one or more immunogenic polypeptides, polynucleotides encoding immunogenic polynucleotides or fragments, vectors comprising polypeptides encoding immunogenic polypeptides, or any combination thereof, intentionally administered to a recipient (e.g., a subject) to induce adaptive immunity.

[0067] "Vector" refers to a polynucleotide that can replicate in a biological system or can be transferred between such systems. Vector polynucleotides typically contain elements such as an origin of replication, a polyadenylation signal, or a selection marker that function to facilitate replication or maintenance of these polynucleotides in a biological system. Examples of such biological systems include cells, viruses, animals, plants, and reconstituted biological systems that utilize biological components capable of replicating the vector. The polynucleotides that make up the vector can be DNA or RNA molecules, or hybrid molecules thereof.

[0068] "Viral vector" refers to a vector construct that contains at least one polynucleotide element of viral origin and has the capacity to be packaged into a viral vector particle.

[0069] "Mutant," "mutation," or "altered" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, e.g., one or more substitutions, insertions, or deletions.

[0070] Polypeptides Disclosed herein are polypeptides comprising epitope sequences of mutant CALR and mutant JAK2 that can elicit an immune response in a subject. In some embodiments, the polypeptide comprises: the CALR epitope of SEQ ID NO: 1 (MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE) or having at least 90% sequence identity with SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2 (EEAEDNCRRMMRTK) or having at least 90% sequence identity with SEQ ID NO: 2, a JAK2 epitope of SEQ ID NO: 4 (KLSHKHLVLNYGVCFCGDENILVQEFVKFG) or having at least 90% sequence identity with SEQ ID NO: 4, a JAK2 epitope of SEQ ID NO: 5 (VLNYGVCFC) or having at least 90% sequence identity with SEQ ID NO: 5, It may comprise at least two or more epitope sequences selected from the group consisting of SEQ ID NO: 6 (FCGDENILV) or having at least 90% sequence identity with SEQ ID NO: 6, the JAK2 epitope, and combinations thereof.

[0071] In some embodiments, the disclosure provides a polypeptide comprising an epitope sequence of the CALR epitope of SEQ ID NO: 1 or having at least 90% sequence identity with SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2 or having at least 90% sequence identity with SEQ ID NO: 2, and the JAK2 epitope of SEQ ID NO: 4 or having at least 90% sequence identity with SEQ ID NO: 4. The epitope sequences of SEQ ID NOs: 1, 2, and 4 may be present in any order and separated by a linker. Exemplary linker sequences include AAY, RR, DPP, HHAA ... (SEQ ID NO:56) , HHA, HHL, RKSYL (SEQ ID NO:57) , R.K.S.Y. (SEQ ID NO:58) , SSL, or REKR (SEQ ID NO:59) Includes:

[0072] In some embodiments, the disclosure provides a polypeptide comprising an epitope sequence of a Janus kinase 2 (JAK2) epitope of SEQ ID NO:6 or having at least 90% sequence identity with SEQ ID NO:6, a calreticulin (CALR) epitope of SEQ ID NO:1 or having at least 90% sequence identity with SEQ ID NO:1, and a CARL epitope of SEQ ID NO:2 or having at least 90% sequence identity with SEQ ID NO:2. The epitope sequences of SEQ ID NOs:6, 1, and 2 may be present in any order and separated by a linker. Exemplary linker sequences include AAY, RR, DPP, HHAA ... (SEQ ID NO:56) , HHA, HHL, RKSYL (SEQ ID NO:57) , R.K.S.Y. (SEQ ID NO:58) , SSL, or REKR (SEQ ID NO:59) Includes:

[0073] In some embodiments, the present disclosure also provides a polypeptide comprising an epitope sequence of a Janus kinase 2 (JAK2) epitope of SEQ ID NO:6 or having at least 90% sequence identity with SEQ ID NO:6, a JAK2 epitope of SEQ ID NO:5 or having at least 90% sequence identity with SEQ ID NO:5, a calreticulin (CALR) epitope of SEQ ID NO:1 or having at least 90% sequence identity with SEQ ID NO:1, and a CARL epitope of SEQ ID NO:2 or having at least 90% sequence identity with SEQ ID NO:2. The epitope sequences of SEQ ID NOs:6, 5, 1, and 2 may be present in any order and are AAY, RR, DPP, HHAA ... (SEQ ID NO:56) , HHA, HHL, RKSYL (SEQ ID NO:57) , R.K.S.Y. (SEQ ID NO:58) , SSL, or REKR (SEQ ID NO:59) The aryl groups may be separated by a linker such as

[0074] The disclosure also provides polypeptides or immunogenic fragments thereof comprising one or more of the amino acid sequences of SEQ ID NO:12, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:28, SEQ ID NO:31.

[0075] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:3, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:3.

[0076] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:4, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:4.

[0077] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:7, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:7.

[0078] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:9, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:9.

[0079] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:10, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:10.

[0080] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:11, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:11.

[0081] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:12, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:12.

[0082] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:13, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:13.

[0083] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:14, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:14.

[0084] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:15, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:15.

[0085] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:28, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:28.

[0086] In some embodiments, the disclosure provides a polypeptide comprising the amino acid sequence of SEQ ID NO:31, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:31.

[0087] In some embodiments, the polypeptide comprises a polypeptide comprising an amino acid sequence of SEQ ID NO:6 or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:6. The present disclosure also provides polypeptides comprising two or more repeats of SEQ ID NO:6. In some embodiments, the polypeptide comprises 2, 3, 4, 5, or more than 5 repeats of SEQ ID NO:6. In some embodiments, the repeats of SEQ ID NO:6 can be separated by a linker. Exemplary linker sequences include AAY, RR, or DPP, HHAA. (SEQ ID NO:56) , HHA, HHL, RKSYL (SEQ ID NO:57) , R.K.S.Y. (SEQ ID NO:58) , SSL, or REKR (SEQ ID NO:59) Includes:

[0088] In some embodiments, the linkers disclosed herein may contain a protease cleavage site such that the polypeptide can be cleaved into peptide fragments comprising the epitope sequence in vivo in a subject, resulting in an improved immune response.

[0089] In some embodiments, the polypeptides of the present disclosure may further comprise a leader sequence or T-cell enhancer sequence (TCE) at the N-terminus. Leader sequences can increase expression and / or increase immunological responses. Exemplary leader sequences include T 2 Lymphocyte TCR receptor α chain (HAVT20) (MACPGFLWALVISTCLEFSMA; SEQ ID NO: 8), ubiquitin signal sequence (Ubiq) (MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRG V; SEQ ID NO: 54), or a 28 aa long peptide fragment from the Mandarin fish invariant chain (MGQKEQIHTLQKNSERMSKQLTRSSQAV; SEQ ID NO: 29). It is believed that leader sequences may be useful in increasing the immune response to the epitopes disclosed herein.

[0090] Polynucleotides The present disclosure also provides polynucleotides that encode any of the polypeptides disclosed herein.

[0091] In some embodiments, the polynucleotide comprises: the CALR epitope of SEQ ID NO: 1 (MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE) or having at least 90% sequence identity with SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2 (EEAEDNCRRMMRTK) or having at least 90% sequence identity with SEQ ID NO: 2, a JAK2 epitope of SEQ ID NO: 4 (KLSHKHLVLNYGVCFCGDENILVQEFVKFG) or having at least 90% sequence identity with SEQ ID NO: 4, a JAK2 epitope of SEQ ID NO: 5 (VLNYGVCFC) or having at least 90% sequence identity with SEQ ID NO: 5, - Encoding a polypeptide comprising at least two or more epitope sequences selected from the group consisting of SEQ ID NO:6 (FCGDENILV), or having at least 90% sequence identity to SEQ ID NO:6, JAK2 epitopes, and combinations thereof.

[0092] In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide comprising the epitope sequence of the CALR epitope of SEQ ID NO:1 or having at least 90% sequence identity with SEQ ID NO:1, the CALR epitope of SEQ ID NO:2 or having at least 90% sequence identity with SEQ ID NO:2, and the JAK2 epitope of SEQ ID NO:4 or having at least 90% sequence identity with SEQ ID NO:4. The epitope sequences of SEQ ID NOs:1, 2, and 4 may be present in any order and may be separated by a linker.

[0093] In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide that includes an epitope sequence of a Janus kinase 2 (JAK2) epitope of SEQ ID NO: 6 (FCGDENILV), or having at least 90% sequence identity to SEQ ID NO: 6, a calreticulin (CALR) epitope of SEQ ID NO: 1, or having at least 90% sequence identity to SEQ ID NO: 1, and a CARL epitope of SEQ ID NO: 2, or having at least 90% sequence identity to SEQ ID NO: 2. The epitope sequences of SEQ ID NOs: 6, 1, and 2 may be present in any order and may be separated by a linker.

[0094] In some embodiments, the disclosure also provides a polynucleotide encoding a polypeptide comprising an epitope sequence of a Janus kinase 2 (JAK2) epitope of SEQ ID NO:6 or having at least 90% sequence identity with SEQ ID NO:6, a JAK2 epitope of SEQ ID NO:5 or having at least 90% sequence identity with SEQ ID NO:5, a calreticulin (CALR) epitope of SEQ ID NO:1 or having at least 90% sequence identity with SEQ ID NO:1, and a CARL epitope of SEQ ID NO:2 or having at least 90% sequence identity with SEQ ID NO:2. The epitope sequences of SEQ ID NOs:6, 5, 1, and 2 can be present in any order and can be separated by a linker.

[0095] In some embodiments, the isolated polynucleotide encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:3, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:3.

[0096] In some embodiments, the isolated polynucleotide encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:4, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:4.

[0097] In some embodiments, the isolated polynucleotide encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:7, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:7.

[0098] In some embodiments, the isolated polynucleotide encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:12, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:12.

[0099] In some embodiments, the isolated polynucleotide encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:13, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:13.

[0100] In some embodiments, the isolated polynucleotide encodes a polypeptide comprising an amino acid sequence of SEQ ID NO:6, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:6.

[0101] The disclosure also provides isolated polynucleotides encoding polypeptides comprising two or more repeats of SEQ ID NO: 6. In some embodiments, the polynucleotides encode polypeptides comprising 2, 3, 4, 5, or more than 5 repeats of SEQ ID NO: 6. In some embodiments, the repeats of SEQ ID NO: 6 may be separated by a linker.

[0102] In some embodiments, the polynucleotide comprises: a nucleic acid sequence of SEQ ID NO:16 or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:16; a nucleic acid sequence of SEQ ID NO:17 or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:17; a nucleic acid sequence of SEQ ID NO:18 or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:18; a nucleic acid sequence of SEQ ID NO:19 or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:19; a nucleic acid sequence of SEQ ID NO:20 or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:20; a nucleic acid sequence of SEQ ID NO:21 or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:21; a nucleic acid sequence of SEQ ID NO:22 or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:22; A nucleic acid sequence of SEQ ID NO:26 or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:26; The nucleic acid sequence is selected from the group consisting of SEQ ID NO:27 or a nucleic acid sequence having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:27.

[0103] In some embodiments, the polynucleotide comprises DNA.

[0104] In some embodiments, the polynucleotide comprises RNA.

[0105] In some embodiments, the RNA is mRNA or self-replicating RNA.

[0106] In some embodiments, the polynucleotide comprises a promoter, an enhancer, a polyadenylation site, a Kozak sequence, a stop codon, or any combination thereof.

[0107] Methods for making polynucleotides of the present disclosure are known in the art and include chemical synthesis, enzymatic synthesis (e.g., in vitro transcription), enzymatic or chemical cleavage of longer precursors, chemical synthesis of smaller fragments of polynucleotides followed by ligation of the fragments, or known PCR methods. The polynucleotide sequence to be synthesized can be designed with the appropriate codons for the desired amino acid sequence. In general, preferred codons may be selected for the intended host that the sequence will be used for expression.

[0108] vector The present disclosure also provides a vector comprising any of the polynucleotides disclosed herein.The present disclosure also provides a vector comprising a polynucleotide encoding any of the polypeptides disclosed herein.

[0109] In some embodiments, the vector comprises: the CALR epitope of SEQ ID NO: 1 (MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE) or having at least 90% sequence identity with SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2 (EEAEDNCRRMMRTK) or having at least 90% sequence identity with SEQ ID NO: 2, a JAK2 epitope of SEQ ID NO: 4 (KLSHKHLVLNYGVCFCGDENILVQEFVKFG) or having at least 90% sequence identity with SEQ ID NO: 4, a JAK2 epitope of SEQ ID NO: 5 (VLNYGVCFC) or having at least 90% sequence identity with SEQ ID NO: 5, - comprising a polynucleotide encoding a polypeptide comprising at least two or more epitope sequences selected from the group consisting of SEQ ID NO:6 (FCGDENILV), or a JAK2 epitope having at least 90% sequence identity to SEQ ID NO:6, and combinations thereof.

[0110] The vector may be a vector intended for expressing the polynucleotide of the present disclosure in any host, such as bacteria, yeast, or mammal.Suitable expression vectors are typically replicable in the host organism, either as episomes or as an integral part of the host chromosomal DNA.Usually, expression vectors contain a selection marker, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance, to allow detection of cells transformed or transduced with the desired DNA sequence.Exemplary vectors are plasmids, cosmids, phages, viral vectors, or artificial chromosomes.

[0111] Suitable vectors that can be used are: bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0112] The present disclosure provides an expression vector comprising a polynucleotide of the present disclosure. The present disclosure also provides an expression vector comprising a polynucleotide encoding a polypeptide of the present disclosure.

[0113] The present disclosure also provides viral vectors comprising any of the polynucleotides of the present disclosure.

[0114] The present disclosure also provides viral vectors comprising a polynucleotide encoding any of the polypeptides of the present disclosure.

[0115] Viral vectors are derived from naturally occurring viral genomes and are typically modified to be replication-deficient, e.g., non-replicating. Non-replicating viruses require proteins to be provided in trans for replication. Typically, these proteins are stably or transiently expressed in virus-producing cell lines, thereby allowing the virus to replicate. Thus, viral vectors are typically infectious and non-replicating. Viral vectors include adenovirus vectors, adeno-associated virus (AAV) vectors (e.g., AAV types 5 and 2), great ape adenovirus vectors (GAd), alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), Sindbis virus (SIN), Semliki forest virus (SFV), and VEE-SIN chimeras), herpesvirus vectors (e.g., vectors derived from cytomegaloviruses such as rhesus cytomegalovirus (RhCMV)), arenavirus vectors (e.g., lymphocytic choriomeningitis virus (LCMV) vectors), measles virus vectors, poxvirus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara, modified vaccinia virus (MAV), and avian encephalitis virus (AVV) vectors), and the like. vaccinia virus Ankara (MVA), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), vesicular stomatitis virus vectors, retroviral vectors, lentiviral vectors, virus-like particles, and bacterial spores.

[0116] In some embodiments, the viral vector is derived from an adenovirus, a poxvirus, an alphavirus, an adeno-associated virus, a retrovirus, or a self-replicating RNA molecule.

[0117] Adenovirus Vectors In some embodiments, the viral vector is derived from an adenovirus.

[0118] Adenoviral vectors may be derived from human adenoviruses (Ad), but also from bovine adenoviruses (e.g., bovine adenovirus 3, BAdV3), canine adenoviruses (e.g., CAdV2), porcine adenoviruses (e.g., PAdV3 or 5), or adenoviruses that infect other species, such as great apes, such as chimpanzees (Pan), gorillas (Gorilla), orangutans (Pongo), bonobos (Pan paniscus), and mongooses (Pan troglodytes). Typically, naturally occurring great ape adenoviruses are isolated from fecal samples of the respective great apes.

[0119] Human adenoviral vectors may be derived from various adenovirus serotypes, for example, human adenovirus serotypes hAd5, hAd7, hAd11, hAd26, hAd34, hAd35, hAd48, hAd49, or hAd50 (serotypes are also referred to as Ad5, Ad7, Ad11, Ad26, Ad34, Ad35, Ad48, Ad49, or Ad50).

[0120] Great ape adenovirus (GAd) vectors can be derived from various adenovirus serotypes, for example, great ape adenovirus serotypes GAd20, Gad19, GAd21, GAd25, GAd26, GAd27, GAd28, GAd29, GAd30, GAd31, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAdI7, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd55, ChAd63, ChAd73, ChAd82, ChAd83, ChAd146, ChAd147, PanAd1, PanAd2, or PanAd3.

[0121] Adenoviral vectors are known in the art. Most sequences of human and non-human adenoviruses are known, and others can be obtained using routine methods. An exemplary genomic sequence of Ad26 can be found in GenBank Accession No. EF153474 and SEQ ID NO: 1 of WO 2007 / 104792. An exemplary genomic sequence of Ad35 can be found in FIG. 6 of WO 2000 / 70071. Vectors based on Ad26 are described, for example, in WO 2007 / 104792. Vectors based on Ad35 are described, for example, in U.S. Patent No. 7,270,811 and WO 2000 / 70071. Vectors based on ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63, and ChAd82 are described in WO 2005 / 071093. Vectors based on PanAd1, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAd146, and ChAd147 are described in WO 2010 / 086189.

[0122] The adenoviral vector is engineered to have at least one gene product essential for viral replication, such as one or more of the adenoviral regions E1, E2, and E4, functionally deleted or completely removed, thereby rendering the adenovirus unable to replicate. The deletion of the El region may include deletion of EIA, EIB 55K, or EIB 21K, or any combination thereof. Replication-deficient adenoviruses are propagated by providing in trans the proteins encoded in the deleted region by the producer cell using a helper plasmid, or by genetically engineering the producer cell to express the required proteins. The adenoviral vector may also have a deletion in the E3 region, which is not required for replication, and therefore such a deletion does not need to be complemented. The adenoviral vector of the present disclosure may have at least a portion of the E1 and E3 regions functionally deleted or completely removed. The adenoviral vector of the present disclosure may further have the E4 and / or E2 regions functionally deleted or completely removed. Suitable producer cells that can be utilized are human retinal cells immortalized with El, such as 911 or PER.C6 cells (see, e.g., U.S. Pat. No. 5,994,128), El-transformed amniotic cells (see, e.g., European Patent No. 1230354), E1-transformed A549 cells (see, e.g., WO 1998 / 39411, U.S. Pat. No. 5,891,690). An exemplary vector that can be used is Ad26, which contains a functional E1 coding region sufficient for viral replication, has a deleted E3 coding region, and has a deleted E4 coding region, so long as the E4 open reading frame 6 / 7 is not deleted (see, e.g., U.S. Pat. No. 9,750,801).

[0123] In some embodiments, the adenovirus vector is a human adenovirus (Ad) vector. In some embodiments, the Ad vector is derived from Ad5. In some embodiments, the Ad vector is derived from Ad11. In some embodiments, the Ad vector is derived from Ad7. In some embodiments, the Ad vector is derived from Ad26. In some embodiments, the Ad vector is derived from Ad34. In some embodiments, the Ad vector is derived from Ad35. In some embodiments, the Ad vector is derived from Ad48. In some embodiments, the Ad vector is derived from Ad49. In some embodiments, the Ad vector is derived from Ad50.

[0124] In some embodiments, the adenovirus vector is a great ape adenovirus (GAd) vector. In some embodiments, the GAd vector is derived from GAd20. In some embodiments, the GAd vector is derived from GAd19. In some embodiments, the GAd vector is derived from GAd21. In some embodiments, the GAd vector is derived from GAd25. In some embodiments, the GAd vector is derived from GAd26. In some embodiments, the GAd vector is derived from GAd27. In some embodiments, the GAd vector is derived from GAd28. In some embodiments, the GAd vector is derived from GAd29. In some embodiments, the GAd vector is derived from GAd30. In some embodiments, the GAd vector is derived from GAd31. In some embodiments, the GAd vector is derived from ChAd3. In some embodiments, the GAd vector is derived from ChAd4. In some embodiments, the GAd vector is derived from ChAd5. In some embodiments, the GAd vector is derived from ChAd6. In some embodiments, the GAd vector is derived from ChAd7. In some embodiments, the GAd vector is derived from ChAd8. In some embodiments, the GAd vector is derived from ChAd9. In some embodiments, the GAd vector is derived from ChAd9. In some embodiments, the GAd vector is derived from ChAd10. In some embodiments, the GAd vector is derived from ChAd11. In some embodiments, the GAd vector is derived from ChAd16. In some embodiments, the GAd vector is derived from ChAd17. In some embodiments, the GAd vector is derived from ChAd19. In some embodiments, the GAd vector is derived from ChAd20. In some embodiments, the GAd vector is derived from ChAd22. In some embodiments, the GAd vector is derived from ChAd24. In some embodiments, the GAd vector is derived from ChAd26. In some embodiments, the GAd vector is derived from ChAd30. In some embodiments, the GAd vector is derived from ChAd31.In some embodiments, the GAd vector is derived from ChAd32. In some embodiments, the GAd vector is derived from ChAd31. In some embodiments, the GAd vector is derived from ChAd33. In some embodiments, the GAd vector is derived from ChAd37. In some embodiments, the GAd vector is derived from ChAd38. In some embodiments, the GAd vector is derived from ChAd44. In some embodiments, the GAd vector is derived from ChAd55. In some embodiments, the GAd vector is derived from ChAd63. In some embodiments, the GAd vector is derived from ChAd68. In some embodiments, the GAd vector is derived from ChAd73. In some embodiments, the GAd vector is derived from ChAd82. In some embodiments, the GAd vector is derived from ChAd83. GAd19-21 and GAd25-31 are described in WO 2019 / 008111 and represent strains that are highly immunogenic in the general human population and have no pre-existing immunity. The polynucleotide sequence of the GAd20 genome is disclosed in WO 2019 / 008111.

[0125] The polynucleotide of the present disclosure can be inserted into a site or region (insertion region) within the vector that does not affect the viral viability of the resulting recombinant virus. The polynucleotide of the present disclosure can be inserted in a parallel (transcribed 5' to 3') or antiparallel (transcribed 3' to 5' relative to the vector backbone) orientation to the deleted E1 region. In addition, suitable transcriptional regulatory elements capable of directing expression of a polypeptide or a polypeptide of the present disclosure in a mammalian host cell in which the vector is prepared for use can be operably linked to the polypeptide or a polypeptide of the present disclosure.

[0126] Recombinant adenoviral particles can be prepared and propagated according to any conventional technique in the art (e.g., WO 1996 / 17070) using a complementing cell line or helper virus that provides in trans the defective viral genes necessary for viral replication. The cell lines 293, PER.C6, E1 A549 and 911 are commonly used to complement the El deletion. Other cell lines have been genetically engineered to complement defective vectors (Yeh, et al., 1996, J. Virol. 70:559-565; Kroughak and Graham, 1995, Human Gene Ther. 6:1575-1586; Wang, et al., 1995, Gene Ther. 2:775-783; Lusky, et al., 1998, J. Virol. 72:2022-203; European Patent No. 919627, and International Publication No. WO 1997 / 04119). Adenoviral particles can be recovered from the culture supernatant as well as from the cells after lysis and, optionally, further purified according to standard techniques (e.g., chromatography, ultracentrifugation as described in WO 1996 / 27677, WO 1998 / 00524, WO 1998 / 26048 and WO 2000 / 50573). Methods for constructing and propagating adenovirus vectors are described, for example, in U.S. Patent Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191 and 6,113,913.

[0127] Provided herein are viral vectors comprising any of the polynucleotides of the present disclosure, where the vectors are derived from hAd26 (also referred to as Ad26-bearing).

[0128] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:16, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:16.

[0129] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:17, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:17.

[0130] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:18, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:18.

[0131] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:19, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:19.

[0132] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:20, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:20.

[0133] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:21, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:21.

[0134] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:22, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:22.

[0135] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:23, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:23.

[0136] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:24, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:24.

[0137] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:25, or having at least 90% sequence identity to SEQ ID NO:25.

[0138] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:26, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:26.

[0139] In some embodiments, the Ad26 vector comprises a polynucleotide of SEQ ID NO:27, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:27.

[0140] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:1, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:1.

[0141] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:2, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:2.

[0142] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:3, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:3.

[0143] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:4, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:4.

[0144] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:5, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:5.

[0145] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:6, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:6.

[0146] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:7, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:7.

[0147] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:9, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:9.

[0148] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:10, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:10.

[0149] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:11, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:11.

[0150] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:12, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:12.

[0151] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:13, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:13.

[0152] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:14, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:14.

[0153] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:15, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:15.

[0154] In some embodiments, the Ad26 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:31, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:31.

[0155] Provided herein is a viral vector comprising any of the polynucleotides of the present disclosure, wherein the vector is derived from GAd20.

[0156] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:16, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:16.

[0157] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:17, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:17.

[0158] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:18, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:18.

[0159] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:19, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:19.

[0160] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:20, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:20.

[0161] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:21, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:21.

[0162] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:22, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:22.

[0163] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:23, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:23.

[0164] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:24, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:24.

[0165] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:25, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:25.

[0166] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:26, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:26.

[0167] In some embodiments, the GAd20 vector comprises a polynucleotide of SEQ ID NO:27, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:27.

[0168] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:1, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:1.

[0169] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:2, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:2.

[0170] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:3, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:3.

[0171] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:4, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:4.

[0172] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:5, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:5.

[0173] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:6, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:6.

[0174] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:7, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:7.

[0175] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:9, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:9.

[0176] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:10, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:10.

[0177] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:11, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:11.

[0178] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:12, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:12.

[0179] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:13, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:13.

[0180] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:14, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:14.

[0181] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:15, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:15.

[0182] In some embodiments, the GAd20 vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:31, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:31.

[0183] Poxvirus Vectors Poxvirus (Poxviridae) vectors may be derived from Variola virus (variola), Vaccinia virus, Cowpox virus, or Monkeypox virus. Exemplary vaccinia viruses are Copenhagen Vaccinia Virus (W), New York Attenuated Vaccinia Virus (NYVAC), ALVAC, TROVAC, or Modified Vaccinia Ankara (MVA).

[0184] MVA originates from the cutaneous vaccinia strain Ankara (Ankara chorioallantoic vaccinia (CVA) virus), which has been maintained for many years at the Vaccination Institute (Ankara, Turkey) and used as the basis for human vaccination. However, the high incidence of severe post-vaccination complications associated with vaccinia virus (VACV) has led to several attempts to create a more attenuated and safer smallpox vaccine.

[0185] MVA has been produced by serial passage of CVA virus 516 times in chicken embryo fibroblast cells (see Meyer et al., J. Gen. Virol., 72:1031-1038 (1991) and U.S. Patent No. 10,035,832). As a result of these long-term passages, the resulting MVA virus was deleted of approximately 31 kilobases of its genomic sequence and was therefore described as highly avian-cell-restricted host cell (Meyer, H. et al., Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence, J. Gen. Virol. 72, 1031-1038, 1991; Meisinger-Henschel et al., Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara, J. Gen. Virol. 88:3249-3259, 2007). Comparison of the MVA genome with its parent CVA revealed six major deletions (deletions I, II, III, IV, V, and VI) totaling 31,000 base pairs of genomic DNA. (Meyer et al., J. Gen. Virol. 72:1031-8 (1991)). The resulting MVA was shown in various animal models to be significantly attenuated (Mayr, A. & Danner, K. Vaccination against pox diseases under immunosuppressive conditions, Dev. Biol. Stand. 41:225-34, 1978). Many passages were used to attenuate MVA, resulting in a large number of different strains or isolates depending on the number of passages in CEF cells, such as MVA 476 MG / 14 / 78, MVA-571, MVA-572, MVA-574, MVA-575, and MVA-BN. MVA 476 MG / 14 / 78 is described, for example, in WO 2019 / 115816 (A1).The MVA-572 strain was deposited at the European Collection of Animal Cell Cultures ("ECACC"), Health Protection Agency, Microbiology Services, Porton Down, Salisbury SP4 0JG, United Kingdom ("UK"), on January 27, 1994 under accession number ECACC 94012707. The MVA-575 strain was deposited at the ECACC on December 7, 2000 under accession number ECACC 00120707. The MVA-Bavarian Nordic ("MVA-BN") strain was deposited at the ECACC on August 30, 2000 under accession number V00080038. The genomic sequences of MVA-BN and MVA-572 are available in GenBank (accession numbers DQ983238 and DQ983237, respectively). The genomic sequences of other MVA strains can be obtained using standard sequencing methods.

[0186] The vectors and viruses of the present disclosure can be derived from any MVA strain or further derivatives of MVA strains. A further exemplary MVA strain is deposit VR-1508 deposited with the American Type Culture collection (ATCC) (Manassas, Va. 20108, USA).

[0187] A derivative of MVA refers to a virus that exhibits essentially the same characteristics as the parent MVA, but exhibits differences in one or more parts of its genome.

[0188] In some embodiments, the MVA vector is derived from MVA476MG / 14 / 78. In some embodiments, the MVA vector is derived from MVA-571. In some embodiments, the MVA vector is derived from MVA-572. In some embodiments, the MVA vector is derived from MVA-574. In some embodiments, the MVA vector is derived from MVA-575. In some embodiments, the MVA vector is derived from MVA-BN.

[0189] The polynucleotides of the present disclosure can be inserted into sites or regions (insertion regions) within the MVA vector that do not affect the viral viability of the resulting recombinant virus. Such regions can be easily identified by testing segments of viral DNA for regions that allow recombinant formation without severely affecting the viral viability of the recombinant virus. The thymidine kinase (TK) gene is an insertion region that can be used and is present in many viruses, for example in all examined poxvirus genomes. In addition, MVA contains six naturally occurring deletion sites, each of which can be used as an insertion site (e.g., deletions I, II, III, IV, V, and VI; see, e.g., U.S. Pat. Nos. 5,185,146 and 6,440,442). One or more intergenic regions (IGRs) of MVA may be used as insertion sites, such as IGRs IGR07 / 08, IGR 44 / 45, IGR 64 / 65, IGR 88 / 89, IGR 136 / 137, and IGR 148 / 149 (see, for example, U.S. Patent Application Publication No. 2018 / 0064803). Further suitable insertion sites are described in WO 2005 / 048957.

[0190] Recombinant poxvirus particles such as rMVA are prepared as described in the art (see Piccini, et al., 1987, Methods of Enzymology 153:545-563; U.S. Pat. Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587, and 5,179,993). In an exemplary method, the DNA sequence to be inserted into the virus can be placed into an E. coli plasmid construct into which DNA homologous to a portion of the DNA of MVA has been inserted. Separately, the DNA sequence to be inserted can be ligated to a promoter. The promoter gene linkage can be located in the plasmid construct such that it is flanked on both ends by DNA homologous to DNA sequences flanking the MVA DNA region containing the non-essential gene locus. The resulting plasmid construct can be amplified and isolated by growth in E. coli bacteria. The isolated plasmid containing the DNA gene sequence to be inserted may be transfected into a cell culture, for example, of chicken embryo fibroblasts (CEF), and the culture is simultaneously infected with MVA. Recombination between homologous MVA DNA in the plasmid and viral genome, respectively, can produce MVA modified by the presence of the foreign DNA sequence. rMVA particles can be recovered from the culture supernatant or cultured cells after a lysis step (e.g., chemical lysis, freeze / thaw, osmotic shock, sonication, etc.). Successive purification rounds of plaques can be used to remove contaminating wild-type virus. The viral particles can then be purified using techniques known in the art (e.g., chromatographic methods or ultracentrifugation in cesium chloride or sucrose gradients).

[0191] Provided herein are viral vectors comprising any of the polynucleotides of the present disclosure, the vectors being derived from MVA.

[0192] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:16, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:16.

[0193] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:17, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:17.

[0194] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:18, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:18.

[0195] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:19, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:19.

[0196] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:20, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:20.

[0197] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:21, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:21.

[0198] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:22, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:22.

[0199] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:23, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:23.

[0200] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:24, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:24.

[0201] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:25, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:25.

[0202] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:26, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:26.

[0203] In some embodiments, the MVA vector comprises a polynucleotide of SEQ ID NO:27, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:27.

[0204] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:1, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:1.

[0205] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:2, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:2.

[0206] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:3, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:3.

[0207] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:4, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:4.

[0208] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:5, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:5.

[0209] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:6, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:6.

[0210] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:7, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:7.

[0211] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:9, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:9.

[0212] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:10, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:10.

[0213] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:11, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:11.

[0214] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:12, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:12.

[0215] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:13, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:13.

[0216] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:14, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:14.

[0217] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:15, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:15.

[0218] In some embodiments, the MVA vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:31, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:31.

[0219] self-replicating RNA molecule The self-replicating RNA may be derived from an alphavirus. The alphavirus may belong to the VEEV / EEEV group, or the SF group, or the SIN group. Non-limiting examples of SF group alphaviruses include Semliki Forest virus, O'nyong-nyong virus, Ross River virus, Middleburg virus, Chikungunya virus, Barmah Forest virus, Getah virus, Mayaro virus, Sagiyama virus, Bebaru virus, and Una virus. Non-limiting examples of SIN group alphaviruses include Sindbis virus, Girdwood SA virus, South African arbovirus No. 86, Okerubo virus, Aura virus, Babanki virus, Wataroa virus, and Kijiraga virus. Non-limiting examples of VEEV / EEEV group alpha viruses include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MIDV), and the like. virus (MAYV), and Una virus (UNAV).

[0220] The self-replicating RNA molecules may be derived from an alphavirus genome, meaning that they have or are similar to some of the structural features of the alphavirus genome. The self-replicating RNA molecules may be derived from a modified alphavirus genome.

[0221] Self-replicating RNA molecules include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki Forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), Onyong-nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYV), and Sarcovirus (SAGV). The alphavirus may be derived from KYZV, WEEV, Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Boggy Creek virus. Both pathogenic and non-pathogenic alphavirus strains are suitable. In some embodiments, the alphavirus RNA replicon is from Sindbis virus (SIN), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), or Eastern equine encephalitis virus (EEEV).

[0222] In some embodiments, the self-replicating RNA molecule derived from an alphavirus is Venezuelan Equine Encephalitis Virus (VEEV).

[0223] A self-replicating RNA molecule can contain an RNA sequence from a wild-type New World or Old World alphavirus genome (or an amino acid sequence encoded thereby). Any of the self-replicating RNA molecules disclosed herein can include an RNA sequence "derived from" or "based on" a wild-type alphavirus genome sequence, meaning that it has a sequence that is at least 60%, or at least 65%, or at least 68%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or 100%, or 80-99%, or 90-100%, or 95-99%, or 95-100%, or 97-99%, or 98-99% identical to an RNA sequence from a wild-type RNA alphavirus genome (which may be a corresponding RNA sequence), which may be a New World or Old World alphavirus genome.

[0224] Self-replicating RNA molecules contain all the genetic information necessary to direct their own amplification or self-replication in permissive cells. To direct their own replication, they encode polymerases, replicases, or other proteins that can interact with proteins, nucleic acids, or ribonucleoproteins derived from viruses or host cells to catalyze the RNA amplification process, and contain the cis-acting RNA sequences necessary for the replication and transcription of the replicon-encoding RNA. Thus, RNA replication leads to the generation of multiple daughter RNAs. These daughter RNAs, and collinear subgenomic transcripts, can be translated to provide in situ expression of the gene of interest, or can be transcribed to provide further transcripts of the same sense as the delivered RNA, which are translated to provide in situ expression of the gene of interest. The overall result of this transcription sequence is that the number of introduced replicon RNAs is greatly amplified, so that the encoded gene of interest becomes the major polypeptide product of the cell.

[0225] There are two open reading frames (ORFs) in the genome of alphaviruses, nonstructural (ns) and structural genes. The nsORFs encode proteins (nsP1-nsP4) required for viral RNA transcription and replication, produced as a polyprotein, and are the viral replication machinery. The structural ORFs encode three structural proteins, the core nucleocapsid protein C, and the envelope proteins P62 and El, which assemble as a heterodimer. The viral membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells by membrane fusion. The four ns protein genes are encoded by genes in the 5'2 / 3 of the genome, and the three structural proteins are translated from a collinear subgenomic mRNA in the 3'1 / 3 of the genome. An exemplary depiction of an alphavirus genome is shown in Figure 18A.

[0226] Self-replicating RNA molecules can be used as a basis for the introduction of foreign sequences into host cells by replacing viral sequences encoding structural genes or by inserting foreign sequences 5' or 3' of sequences encoding structural genes. They can be engineered to replace viral structural genes (GOIs) downstream of a replicase under the control of a subgenomic promoter, e.g., a polynucleotide encoding a polypeptide of the present disclosure. Upon transfection, the immediately translated replicase interacts with the 5' and 3' ends of the genomic RNA and synthesizes complementary genomic RNA copies. These serve as templates for the synthesis of new positive strand capped and polyadenylated genomic copies and subgenomic transcripts (Figure 18B). Amplification ultimately reaches up to 2 x 10 per cell. 5This results in a very high RNA copy number of 100 copies. This results in uniform and / or enhanced expression of the GOI (e.g., the polynucleotide encoding the polypeptide of the present disclosure), which may affect the efficacy of the vaccine or the therapeutic impact of the treatment. Thus, vaccines based on self-replicating RNA molecules can be administered at very low levels compared to traditional viral vectors due to the very high number of copies of RNA produced. One of the important values ​​of the compositions and methods disclosed herein is that they can enhance vaccine efficacy in individuals with chronic or acute conditions of immune activation.

[0227] The self-replicating RNA molecules of the present disclosure, including RNA encoding the CALR / JAK2 or JAK2 epitope 2 polypeptides of the present disclosure, can be utilized as therapeutic agents by delivering them to subjects having myeloproliferative neoplasms using a variety of techniques, including viral vectors described herein or other delivery techniques described herein.

[0228] The present disclosure provides a self-replicating RNA molecule that contains all the genetic information necessary to direct its own amplification or self-replication in a permissive cell.

[0229] The present disclosure also provides self-replicating RNA molecules that can be used as the basis for introducing foreign sequences (e.g., the CALR / JAK2 or JAK2 epitope 2 polypeptides of the present disclosure) into a host cell by replacing viral sequences encoding structural genes.

[0230] Provided herein are viral vectors comprising any of the polynucleotides of the present disclosure, where the vectors are derived from self-replicating RNA molecules.

[0231] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:16, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:16.

[0232] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:17, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:17.

[0233] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:18, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:18.

[0234] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:19, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:19.

[0235] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:20, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:20.

[0236] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:21, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:21.

[0237] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:22, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:22.

[0238] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:23, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:23.

[0239] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:24, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:24.

[0240] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:25, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:25.

[0241] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:26, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:26.

[0242] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence derived from a polynucleotide of SEQ ID NO:27, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:27.

[0243] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence that encodes an amino acid sequence of SEQ ID NO:1, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:1.

[0244] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence that encodes an amino acid sequence of SEQ ID NO:2, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:2.

[0245] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence that encodes an amino acid sequence of SEQ ID NO:3, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:3.

[0246] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence that encodes an amino acid sequence of SEQ ID NO:4, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:4.

[0247] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence that encodes an amino acid sequence of SEQ ID NO:5, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:5.

[0248] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence that encodes an amino acid sequence of SEQ ID NO:6, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:6.

[0249] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence that encodes the amino acid sequence of SEQ ID NO:7, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:7.

[0250] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence that encodes an amino acid sequence of SEQ ID NO:9, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:9.

[0251] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence encoding an amino acid sequence of SEQ ID NO:10, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:10.

[0252] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence encoding the amino acid sequence of SEQ ID NO:11, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:11.

[0253] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence encoding an amino acid sequence of SEQ ID NO:12, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:12.

[0254] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence encoding the amino acid sequence of SEQ ID NO:13, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:13.

[0255] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence encoding the amino acid sequence of SEQ ID NO:14, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:14.

[0256] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence encoding the amino acid sequence of SEQ ID NO:15, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:15.

[0257] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence encoding the amino acid sequence of SEQ ID NO:31, or having at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:31.

[0258] Any of the above self-replicating RNA molecules may be one or more of the nonstructural genes, nsP1, nsP2, nsP3, and nsP4; at least one of a DLP motif, a 5'UTR, a 3'UTR, and a polyA, and may further comprise one or more subgenomic promoters.

[0259] In some embodiments, for example, the self-replicating RNA molecule is one or more of the nonstructural genes, nsP1, nsP2, nsP3, and nsP4; at least one of a DLP motif, a 5'UTR, a 3'UTR, and a polyA, and Subgenomic promoters, and an RNA encoding the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, or 31, operably linked to a subgenomic promoter.

[0260] In some embodiments, the self-replicating RNA molecule comprises an RNA sequence encoding a protein or peptide, 5' and 3' alphavirus untranslated regions, an RNA sequence encoding an amino acid sequence derived from New World alphavirus VEEV nonstructural proteins nsP1, nsP2, nsP3, and nsP4, a subgenomic promoter operably linked to the RNA sequence encoding the protein and regulating its translation, a 5' cap and a 3' polyA tail, a positive-sense single-stranded RNA, a DLP from Sindbis virus upstream of nonstructural protein 1 (nsP1), a 2A ribosomal skipping element, and nsp1 nucleotide repeats downstream of the 5'-UTR and upstream of the DLP.

[0261] In some embodiments, the self-replicating RNA molecule may be at least 1 kb, or at least 2 kb, or at least 3 kb, or at least 4 kb, or at least 5 kb, or at least 6 kb, or at least 7 kb, or at least 8 kb, or at least 10 kb, or at least 12 kb, or at least 15 kb, or at least 17 kb, or at least 19 kb, or at least 20 kb in size, or may be 100 bp to 8 kb, or 500 bp to 8 kb, or 500 bp to 7 kb, or 1 to 7 kb, or 1 to 8 kb, or 2 to 15 kb, or 2 to 20 kb, or 5 to 15 kb, or 5 to 20 kb, or 7 to 15 kb, or 7 to 18 kb, or 7 to 20 kb in size.

[0262] Any of the self-replicating RNA molecules disclosed above can further comprise a coding sequence for an autoprotease peptide (e.g., an autocatalytic self-cleaving peptide), where the coding sequence for the autoprotease is optionally operably linked upstream of a second nucleic acid sequence.

[0263] Generally, any proteolytic cleavage site known in the art can be incorporated into the nucleic acid molecule of the present disclosure, for example, a proteolytic cleavage sequence that is cleaved after production by a protease. Further suitable proteolytic cleavage sites also include proteolytic cleavage sequences that can be cleaved after the addition of an external protease. As used herein, the term "autoprotease" refers to a "self-cleaving" peptide that has autoproteolytic activity and can cleave itself from a larger polypeptide moiety. Several autoproteases, such as the "2A-like" peptide from Equine Rhinitis A Virus (E2A), Porcine Teschovirus-1 (P2A), and Thosea asigna virus (T2A), which were first identified in foot-and-mouth disease virus (FMDV) and are members of the picornavirus group, have since been identified, and their activity in proteolytic cleavage has been shown in various ex vitro and in vivo eukaryotic systems. Thus, the concept of autoprotease is available to those skilled in the art, since many naturally occurring autoprotease systems have been identified. Well-studied autoprotease systems are, for example, viral proteases, developmental proteins (e.g., HetR, hedgehog proteins), RumA autoprotease domain, UmuD, etc. Non-limiting examples of autoprotease peptides suitable for the compositions and methods of the present disclosure include peptide sequences from Porcine Teschovirus-1 2A (P2A), Foot and Mouth Disease Virus (FMDV) 2A (F2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), Thosea asigna Virus 2A (T2A), Cytoplasmic Polyhedrosis Virus 2A (BmCPV2A), Flacheria Disease Virus 2A (BmIFV2A), or combinations thereof.

[0264] In some embodiments, the coding sequence for the autoprotease peptide is operably linked downstream of the DLP motif and upstream of the first and second polynucleotides.

[0265] In some embodiments, the autoprotease peptide comprises or consists of a peptide sequence selected from the group consisting of Porcine Teschovirus-1 2A (P2A), Foot and Mouth Disease Virus (FMDV) 2A (F2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), Cytoplasmic Polyhedrosis Virus 2A (BmCPV2A), Flacheria Disease Virus 2A (BmIFV2A), and combinations thereof. In some embodiments, the autoprotease peptide comprises the peptide sequence of Porcine Teschovirus-1 2A (P2A).

[0266] In some embodiments, the autoprotease peptide is selected from the group consisting of Porcine Teschovirus-1 2A (P2A), Foot and Mouth Disease Virus (FMDV) 2A (F2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), Cytoplasmic Polyhedrosis Virus 2A (BmCPV2A), Flacheria Disease Virus 2A (BmIFV2A), and combinations thereof.

[0267] In some embodiments, the autoprotease peptide is porcine teschovirus-1 2A (P2A).

[0268] Incorporation of the P2A peptide in the modified viral RNA replicon of the present disclosure allows for release of the protein encoded by the GOI (eg, the CALR-JAK2 or JAK2 epitope 2 polypeptide of the present disclosure) from the capsid-GOI fusion.

[0269] In some embodiments disclosed herein, the porcine teschovirus-1 2A (P2A) peptide sequence is engineered in frame immediately following the DLP sequence and in frame immediately upstream of all GOIs.

[0270] Any of the self-replicating RNA molecules disclosed above may further comprise a coding sequence downstream Loop (DLP) motif.

[0271] Some viruses have sequences that can form one or more stem-loop structures that modulate, e.g., increase, capsid gene expression. Viral capsid enhancer as used herein refers to a regulatory element that includes a sequence that can form such a stem-loop structure. In some instances, the stem-loop structure is formed by a sequence within the coding sequence of a capsid protein, and is referred to as a downstream loop (DLP) sequence. As disclosed herein, these stem-loop structures or variants thereof can be used, for example, to modulate, e.g., increase, the expression level of a gene of interest. For example, these stem-loop structures or variants can be used in a recombinant vector (e.g., in a heterologous virus genome) to enhance the transcription and / or translation of a coding sequence operably linked downstream thereof.

[0272] Alphavirus replication in host cells is known to induce double-stranded RNA-dependent protein kinase (PKR). PKR phosphorylates eukaryotic translation initiation factor 2α (eIF2α). Phosphorylation of eIF2α inhibits translation initiation of mRNAs, thereby preventing the virus from completing a productive replication cycle. Infection of cells with Sindbis virus induces PKR, which leads to phosphorylation of eIF2α, but viral subgenomic mRNAs are efficiently translated and translation of all other cellular mRNAs is restricted. Efficient translation of viral subgenomic mRNAs in Sindbis virus is enabled by the presence of a stable RNA hairpin loop (or DLP motif) downstream of the wild-type AUG start codon of the viral capsid protein (e.g., capsid enhancer). It has been reported that the DLP structure can stall the ribosome on the wild-type AUG, which supports translation of subgenomic mRNAs without the need for functional eIF2α. Thus, the subgenomic mRNAs of Sindbis virus (SINV) and other alphaviruses are efficiently translated even in cells with highly active PKR, leading to full phosphorylation of eIF2α.

[0273] DLP structures were first characterized in the Sindbis virus (SINV) 26S mRNA and were also detected in Semliki Forest virus (SFV). Similar DLP structures have been reported to exist in at least 14 other members of the alphavirus genus, including New World (e.g., MAYV, UNAV, EEEV (NA), EEEV (SA), AURAV) and Old World (SV, SFV, BEBV, RRV, SAG, GETV, MIDV, CHIKV, and ONNV) members. The predicted structures of these alphavirus 26S mRNAs were constructed based on SHAPE (selective 2'-hydroxyl acylation and primer extension) data (Toribio et al., Nucleic Acids Res. May 19; 44(9): 4368-80, 2016), the contents of which are incorporated herein by reference). Stable stem-loop structures were detected in all cases except for CHIKV and ONNV, whereas MAYV and EEEV showed less stable DLPs (Toribio et al., 2016, supra). The highest DLP activity was reported for alphaviruses, which contain the most stable DLP structures.

[0274] As an example, members of the alphavirus genus can resist activation of the antiviral RNA-activated protein kinase (PKR) by a downstream loop (DLP) present in the viral 26S transcript, allowing eIF2-independent translation initiation of these mRNAs. The DLP is located downstream of the AUG in the SINV26S mRNA and other members of the alphavirus genus.

[0275] In some embodiments, a nucleic acid molecule of the present disclosure may comprise a DLP motif(s) and / or a coding sequence for a gene of interest (GOI) operably linked to a coding sequence for a DLP motif.

[0276] In some embodiments, the self-replicating RNA molecules of the present disclosure comprise a downstream loop (DLP).

[0277] In some embodiments, the downstream loop (DLP) comprises at least one RNA-stem loop.

[0278] In some cases, DLP activity depends on the distance between the DLP motif and the start codon AUG (AUGi). The AUG-DLP spacing of alphavirus 26S mRNA is adjusted to the topology of the ES6S region of the ribosomal 18SrRNA in a way that positions the AUGi at the P site of the 40S subunit stalled by DLP, allowing the incorporation of Met-tRNA without the involvement of eIF2. In the case of Sindbis virus, the DLP motif is found in the first ~150 nt of the Sindbis subgenomic RNA. The hairpin is located downstream of the Sindbis capsid AUG start codon (AUG at nt 50 of the Sindbis subgenomic RNA), thus pausing the ribosome to begin translation using the correct capsid gene AUG. Previous studies of sequence comparison and structural RNA analysis have revealed evolutionary conservation of the DLP in SINV and predicted the presence of equivalent DLP structures in many members of the alphavirus genus (see, e.g., Ventoso, J. Virol. 9484-9494, Vol. 86, September 2012).

[0279] Without wishing to be bound by any particular theory, it is believed that placement of a DLP motif upstream of the coding sequence of any GOI will generate a fusion protein between the N-terminal capsid amino acids, usually encoded in the hairpin region, and the GOI-encoded protein, because initiation occurs at the capsid AUG and not the GOI AUG.

[0280] In some embodiments, the self-replicating RNA molecule comprises a downstream loop located upstream of nonstructural protein 1 (nsP1).

[0281] In some embodiments, the downstream loop is positioned upstream of nonstructural protein 1 (nsP1) and is bound to nsP1 by the porcine teschovirus-1 2A (P2A) ribosomal skipping element.

[0282] The DLP-containing self-replicating RNA of the present disclosure can be useful for conferring resistance to the innate immune system in a subject. Unmodified RNA replicons are sensitive to the initial innate immune system state of the cells into which they are introduced. When a cell / individual is in a highly active innate immune system state, the performance of the RNA replicon (e.g., replication and expression of GOI) may be adversely affected. By manipulating DLP to control the initiation of protein translation, especially of nonstructural proteins, the effect of the existing activation state of the innate immune system on efficient RNA replicon replication is eliminated or reduced. This results in a more uniform and / or enhanced expression of the GOI, which may affect the efficacy of a vaccine or the therapeutic impact of a treatment.

[0283] The DLP motif of the self-replicating RNA of the present disclosure can confer efficient mRNA translation in cellular environments where cellular mRNA translation is inhibited. When DLP is coupled to the translation of nonstructural protein genes of the replicon vector, replicase and transcriptase proteins can initiate functional replication in PKR-activated cellular environments. When DLP is coupled to the translation of subgenomic mRNA, robust GOI expression is possible even when intracellular mRNA is restricted by innate immune activation. Thus, engineering self-replicating RNAs that contain DLP structures to help drive the translation of both nonstructural protein genes and subgenomic mRNAs provides a powerful method to overcome innate immune activation.

[0284] Examples of self-replicating RNA vectors containing DLP motifs are described in U.S. Patent Application Publication No. 2018 / 0171340 and WO 2018106615, the contents of which are incorporated by reference in their entireties.

[0285] Any of the self-replicating RNA molecules disclosed above may further comprise the non-structural genes nsP1, nsP2, nsP3, and / or nsP4. In some embodiments, the self-replicating RNA molecule does not encode a functional viral structural protein.

[0286] The alphavirus genome encodes the nonstructural proteins nsP1, nsP2, nsP3 and nsP4, which are produced as a single polyprotein precursor, sometimes called P1234 (or nsP1-4 or nsP1234), and are cleaved into mature proteins by proteolytic processing (Figure 18B). nsP1 is approximately 60 kDa in size, may have methyltransferase activity and may be involved in the viral capping reaction. nsP2 is approximately 90 kDa in size, may have helicase and protease activity, and nsP3 is approximately 60 kDa in size and contains three domains: a macrodomain, a central (or alphavirus-specific) domain, and a hypervariable domain (HVD). nsP4 is approximately 70 kDa in size and contains a core RNA-dependent RNA polymerase (RdRp) catalytic domain. After infection, the alphavirus genome RNA is translated to yield the P1234 polyprotein, which is cleaved into individual proteins.

[0287] The alphavirus genome also encodes three structural proteins: the core nucleocapsid protein C, and the envelope proteins P62 and E1, which associate as heterodimers. The structural proteins are under the control of a subgenomic promoter and can be replaced by a gene of interest (GIO).

[0288] In some embodiments of the present disclosure, the self-replicating RNA can lack (or not include) the sequence of at least one (or all) of the structural viral proteins (e.g., nucleocapsid protein C, envelope proteins P62, 6K, and E1). In these embodiments, the sequence encoding one or more structural genes can be replaced with, for example, one or more sequences of a coding sequence for at least one protein or peptide (or other gene of interest (GOI)), such as, for example, a CALR / JAK2 polypeptide or a JAK2 epitope 2 polypeptide of the present disclosure.

[0289] In some embodiments, the self-replicating RNA lacks sequences encoding alphavirus structural proteins or does not encode alphavirus (or, optionally, any other) structural proteins. In some embodiments, the self-replicating RNA molecule further lacks part or all of the coding regions for one or more viral structural proteins. For example, the alphavirus expression system may lack part or all of the coding sequences for one or more of the viral capsid protein C, E1 glycoprotein, E2 glycoprotein, E3 protein, and 6K protein.

[0290] In some embodiments, the self-replicating RNA molecule does not contain a coding sequence for at least one of the structural viral proteins. In these instances, the coding sequence for a structural gene can be replaced with one or more sequences, such as, for example, a coding sequence for a GOI, such as the CALR / JAK2 or JAK2 epitope 2 polynucleotides of FIG. 18 of the present disclosure.

[0291] The present disclosure also provides a self-replicating RNA molecule comprising the non-structural genes nsP1, nsP2, nsP3 and nsP4, wherein the self-replicating RNA molecule does not encode a functional viral structural protein.

[0292] In some embodiments, the present disclosure provides a self-replicating RNA molecule that includes coding sequences for at least one, at least two, at least three, or at least four nonstructural viral proteins (e.g., nsP1, nsP2, nsP3, nsP4). The nsP1, nsP2, nsP3, and nsP4 proteins encoded by the replicon are functional or biologically active proteins.

[0293] In some embodiments, the self-replicating RNA molecule comprises a coding sequence of a portion of at least one nonstructural viral protein. For example, the self-replicating RNA molecule may comprise about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% of the coding sequence of at least one nonstructural viral protein, or a range between any two of these values. In some embodiments, the self-replicating RNA molecule may comprise a coding sequence of a substantial portion of at least one nonstructural viral protein. As used herein, a "substantial portion" of a nucleic acid sequence encoding a nonstructural viral protein includes sufficient nucleic acid sequence encoding a nonstructural viral protein to allow for putative identification of that protein, either by manual evaluation of the sequence by a person skilled in the art, or by computer-based automated sequence comparison and identification using algorithms such as BLAST (see, e.g., "Basic Local Alignment Search Tool"; Altschul SF et al., J. Mol. Biol. 215:403-410, 1993).

[0294] In some embodiments, the self-replicating RNA molecule may comprise the entire coding sequence of at least one nonstructural protein. In some embodiments, the self-replicating RNA molecule comprises substantially all of the coding sequence of a native viral nonstructural protein. In certain embodiments, the one or more nonstructural viral proteins are derived from the same virus.

[0295] In some embodiments, the downstream loop DLP of the self-replicating RNA molecule located upstream of nonstructural protein 1 (nsP1) is derived from Sindbis virus.

[0296] In some embodiments, the self-replicating RNA molecule comprises nsP1, nsP2, nsP3 and nsP4 sequences from Venezuelan Equine Encephalitis Virus (VEEV) and a DLP motif from Sindbis Virus (SIN).

[0297] In some embodiments, the self-replicating RNA molecule also has an RNA subsequence that encodes an amino acid sequence derived from an alphavirus nsP3 macrodomain and an RNA subsequence that encodes an amino acid sequence derived from an alphavirus nsP3 central domain. The self-replicating RNA molecule can also have an RNA subsequence that encodes an amino acid sequence derived entirely from an Old World alphavirus nsP3 hypervariable domain, or it can have an amino acid sequence that has a portion derived from a New World alphavirus nsP3 hypervariable domain and a portion derived from an Old World alphavirus nsP3 hypervariable domain, i.e., the hyper variable domain (HVD) can be a hybrid or chimeric New World / Old World sequence.

[0298] In some embodiments, the self-replicating RNA molecule can have an RNA sequence that encodes an amino acid sequence derived from the wild-type New World alphavirus nsP1, nsP2, nsP3, and nsP4 protein sequences, hi other embodiments, one or more of the nonstructural proteins are derived from a different virus.

[0299] In some embodiments, the self-replicating RNA molecule may have an RNA sequence encoding an nsP3 macrodomain derived from a wild-type alphavirus nsP3 and an nsP3 central domain derived from a wild-type alphavirus nsP3. In various embodiments, the macrodomain and central domain(s) may both be derived from a New World wild-type alphavirus nsP3 or both may be derived from an Old World wild-type alphavirus nsP3 protein. In other embodiments, the macrodomain may be derived from a New World wild-type alphavirus macrodomain and the central domain may be derived from an Old World wild-type alphavirus central domain, or vice versa. The various domains may be of any sequence as described herein.

[0300] In some embodiments, the self-replicating RNA molecule contains the non-VEEV nonstructural proteins nsP1, nsP2, nsP3, and nsP4.

[0301] Accumulating experimental evidence indicates that the replication / amplification of VEEV and other alphavirus genomes and their defective interfering (DI) RNAs is determined by three promoter elements: (i) a conserved 3' terminal sequence element (3'CSE) followed by a poly(A) tail, (ii) the 5'UTR, which functions as a key promoter element for both negative- and positive-strand RNA synthesis, and (iii) a 51-nt conserved sequence element (51-ntCSE) present in the nsP1 coding sequence, which functions as an enhancer of alphavirus genome replication (Kim et al., PNAS, 2014, 111:10708-10713, and references therein).

[0302] Any of the self-replicating RNA molecules disclosed above may further comprise an unmodified 5' untranslated region (5'UTR).

[0303] Previous studies have revealed that only a small portion of viral nonstructural proteins (nsPs) colocalize with dsRNA replication intermediates during VEEV and Sindbis virus infection. Thus, the majority of nsPs are not involved in RNA replication (Gorchakov R, et al. (2008) A new role for ns polyprotein cleavage in Sindbis virus replication. J Virol 82 (13): 6218-6231). This provides an opportunity to utilize the underutilized ns proteins for the amplification of subgenomic RNAs encoding proteins of interest that are usually transcribed from subgenomic promoters and not further amplified.

[0304] In some embodiments, a fragment of nsP1 of a self-replicating RNA molecule of the present disclosure is replicated downstream of the 5'-UTR and upstream of the DLP.

[0305] In some embodiments, the first 193 nucleotides of nsP1 are duplicated downstream of the 5'UTR and upstream of the DLP.

[0306] In some embodiments, the self-replicating RNA molecule comprises a modified 5' untranslated region (5'-UTR). For example, the modified 5'-UTR may comprise one or more nucleotide substitutions at position 1, position 2, position 4, or a combination thereof. Preferably, the modified 5'-UTR comprises a nucleotide substitution at position 2, and more preferably, the modified 5'-UTR has a U->G substitution at position 2. Examples of such self-replicating RNA molecules are described in US Patent Application Publication No. 2018 / 0104359 and WO20181075235, the contents of which are incorporated herein by reference in their entireties.

[0307] In some embodiments, the UTR can be a wild-type New World or Old World alphavirus UTR sequence, or a sequence derived from either. The 5'UTR can be any suitable length, such as about 60 nt, or 50-70 nt, or 40-80 nt. In some embodiments, the 5'UTR can also have a conserved primary or secondary structure (e.g., one or more stem-loop(s)) and can be involved in the replication of the alphavirus or replicon RNA. The 3'UTR can be up to several hundred nucleotides, for example, 50-900, or 100-900, or 50-800, or 100-700, or 200 nt-700 nt. The 3'UTR can also have a secondary structure, such as a step loop, followed by a polyadenylation region or a polyA tail.

[0308] The 5' and 3' untranslated regions may be operably linked to any other sequence encoded by the replicon. The UTRs may be operably linked to a promoter and / or sequences encoding a protein or peptide by providing sequences and spacing necessary for recognition and transcription of other coding sequences.

[0309] The GOI, e.g., the CALR / JAK2 or JAK2 epitope 2 polynucleotide of the present disclosure, can be expressed under the control of a subgenomic promoter. In certain embodiments, instead of the native subgenomic promoter, the subgenomic RNA can be placed under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV), Bovine Viral Diarrhea Virus (BVDV), poliovirus, foot and mouth disease virus (FMD), enterovirus 71, or hepatitis C virus. Subgenomic promoters range from 24 nucleotides (Sindbis virus) to more than 100 nucleotides (Beet Necrotic Yellow Vein Virus) and are usually found upstream of the transcription start.

[0310] The self-replicating RNA molecule can have a 3' poly A tail. It can also contain a poly A polymerase recognition sequence (e.g., AAUAAA) at its 3' end.

[0311] When a self-replicating RNA molecule is packaged into a recombinant alphavirus particle, it may contain one or more sequences, so-called packaging signals, that serve to initiate interactions with alphavirus structural proteins that lead to particle formation, hi some embodiments, the alphavirus particle comprises RNA from one or more alphaviruses and structural proteins, at least one of the structural proteins being derived from two or more alphaviruses.

[0312] In some embodiments, the self-replicating RNA molecule comprises a VEEV-derived vector in which the structural viral proteins (e.g., nucleocapsid protein C, and envelope proteins P62, 6K, and E1) have been removed and replaced by coding sequences for the CARL.JAK2 or JAK2 epitope 2 polypeptides of the present disclosure.

[0313] Other viral vectors and recombinant viruses Viral vectors comprising the polynucleotides of the present disclosure may be derived from human adeno-associated viruses, such as AAV-2 (adeno-associated virus type 2). An attractive feature of AAV vectors is that they do not express any viral genes. The only viral DNA sequence contained in AAV vectors is the 145 bp inverted terminal repeat (ITR). Thus, as in naked DNA immunization, the only gene expressed is that of the antigen or antigen chimera. In addition, AAV vectors are known to transduce both dividing and non-dividing cells, such as human peripheral blood monocyte-derived dendritic cells, for sustained transgene expression, as well as allowing oral and intranasal delivery to generate mucosal immunity. Furthermore, the amount of DNA required is likely to be several orders of magnitude less, at 50 μg or about 10 15 As opposed to a dose of naked DNA of 10 copies 10 ~10 n A dose of 1000 particles or copies of DNA produces the maximum response. AAV vectors are packaged by co-transfecting a suitable cell line (e.g., human 293 cells) with DNA contained in a construct encoding an AAV ITR chimeric protein and the AAV helper plasmid ACG2, which contains the AAV coding region (AAV rep and cap genes) without the ITRs. The cells are then infected with adenovirus Ad5. The vectors can be purified from cell lysates using methods known in the art (e.g., cesium chloride density gradient ultracentrifugation, etc.) and verified to be free of detectable replication-competent AAV or adenovirus (e.g., by cytopathic effect bioassay).

[0314] Viral vectors comprising the polynucleotides of the present disclosure also include retroviral vectors. Retroviruses are a class of integrative viruses that replicate using a virally encoded reverse transcriptase enzyme to replicate the viral RNA genome into double-stranded DNA that is integrated into the chromosomal DNA of infected cells (e.g., target cells). Such vectors include those derived from murine leukemia viruses, particularly Moloney (Gilboa, et al., 1988, Adv. Exp. Med. BiBiol 241:29) or Friend FB29 strains (WO 1995 / 01447). In general, retroviral vectors are deleted of all or part of the viral genes gag, pol, and env, while retaining the 5' and 3' LTRs and encapsidation sequences. These elements can be modified to increase the expression level or stability of the retroviral vector. Such modifications include replacement of the retroviral encapsidation sequence with one of the retrotransposons, such as VL30 (see, eg, US Pat. No. 5,747,323).

[0315] A polynucleotide encoding a polypeptide of the present disclosure may be inserted downstream of the encapsidation sequence, such as in the opposite orientation relative to the retroviral genome. Retroviral particles are prepared in the presence of a helper virus or in a suitable complementing (packaging) cell line in which the retroviral genes (e.g., gag / pol and env) that the retroviral vector lacks are integrated into the genome. Such cell lines have been described in the prior art (Miller and Rosman, 1989, BioTechniques 7:980; Danos and Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:6460; Markowitz, et al., 1988, Virol. 167:400). The product of the env gene is involved in binding of the virus particle to the viral receptor present on the surface of the target cell, thus determining the host range of the retroviral particle. Thus, packaging cell lines such as PA317 cells (ATCC CRL 9078) or 293EI6 (WO 97 / 35996), which contain amphotropic envelope proteins, can be used to allow infection of target cells in humans and other species. Retroviral particles are recovered from the culture supernatant and can optionally be further purified according to standard techniques (e.g., chromatography, ultracentrifugation).

[0316] Regulatory elements The polynucleotide encoding the polypeptide of the present disclosure may be operably linked to one or more regulatory elements in a vector. Regulatory elements may include promoters, enhancers, polyadenylation signals, repressors, etc. As used herein, the term "operably linked" should be interpreted in its broadest reasonable context and refers to the linkage of polynucleotide elements in a functional relationship. A polynucleotide is "operably linked" when it is placed in a functional relationship with another polynucleotide. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0317] Some of the enhancer and promoter sequences commonly used in expression vectors and viral vectors are, for example, human cytomegalovirus (hCMV), vaccinia P7.5 early / late promoter, CAG, SV40, mouse CMV (mCMV), EF-1, and hPGK promoter. Due to its high efficiency and moderate size of about 0.8 kB, the hCMV promoter is one of the most commonly used of these promoters. The hPGK promoter is characterized by its small size (about 0.4 kB), but is less effective than the hCMV promoter. On the other hand, the CAG promoter, which consists of the cytomegalovirus early enhancer element, promoter, first exon and intron of the chicken beta-actin gene, and the splice acceptor of the rabbit beta-globin gene, can direct very strong gene expression comparable to the hCMV promoter, but due to its large size it is less suitable for viral vectors where spatial constraints can be a significant concern, e.g., in adenoviral vectors (AdV), adeno-associated viral vectors (AAV), or lentiviral vectors (LV).

[0318] An additional promoter that can be used is the AoHV-1 promoter, described in WO 2018 / 146205. The promoter can be operably linked to a repressor operating sequence to which a repressor protein can bind to repress expression of the promoter in the presence of the repressor protein. In certain embodiments, the repressor operating sequence is a TetO sequence or a CuO sequence (see, e.g., U.S. Pat. No. 9,790,256).

[0319] In certain cases, it may be desirable to express at least two separate polypeptides from the same vector. In this case, each polynucleotide may be operably linked to the same or different promoter and / or enhancer sequences, or a well-known bicistronic expression system may be used, for example by utilizing an internal ribosome entry site (IRES) from encephalomyocarditis virus. Alternatively, a bidirectional synthetic promoter may be used, such as the hCMV-rhCMV promoter and other promoters described in WO 2017 / 220499. The polyadenylation signal may be derived from SV40 or bovine growth hormone (BGH).

[0320] A self-replicating RNA vector comprising a polynucleotide encoding a polypeptide of the present disclosure may further comprise any regulatory element for establishing the conventional function(s) of the vector, including but not limited to the replication and expression of the polypeptide of the present disclosure encoded by the polynucleotide sequence of the vector. Regulatory elements include but are not limited to promoters, enhancers, polyadenylation signals, translation stop codons, ribosome binding elements, transcription terminators, selection markers, origins of replication, and the like. A vector may comprise one or more expression cassettes. An "expression cassette" is a portion of a vector that directs the cellular machinery to make RNA and proteins. An expression cassette typically comprises three components: a promoter sequence, an open reading frame, and a 3' untranslated region (UTR) that optionally contains a polyadenylation signal. An open reading frame (ORF) is a reading frame that contains the coding sequence of a protein of interest (e.g., a polypeptide of the present disclosure) from a start codon to a stop codon. The regulatory element of an expression cassette may be operably linked to a polynucleotide sequence encoding a polypeptide of interest. Any of the components suitable for use in the expression cassettes described herein can be used in any combination, and in any order, to prepare the vectors of the present application.

[0321] The vector may contain a promoter sequence, preferably in an expression cassette, to control the expression of the polypeptide of the present disclosure. The term "promoter" is used in its conventional sense and refers to a nucleotide sequence that initiates transcription of an operably linked nucleotide sequence. A promoter is located near and on the same strand as the transcribed nucleotide sequence. A promoter may be constitutive, inducible, or repressible. A promoter may be natural or synthetic. A promoter may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). Preferably, a promoter is located upstream of a polynucleotide encoding a polypeptide of the present disclosure in an expression cassette. For example, in a self-replicating RNA, the promoter may be an alphavirus subgenomic promoter.

[0322] In self-replicating RNA, the vector may further comprise additional polynucleotide sequences that stabilize the expressed transcript, improve nuclear transport of the RNA transcript, and / or improve transcription-translation coupling. Examples of such sequences include polyadenylation signals and enhancer sequences. Polyadenylation signals are usually located downstream of the coding sequence of a protein of interest (e.g., a polypeptide of the present disclosure) in the expression cassette of the vector. Enhancer sequences are regulatory DNA sequences that enhance the transcription of an associated gene when bound by a transcription factor. Enhancer sequences are preferably located upstream of the polynucleotide sequence that codes for a polypeptide of the present disclosure, but downstream of the promoter sequence in the expression cassette of the vector.

[0323] Any enhancer sequence known to those of skill in the art in light of the present disclosure can be used.

[0324] Any of the components or sequences of the self-replicating RNA vectors of the present disclosure may be functionally or operably linked to any other of the components or sequences. The components or sequences of the self-replicating RNA molecule may be operably linked for expression of at least one protein or peptide (or biological drug) in a host cell or engineered organism and / or for the ability of the replicon to self-replicate.

[0325] A promoter or UTR operably linked to a coding sequence can affect the transcription and expression of the coding sequence when the appropriate enzymes are present. The promoter need not be adjacent to the coding sequence, so long as it functions to direct its expression. Thus, an operable link between an RNA sequence encoding a protein or peptide and a regulatory sequence (e.g., a promoter or UTR) is an operable linkage that allows expression of a polynucleotide of interest. Operable linkage can also refer to sequences such as RdRp (e.g., nsP4), nsP1-4, UTR, promoter-encoding sequences, and other sequences encoded in an RNA replicon that are linked to allow transcription and translation of a biological drug molecule and / or replication of the replicon. UTRs can be operably linked by providing sequences and spacing necessary for ribosomal recognition and translation of other coding sequences.

[0326] A molecule is functional or biologically active if it performs at least 50% of the same activity as its native (or wild-type) counterpart molecule, although a functional molecule can also perform at least 60%, or at least 70%, or at least 90%, or at least 95%, or 100% of the same activity as its native (or wild-type) counterpart molecule. The self-replicating RNA molecule can also encode an amino acid sequence derived from or based on a wild-type alphavirus amino acid sequence, meaning that it has a sequence that is at least 60%, or at least 65%, or at least 68%, or at least 70%, or at least 80%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or 100%, or 80-99%, or 90-100%, or 95-99%, or 95-100%, or 97-99%, or 98-99% identical to the amino acid sequence encoded by a wild-type RNA alphavirus genome, which may be a New World or Old World alphavirus genome. The sequence derived from the other sequence may be up to 5%, or up to 10%, or up to 20%, or up to 30% longer or shorter than the original sequence. In any of the embodiments, the sequence identity may be at least 95%, or at least 97%, or at least 98%, or at least 99%, or 100% for any nucleotide sequence encoding a G3BP or FXR binding site (or an amino acid sequence having a G3BP or FXR binding site) above. These sequences may also be up to 5%, or up to 10%, or up to 20%, or up to 30% longer or shorter than the original sequence.

[0327] host cell The present disclosure also provides a host cell comprising any of the above-mentioned vectors of the present disclosure. In some embodiments, the host cell is a prokaryotic or eukaryotic host cell. In some embodiments, the host cell is a PER.C6, PER.C6TetO, chicken embryo fibroblast (CEF), CHO, HEK293, HT-1080, HKB-11, CAP, HuH-7, or Age1 cell line.

[0328] composition The present disclosure also provides compositions comprising any of the polynucleotides, any of the polypeptides, and any of the vectors disclosed herein. In some embodiments, the compositions may comprise a vector comprising any of the nucleotides disclosed herein, the vector being selected from Ad26, GAd20, MVA, or a self-replicating RNA molecule.

[0329] Any of the above compositions may comprise a pharmaceutical composition comprising the composition and a pharma- ceutically acceptable excipient, or may be formulated into a pharmaceutical composition. "Pharmaceutically acceptable" refers to an excipient that, at the dosages and concentrations employed, does not cause undesirable or harmful effects in the subject to which it is administered, and includes carriers, buffers, stabilizers, or other materials well known to those skilled in the art. The exact nature of the carrier or other material may depend on the route of administration, e.g., intramuscular, subcutaneous, oral, intravenous, cutaneous, mucosal (e.g., intestinal), intranasal, or intraperitoneal. Liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil may be included. Saline, dextrose or other sugar liquids, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. Exemplary formulations are the Adenovirus World Standard (Hoganson et al, 2002): 20 mM Tris pH 8, 25 mM NaCl, 2.5% glycerol; or 20 mM Tris, 2 mM MgCl2, 25 mM NaCl, sucrose 10% w / v, polysorbate 80 0.02% w / v, or 10-25 mM citrate buffer pH 5.9-6.2, 4-6% (w / w) hydroxypropyl-β-cyclodextrin (HBCD), 70-100 mM NaCl, 0.018-0.035% (w / w) polysorbate-80, and optionally 0.3-0.45% (w / w) ethanol. Many other buffers can be used and examples of suitable formulations for storage and pharmaceutical administration of purified pharmaceutical preparations are known.

[0330] The composition may include one or more adjuvants. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponin or squalene), oil-based adjuvants (Freund's complete and incomplete adjuvants), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g., monophosphoryl lipid A, or muramyl peptide), synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogs, or synthetic lipid A), or synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine). Other non-limiting examples of adjuvants include QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-arretin, MPC-026, Adjuvax, CpG ODN, betafectin, alum, and MF59.

[0331] Other adjuvants that can be used include lectins, growth factors, cytokines and lymphokines such as alpha interferon, gamma interferon, platelet derived growth factor (PDGF), granulocyte-colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), tumor necrosis factor (TNF), epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, or TLR agonists, and particulate adjuvants (immuno-stimulatory complexes (ISCOMs)).

[0332] Nanoparticles In some embodiments, the composition may include nanoparticles. Any of the polynucleotides of the present disclosure may be bound to or contacted with nanoparticles for delivery to a subject. Delivery of any of the polynucleotides or polypeptides of the present disclosure using nanoparticles may eliminate the need to include a virus or adjuvant in the vaccine composition. The polynucleotides of the present disclosure may be DNA or RNA. The nanoparticles may contain immune danger signals that help to effectively induce an immune response to the peptide. The nanoparticles may induce dendritic cell (DC) activation and maturation necessary for a robust immune response. The nanoparticles may contain non-self components that improve uptake of the nanoparticles, and thus the peptides, by cells, such as antigen-presenting cells.

[0333] Nanoparticles are typically about 1 nm to about 100 nm in diameter, for example about 20 nm to about 40 nm. Nanoparticles having an average diameter of 20 to 40 nm may facilitate uptake of the nanoparticles into the cytosol (see, for example, WO 2019 / 135086). Exemplary nanoparticles are polymeric nanoparticles, inorganic nanoparticles, liposomes, lipid nanoparticles (LNPs), immune stimulating complexes (ISCOMs), virus-like particles (VLPs), or self-assembling proteins. The nanoparticles may be calcium phosphate nanoparticles, silicon nanoparticles, or gold nanoparticles. The polymeric nanoparticles may comprise one or more synthetic polymers, such as poly(d,l-lactide-co-glycolide) (PLG), poly(d,l-lactic-co-glycolic acid) (PLGA), poly(g-glutamic acid) (g-PGA)m poly(ethylene glycol) (PEG), or polystyrene, or one or more natural polymers, such as polysaccharides, e.g., pullulan, alginate, inulin, and chitosan. The use of polymeric nanoparticles can be advantageous due to the properties of the polymers that can be included in the nanoparticles. For example, the natural and synthetic polymers listed above can have good biocompatibility and biodegradability, non-toxic properties, and / or the ability to be processed into desired shapes and sizes. Polymeric nanoparticles can also form hydrogel nanoparticles, hydrophilic three-dimensional polymer networks, which have favorable properties including flexible mesh size, large surface area for multivalent conjugation, high water content, and high loading capacity for antigens. Polymers such as poly(L-lactic acid) (PLA), PLGA, PEG, and polysaccharides are suitable for forming hydrogel nanoparticles. Inorganic nanoparticles typically have a rigid structure and include a shell in which the antigen is encapsulated, or a core to which the antigen can be covalently bound. The core may include one or more atoms of gold (Au), silver (Ag), copper (Cu), Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Pd, or Au / Ag / Cu / Pd, or calcium phosphate (CaP).

[0334] In some embodiments, the nanoparticles may be liposomes. Liposomes are typically formed from biodegradable, non-toxic phospholipids and contain a self-assembled phospholipid bilayer shell with an aqueous core. Liposomes may be unilamellar vesicles containing a single phospholipid bilayer or multilamellar vesicles containing several concentric phospholipid shells separated by aqueous layers. As a result, liposomes can be tailored to incorporate hydrophilic molecules into the aqueous core or hydrophobic molecules within the phospholipid bilayer. Liposomes can encapsulate the polynucleotides, or polypeptides, or fragments thereof, of the present disclosure, within the core for delivery. Liposomes and liposomal formulations can be prepared according to standard methods and are well known in the art, see, for example, Remington's; Akimaru, 1995, Cytokines Mol. Ther. 1:197-210; Alving, 1995, Immunol. Rev. 145:5-31; Szoka, 1980, Ann. Rev. Biophys. Bioeng. 9:467; U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. Liposomes may contain targeting molecules to target the liposomal complex to a specific cell type. Targeting molecules may include binding partners (e.g., ligands or receptors) of biomolecules (e.g., receptors or ligands) on the surface of cells found in blood vessels or target tissues. The charge of liposomes is an important determinant in liposome clearance from blood; negatively charged liposomes are taken up more rapidly by the reticuloendothelial system (Juliano, 1975, Biochem. Biophys. Res. Commun. 63:651) and therefore have a shorter half-life in the bloodstream. Incorporation of phosphatidylethanolamine derivatives extends circulation time by preventing liposome aggregation. For example, incorporation of N-(ω-carboxy)acylamidophosphatidylethanolamine into large unilamellar vesicles of L-α-distearoylphosphatidylcholine dramatically increases liposome circulation lifetime in vivo (e.g., Ahl, 1997, Biochim. Biophys. Acta 1329:370-382).Typically, liposomes are prepared with about 5-15 mole percent of a negatively charged phospholipid, such as phosphatidylglycerol, phosphatidylserine, or phosphatidylinositol. The addition of a negatively charged phospholipid, such as phosphatidylglycerol, also functions to prevent spontaneous liposome aggregation, thus minimizing the risk of small-sized liposome aggregate formation. A membrane stiffening agent, such as sphingomyelin or a saturated neutral phospholipid at a concentration of at least about 50 mole percent and monosialyl ganglioside at 5-15 mole percent, may also desirably impart liposomal properties such as rigidity (see, for example, U.S. Pat. No. 4,837,028). In addition, the liposome suspension may include a lipid protective agent that protects lipids from free radical and lipid peroxidative damage during storage. Lipophilic free radical quenchers, such as α-tocopherol, and water-soluble iron-specific chelators, such as ferrioxianine, are preferred.

[0335] The self-replicating RNA molecules and / or compositions comprising same can also be formulated as nanoparticles using combinations of polymers, such as, but not limited to, calcium phosphate, polymers, lipids, and / or other biodegradable agents. The components can be combined in core-shell, hybrid, and / or multi-layer structures to allow fine tuning of the nanoparticles, which can enhance delivery of the molecules and / or compositions of the present disclosure.

[0336] In some embodiments, the nanoparticles may comprise heterogeneously sized multilamellar vesicles. For example, the vesicle-forming lipids can be dissolved in a suitable organic solvent or solvent system and dried under vacuum or inert gas to form a thin lipid film. If necessary, the film can be redissolved in a suitable solvent such as tertiary butanol and then lyophilized to form a more homogenous lipid mixture in a powdered form that hydrates more easily. This film is coated with an aqueous solution of the polypeptide or polynucleotide and typically hydrated for 15-60 minutes with stirring. The size distribution of the resulting multilamellar vesicles can be shifted to smaller sizes by hydrating the lipids under more vigorous stirring conditions or by adding a solubilizing detergent such as deoxycholate. The hydration medium can contain the desired concentration of nucleic acid in the interior volume of the liposomes in the final liposome suspension. Suitable lipids that can be used to form multilamellar vesicles include DOTMA (Feigner, et al., 1987, Proc. Natl. Acad. Sci. USA 84:7413-7417), DOGS or Transfectain™ (Behr, et al., 1989, Proc. Natl. Acad. Sci. USA 86:6982-6986), DNERIE or DORIE (Feigner, et al., Methods 5:67-75), DC-CHOL (Gao and Huang, 1991, BBRC 179:280-285), DOTAP™ (McLachlan, et al., 1995, Gene Therapy 179:280-285), and the like. 2:674-622), Lipofectamine™, and glycerolipid compounds (see, e.g., EP 901463 and WO 098 / 37916).

[0337] In some embodiments, the nanoparticles may be immunostimulating complexes (ISCOMs). ISCOMs are cage-like particles that are typically formed from colloidal saponin-containing micelles. ISCOMs may contain cholesterol, phospholipids (such as phosphatidylethanolamine or phosphatidylcholine), and saponin (such as Quil A from the tree Quillaia saponaria).

[0338] In some embodiments, the nanoparticles may be virus-like particles (VLPs). VLPs are self-assembling nanoparticles without infectious nucleic acid formed by self-assembly of biocompatible capsid proteins. The diameter of VLPs is typically about 20 to about 150 nm, e.g., about 20 to about 40 nm, about 30 to about 140 nm, about 40 to about 130 nm, about 50 to about 120 nm, about 60 to about 110 nm, about 70 to about 100 nm, or about 80 to about 90 nm. VLPs advantageously harness the power of evolved viral structures that are naturally optimized for interaction with the immune system. The naturally optimized nanoparticle size and repetitive structural order means that VLPs induce a strong immune response even in the absence of adjuvants.

[0339] Other molecules suitable for complexing with the polynucleotides of the disclosure include cationic molecules such as polyamidoamines (Haensler and Szoka, 1993, Bioconjugate Chem. 4:372-379), dendritic polylysine (WO 1995 / 24221), polyethylene rinine or polypropylene h-nin (WO 1996 / 02655), polylysine (U.S. Pat. No. 5,595,897), chitosan (U.S. Pat. No. 5,744,166), DNA gelatin coacervates (see, e.g., U.S. Pat. Nos. 6,207,195, 6,025,337, and 5,972,707), or DEAE dextran (Lopata, et al., 1984, Nucleic Acid Res. 12:5707-5717).

[0340] In some embodiments, the self-replicating RNA molecule is coupled to a cationic molecule such as polyamidoamine (Haensler and Szoka, 1993, Bioconjugate Chem. 4:372-379), dendritic polylysine (WO 1995 / 24221), polyethylene linine or polypropylene h-linine (WO 1996 / 02655), polylysine (U.S. Pat. No. 5,595,897), chitosan (U.S. Pat. No. 5,744,166), DNA gelatin coacervates (see, e.g., U.S. Pat. Nos. 6,207,195, 6,025,337, and 5,972,707), or DEAE dextran (Lopata, et al., 1984, Nucleic Acid Res. 12:5707-5717), dendrimers (see, e.g., WO 1996 / 19240), or polyethyleneimine (PEI) (see, e.g., Sun et al., 2014, Mol Med Rep. 10(5):2657-2662).

[0341] Compositions comprising the disclosed self-replicating RNA molecules and / or self-replicating RNA molecules encoding any of the disclosed polypeptides may be encapsulated using one or more liposomes, lipoplexes, and / or lipid nanoparticles. Liposomes are artificially prepared vesicles that may consist primarily of lipid bilayers and may be used as delivery vehicles for administration of polynucleotides and self-replicating RNA molecules. Liposomes may be of different sizes, such as, but not limited to, multilamellar vesicles (MLVs), which may be hundreds of nanometers in diameter, and may contain a series of concentric bilayers separated by narrow aqueous compartments, small unicellular vesicles (SUVs), which may be less than 50 nm in diameter, and large unilamellar vesicles (LUVs), which may be 50 nm to 500 nm in diameter. Liposome designs may include, but are not limited to, opsonins or ligands to improve attachment of liposomes to unhealthy tissues or to activate events such as endocytosis. Liposomes may contain a low or high pH to improve delivery of the polynucleotides and self-replicating RNA molecules disclosed herein.

[0342] The formation of liposomes may depend on, but is not limited to, the pharmaceutical agent and liposomal components to be encapsulated, the nature of the medium in which the lipid vesicles are dispersed, the physicochemical characteristics such as the effective concentration of the encapsulated substance and its potential toxicity, any additional processes involved in the application and / or delivery of the vesicles, the optimized size, polydispersity and shelf life of the vesicles for the intended use, and the possibility of batch-to-batch reproducibility and large-scale production of a safe and efficient liposomal product.

[0343] In some embodiments, the self-replicating RNA molecule is encapsulated, associated with, or adsorbed to a liposome, lipoplex, lipid nanoparticle, or a combination thereof, preferably, the self-replicating RNA molecule is encapsulated in a lipid nanoparticle.

[0344] In some embodiments, the self-replicating RNA molecule encoding any of the polypeptides of the present disclosure can be fully encapsulated within the lipid portion of the particle, thereby protecting the RNA from nuclease degradation. "Fully encapsulated" means that the RNA is not significantly degraded after exposure to serum or nuclease assays that significantly degrade free RNA. When fully encapsulated, preferably less than 25% of the nucleic acid in the particle is degraded, more preferably less than 10%, and most preferably less than 5% of the nucleic acid in the particle is degraded in a process that typically degrades 100% of free nucleic acid. "Fully encapsulated" also means that the nucleic acid-lipid particles are not rapidly degraded into their component parts upon in vivo administration.

[0345] In some embodiments, the self-replicating RNA molecule and / or composition of the present disclosure comprising the same can be formulated in lipid vesicles that may have crosslinks between functionalized lipid bilayers. In some embodiments, the self-replicating RNA molecule and / or composition of the present disclosure can be formulated in lipid-polycation complexes. The formation of lipid-polycation complexes can be achieved by methods known in the art. As non-limiting examples, polycations can include, but are not limited to, cationic peptides or polypeptides, such as polylysine, polyornithine and / or polyarginine, and cationic peptides. In some embodiments, the self-replicating RNA molecule and / or composition disclosed herein can be formulated in lipid-polycation complexes that may further include, but are not limited to, neutral lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE). Lipid nanoparticle formulations can be influenced by biophysical parameters, such as, but not limited to, the choice of cationic lipid component, cationic lipid saturation, nature of PEGylation, ratio of all components, and size.

[0346] In some embodiments, the self-replicating RNA molecules disclosed herein are encapsulated in lipid nanoparticles (LNPs), as shown in FIG. 20. Lipid nanoparticles typically contain four different lipids - ionizable lipids, neutral helper lipids, cholesterol, and diffusible polyethylene glycol (PEG) lipids. LNPs are similar to liposomes, but have slightly different functions and compositions. LNPs are designed to encapsulate polynucleotides such as DNA, mRNA, siRNA, and sRNA. Traditional liposomes contain an aqueous core surrounded by one or more lipid bilayers. LNPs may assume a micelle-like structure that encapsulates the polynucleotide within a non-aqueous core. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs are useful for systemic applications because they exhibit a long circulation lifetime after intravenous (ie) injection and accumulate at distal sites (i.e., sites physically distant from the site of administration). The LNPs may have an average diameter of about 50 nm to about 150 nm, e.g., about 60 nm to about 130 nm, or about 70 nm to about 110 nm, or about 70 nm to about 90 nm, and are substantially non-toxic. Preparation of polynucleotide-loaded LNPs is disclosed, for example, in U.S. Pat. Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, 6,815,432, and WO 96 / 40964. Polynucleotide-containing LNPs are described, for example, in WO 2019 / 191780.

[0347] In some embodiments, the lipid nanoparticles comprise a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid (e.g., one or more PEG-lipid conjugates) that inhibit particle aggregation. The lipid particles may also comprise cholesterol. The lipid particles may encapsulate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more self-replicating RNA molecules that encode one or more polypeptides.

[0348] In some embodiments, LNP formulations comprising polycationic compositions can be used for delivery of the self-replicating RNA molecules described herein in vivo and / or ex vitro. The present disclosure further provides LNP formulations comprising cationic lipids.

[0349] The terms "cationic lipid" and "amino lipid" are used interchangeably to include those lipids and salts thereof that have one, two, three, or more fatty acid or fatty alkyl chains and a pH-tunable amino head group (e.g., an alkylamino or dialkylamino head group). Cationic lipids are typically protonated (i.e., positively charged) at a pH below the pKa of the cationic lipid and are substantially neutral at a pH above the pKa. Cationic lipids may also be referred to as titratable cationic lipids. In some embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH-titrateable) head group, C18 alkyl chains, each alkyl chain independently having 0-3 (e.g., 0, 1, 2, or 3) double bonds, and an ether, ester, or ketal bond between the head group and the alkyl chain. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3), and (DLin-MP-DMA) (also known as 1-B1 1).

[0350] The present disclosure also provides an encapsulated self-replicating RNA molecule, wherein the cationic lipid comprises a protonatable tertiary amine. In some embodiments, the cationic lipid is di((Z)-non-2-en-1-yl) 8,8'-((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate.

[0351] In some embodiments, the cationic lipid compounds are relatively non-cytotoxic. The cationic lipid compounds may be biocompatible and biodegradable. The cationic lipids may have a pKa in the range of about 5.5 to about 7.5, more preferably in the range of about 6.0 to about 7.0.

[0352] The cationic lipid compounds described herein are particularly attractive for drug delivery for several reasons: they incorporate amino groups for interacting with DNA, RNA, other polynucleotides and other negatively charged substances, for pH buffering, for effecting endo-osmolysis, for protection of the self-replicating RNA molecules to be delivered, they can be synthesized from commercially available starting materials, and / or they are pH responsive and can be designed with a desired pKa.

[0353] Lipid nanoparticle formulations can be improved by replacing cationic lipids with biodegradable cationic lipids known as rapidly cleared lipid nanoparticles (reLNPs). Ionizable cationic lipids, such as but not limited to DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity. Rapid metabolism of rapidly cleared lipids can improve the tolerability and therapeutic index of lipid nanoparticles by an order of magnitude from 1 mg / kg to 10 mg / kg doses in rats. The inclusion of enzymatically degraded ester bonds can improve the degradation and metabolic profile of the cationic component while maintaining the activity of reLNP formulations. The ester bonds can be placed internally within the lipid chain or terminally at the end of the lipid chain. The internal ester bonds can replace any carbon in the lipid chain. The lipid particles can include lipid conjugates. Conjugated lipids are useful in that they prevent particle aggregation. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.

[0354] PEG is a linear water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight and include the following: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM), and compounds that contain terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

[0355] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from 550 Daltons to 10,000 Daltons. Examples of PEG-lipids include, but are not limited to, PEG conjugated to dialkyloxypropyl (PEG-DAA), PEG conjugated to diacylglycerol (PEG-DAG), PEG conjugated to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide, PEG conjugated to cholesterol, or derivatives thereof, and mixtures thereof. In some embodiments, the PEG-conjugated lipid is DMG-PEG-2000.

[0356] The self-replicating RNA molecules can also be formulated in particles that include non-cationic lipids. Suitable non-cationic lipids include, for example, neutral uncharged, zwitterionic, or anionic lipids that can form stable complexes. Non-limiting examples of non-cationic lipids include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin, and the like. sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (dioleoylphosphatidylcholine (DPPC)), phosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylethanolamine (palmitoyloleoyl-phosphatidylethanolamine ( leoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (palmitoyloleyol-phosphatidylglycerol (POPG)), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), phosphatidylethanolamine phosphatidylethanolamine phosphatidylethanolamine, phosphatidylethanolamine, phosphatidylethanolamine dipalmitoyl-Phospholipids include dimyristoyl-distearoyl-monomethyl-dimethyl-dielideyl-stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having a carbon chain of C10 to C24, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0357] Additional examples of non-cationic lipids include sterols such as cholesterol and its derivatives.Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5a-cholestan, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof.In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.In some embodiments, the phospholipid is DSPC.In some embodiments, the non-cationic lipid present in the lipid particle comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivatives. In some embodiments in which the lipid particle contains a mixture of phospholipids and cholesterol or a derivative thereof, the mixture may comprise up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid present in the particle.

[0358] In some embodiments, the LNPs may comprise 30-70% cationic lipid compounds, 0-60% cholesterol, 0-30% phospholipids, and 1-10% polyethylene glycol (PEG).

[0359] In some embodiments, the cationic lipid, zwitterionic lipid, cholesterol, and conjugated lipid are combined in a molar ratio of 50:7:40:3, respectively, in the lipid nanoparticle.

[0360] In some embodiments, the LNP formulations described herein can further comprise a permeability enhancer molecule.

[0361] In some embodiments, the nanoparticle formulation may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a self-replicating RNA molecule. By way of non-limiting example, the carbohydrate carrier may include, but is not limited to, anhydrous modified phytoglycogen or glycogen-type materials, phytoglycogen octenyl succinate, phytoglycogen β-dextrin, and anhydrous modified phytoglycogen β-dextrin.

[0362] kit The present disclosure also provides a kit comprising one or more compositions, one or more polynucleotides, one or more polypeptides, or one or more vectors of the present disclosure.The present disclosure also provides a kit comprising one or more recombinant viruses of the present disclosure.The kit can be used to facilitate the implementation of the methods described herein.In some embodiments, the kit further comprises a reagent for facilitating the entry of the vaccine of the present disclosure into cells, such as a lipid-based formulation or a virus packaging material.

[0363] In some embodiments, the kit comprises one or more Ad26 vectors comprising any of the polynucleotides of the present disclosure. In some embodiments, the kit comprises one or more MVA vectors comprising any of the polynucleotides of the present disclosure. In some embodiments, the kit comprises one or more GAd20 vectors comprising any of the polynucleotides of the present disclosure. In some embodiments, the kit comprises one or more self-replicating RNA molecules comprising any of the polynucleotides of the present disclosure.

[0364] In some embodiments, the kit comprises an Ad26 vector of the present disclosure and an MVA vector of the present disclosure. In some embodiments, the kit comprises a GAd20 vector of the present disclosure and an MVA vector of the present disclosure. In some embodiments, the kit comprises an Ad26 vector of the present disclosure and a Gad20 vector of the present disclosure. In some embodiments, the kit comprises a self-replicating RNA molecule of the present disclosure and a Gad20 vector of the present disclosure. In some embodiments, the kit comprises a self-replicating RNA molecule of the present disclosure and an MVA vector of the present disclosure. In some embodiments, the kit comprises a self-replicating RNA molecule of the present disclosure and an Ad26 vector of the present disclosure. In some embodiments, the kit comprises one or more polynucleotides of the present disclosure. In some embodiments, the kit comprises one or more polypeptides of the present disclosure. In some embodiments, the kit comprises one or more cells of the present disclosure.

[0365] Other molecules Peptide-HLA complexes The present disclosure also provides a protein complex comprising a human leukocyte antigen (HLA) and a polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:28, SEQ ID NO:31, or a fragment thereof.

[0366] In some embodiments, the fragment is an immunogenic fragment of any of the polypeptides disclosed herein. In some embodiments, the fragment is about 6-25 amino acids in length. In some embodiments, the fragment is about 6 amino acids in length. In some embodiments, the fragment is about 7 amino acids in length. In some embodiments, the fragment is about 8 amino acids in length. In some embodiments, the fragment is about 9 amino acids in length. In some embodiments, the fragment is about 10 amino acids in length. In some embodiments, the fragment is about 11 amino acids in length. In some embodiments, the fragment is about 12 amino acids in length. In some embodiments, the fragment is about 13 amino acids in length. In some embodiments, the fragment is about 14 amino acids in length. In some embodiments, the fragment is about 15 amino acids in length. In some embodiments, the fragment is about 16 amino acids in length. In some embodiments, the fragment is about 17 amino acids in length. In some embodiments, the fragment is about 18 amino acids in length. In some embodiments, the fragment is about 19 amino acids in length. In some embodiments, the fragment is about 20 amino acids in length. In some embodiments, the fragment is about 21 amino acids in length. In some embodiments, the fragment is about 22 amino acids in length. In some embodiments, the fragment is about 23 amino acids in length. In some embodiments, the fragment is about 24 amino acids in length. In some embodiments, the fragment is about 25 amino acids in length.

[0367] In some embodiments, the HLA is a class I HLA or a class II HLA.

[0368] In some embodiments, the HLA is HLA-A * 02:01, HLA-A * 03:01, HLA-B * 07:02, and HLA-C * 07:02, or any combination thereof.

[0369] In some embodiments, the protein complex is conjugated to a detectable or cytotoxic agent.

[0370] The present disclosure also provides isolated proteinaceous molecules that specifically bind to a polypeptide of the present disclosure, or a complex of an HLA and a polypeptide.

[0371] Proteinaceous molecules In some embodiments, the proteinaceous molecule is an antibody, a selective scaffold, a chimeric antigen receptor (CAR), or a T cell receptor (TCR).

[0372] In some embodiments, the proteinaceous molecule is an antibody.

[0373] In some embodiments, the proteinaceous molecule is an alternative scaffold.

[0374] In some embodiments, the proteinaceous molecule is a chimeric antigen receptor (CAR).

[0375] In some embodiments, the proteinaceous molecule is a T cell receptor (TCR).

[0376] Binding of proteinaceous molecules to the polypeptides or peptide-HLA complexes of the present disclosure can be experimentally determined using any suitable method. Such methods can utilize ProteOn XPR36, Biacore3000, or KinExA instruments, ELISA, or competitive binding assays known to those skilled in the art. The binding measured can be different when measured under different conditions (e.g., osmolality, pH). Thus, affinity and other binding parameters (e.g., K D , K on , K offMeasurements of affinity are typically performed under standard conditions and using standardized buffers, such as those described herein. Those skilled in the art will appreciate that the internal error (measured as standard deviation (SD)) in affinity measurements, e.g., using Biacore 3000 or ProteOn, can typically be in the range of 5-33% when measured within a typical detection range. "Negligible" refers to binding that is less than 100-fold compared to the measured binding of proteinaceous molecules to the polypeptides or HLA / peptides of the present disclosure.

[0377] antibody Antibodies that bind to a polypeptide or HLA / peptide complex can be produced using known methods. For example, monoclonal antibodies can be produced using the hybridoma method of Kohler and Milstein, Nature 256:495, 1975. In the hybridoma method, mice or other host animals, such as hamsters, rats, or monkeys, are immunized with one or more polypeptides or fragments thereof, and then spleen cells from the immunized animals are fused with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Colonies that arise from single immortalized hybridoma cells are screened for the production of antibodies with the desired properties, such as binding specificity and affinity.

[0378] Various host animals can be used to generate antibodies. For example, Balb / c mice, rats, or chickens can be used to generate antibodies containing VH / VL pairs, and llamas and alpacas can be used to generate heavy chain only (VHH) antibodies using standard immunization protocols. Antibodies generated in non-human animals can be humanized using various techniques to generate more human-like sequences.

[0379] Representative humanization techniques involving the selection of a human acceptor framework are known and include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28:489-499), specificity determining residue resurfacing (U.S. Pat. App. Pub. No. 2010 / 0261620), human framework adaptation (U.S. Pat. No. 8,748,356) and superhumanization (U.S. Pat. No. 7,709,226). These methods involve grafting the CDRs of a parent antibody onto a human framework that may be selected based on overall homology to the parent framework, similarity in length of the CDRs or identity of the canonical structure, or any combination thereof.

[0380] Humanized antibodies may be further optimized to improve their selectivity or affinity for the desired antigen by incorporating altered framework support residues that preserve binding affinity (backmutations) or by introducing mutations in any of the CDRs, for example to improve the affinity of the antibody, by techniques such as those described in WO 1090 / 007861 and WO 1992 / 22653.

[0381] Transgenic animals, such as mice or rats, carrying human immunoglobulin (Ig) loci in their genome can be used to generate human antibodies against target proteins, as described, for example, in U.S. Patent No. 6,150,584, WO 99 / 45962, WO 2002 / 066630, WO 2002 / 43478, WO 2002 / 043478, and WO 1990 / 04036. The endogenous immunoglobulin loci of such animals may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the animal's genome using homologous or non-homologous recombination, using a transchromosome, or using a minigene. Companies such as Regeneron (http: / / _www_regeneron_com), Harbour Antibodies (http: / / _www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http: / / _www_omtinc_net), KyMab (http: / / _www_kymab_com), Trianni (http: / / _www.trianni_com), and Ablexis (http: / / _www_ablexis_com) may be working to provide human antibodies targeted to selected antigens using the above technology.

[0382] Human antibodies can be selected from phage display libraries, where phage have been engineered to express human immunoglobulins or portions thereof, such as, for example, Fabs, single chain antibodies (scFvs), or unpaired or paired antibody variable regions. Antibodies of the present disclosure can be isolated, for example, from phage display libraries expressing antibody heavy and light chain variable regions as polypeptides with the bacteriophage pIX coat protein. Libraries are screened for phage binding to the desired antigen, and the resulting positive clones are further characterized, and Fabs can be isolated from lysates of the clones and expressed as full-length IgG. Such phage display methods for isolating human antibodies are described, for example, in U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081.

[0383] Preparation of immunogenic antigens and generation of monoclonal antibodies can be carried out using any suitable technique, such as recombinant protein production or chemical synthesis of peptides. Immunogenic antigens may be administered to an animal in the form of purified proteins or protein mixtures including whole cells or cell or tissue extracts, or antigens may be formed de novo in the animal from nucleic acids encoding the antigen or a portion thereof.

[0384] Selective Scaffolding A variety of scaffolds known in the art and described herein can be used to create selective scaffolds (also referred to as antibody mimetics) that bind to polypeptides or HLA / peptide complexes. The selective scaffolds may be monobodies designed to incorporate the fibronectin type III domain (Fn3) of fibronectin or tenascin as a protein scaffold (US Pat. Nos. 6,673,901 and 6,348,584) or synthetic FN3 domains such as Tencon, as described in US Patent Application Publication Nos. 2010 / 0216708 and 2010 / 0255056. Additional selective scaffolds include Adnectin™, iMab, Anticalin®, EETTI II / AGRP, Kunitz domain, thioredoxin peptide aptamer, Affibody®, DARPin, Affilin, Tetranectin, Fynomer, and Avimer. The selective scaffolds are single chain polypeptide frameworks that contain a highly structured core associated with highly conformationally tolerant variable domains that allow for insertions, deletions, or other substitutions within the variable domains. Libraries that introduce diversity into one or more variable domains, and optionally into the structured core, can be generated using known protocols, the resulting libraries can be screened for binding to the neo-antigens of the present disclosure, and the identified binders can be further characterized for their specificity using known methods.Alternative scaffolds include protein A, particularly its Z domain (affibody), ImmE7 (immunity protein), BPTI / APPI (Kunitz domain), Ras binding protein AF-6 (PDZ-domain), charybdotoxin (scorpion venom), CTLA-4, Min-23 (knottin), lipocalin (anticalin), neokallidinostatin, fibronectin domain, ankyrin consensus repeat domain, or thioredoxin (Skerra, A., "Alternative Non-Antibody Scaffolds for Molecular Recognition," Curr. Opin. Biotechnol. 18:295-304 (2005); Hosse et al., "A New Generation of Protein Display Scaffolds for Molecular Recognition," Protein Sci. 15:14-27 (2006); Nicaise et al., "Affinity Transfer by CDR (see, for example, Nygren and Uhlen, "Grafting on a Nonimmunoglobulin Scaffold," Protein Sci. 13:1882-1891 (2004); Nygren and Uhlen, "Scaffolds for Engineering Novel Binding Sites in Proteins," Curr. Opin. Struc. Biol. 7:463-469 (1997)).

[0385] Chimeric antigen receptors (CARs) CARs can be generated that bind to polypeptides or HLA / peptide complexes. Chimeric antigen receptors (CARs) are genetically engineered receptors. These engineered receptors can be easily inserted into and expressed by immune cells, including T cells, according to techniques known in the art. With CARs, a single receptor can be programmed to recognize a specific antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells that carry that antigen. If these antigens are present on tumor cells, immune cells expressing CARs can target tumor cells and kill them.

[0386] A CAR typically comprises an antigen, an optional linker, a transmembrane domain, and a cytoplasmic domain that contains a costimulatory domain and / or a signaling domain.

[0387] The extracellular domain of the CAR may contain any polypeptide that binds to a desired antigen. The extracellular domain may comprise a portion of an antibody or an alternative scaffold, scFv. The CAR may also be engineered to bind two or more desired antigens, which may be arranged in tandem and separated by a linker sequence. For example, one or more domain antibodies, scFvs, llama VHH antibodies, or other VH-only antibody fragments may be configured in tandem via linkers to provide bispecificity or multispecificity to the CAR.

[0388] The transmembrane domain of the CAR may be the transmembrane domain of CD8, the α, β, or ζ chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDI la, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, ITGAX, CDI lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The IL-16 / IL-16 T cells may be derived from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0389] The intracellular costimulatory domain of the CAR can be derived from the intracellular domain of one or more costimulatory molecules. Costimulatory molecules are well-known cell surface molecules other than antigen receptors or Fc receptors that provide a second signal necessary for efficient activation and function of T lymphocytes upon binding to an antigen. Exemplary costimulatory domains that can be used in the CAR are the intracellular domains of 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0390] The intracellular signaling domain of a CAR can be derived, for example, from the signaling domains of CD3zeta, CD3epsilon, CD22, CD79a, CD66d, or CD39. An "intracellular signaling domain" refers to a portion of a CAR polypeptide that is involved in transducing the message of effective CAR binding to a target antigen inside an immune effector cell to elicit effector cell function, such as activation, cytokine production, proliferation, and cytotoxic activity (including release of cytotoxic factors into a CAR-bound target cell, or other cellular response elicited following antigen binding to the extracellular CAR domain).

[0391] The optional linker of the CAR, located between the extracellular and transmembrane domains, may be a polypeptide of about 2 to 100 amino acids in length. The linker may include or be composed of flexible residues such as glycine and serine, so that adjacent protein domains can move freely relative to each other. Longer linkers can be used if it is desirable to ensure that two adjacent domains do not sterically interfere with each other. The linker may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents and combinations thereof. The linker may also be derived from the hinge region or a portion of the hinge region of any immunoglobulin.

[0392] An exemplary CAR that can be used is, for example, a CAR that contains an extracellular domain that binds to HLA in complex with a fragment of a CALR / JAK2 polypeptide of the present disclosure, a CD8 transmembrane domain, and a CD3 zeta signaling domain. Other exemplary CARs include a CD8 or CD28 transmembrane domain, a CD28, 41BB or OX40 costimulatory domain, and a CD3 zeta signaling domain.

[0393] CARs are produced by standard molecular biology techniques. The extracellular domain that binds to the desired antigen can be derived from an antibody or antigen-binding fragment thereof generated using the techniques described herein.

[0394] T cell receptor (TCR) A TCR can be generated that binds to the HLA / peptide complex of the present disclosure. The TCR can be identified based on T cell binding to the HLA / peptide complex, followed by sequencing of the TCR. The identified TCR can be identified from αβ T cells. The identified TCR can be further engineered to improve its affinity, stability, solubility, etc. For example, the TCR may be cysteine ​​stabilized and may be expressed as a soluble TCR, as a single chain TCR, as a fusion with an N- or C-terminal epitope tag and may also be engineered to improve stability by mutations in the hydrophobic core such as positions 11, 13, 19, 21, 53, 76, 89, 91 or 94 of the alpha chain, by domain swapping with alpha and beta chain variable and / or constant domains as described in U.S. Pat. Nos. 7,329,731, 7,569,664, 9,133,264, 9,624,292, 2016 / 0130319 and 9,884,075.

[0395] Methods of Using Any of the Compositions Herein Methods for treating a subject with the compositions disclosed herein are provided herein. The methods provided herein include administering any of the polynucleotides, polypeptides, vectors, and compositions disclosed herein. The polynucleotides, polypeptides, vectors, compositions, and administration regimens disclosed herein can be used to treat, prevent, or reduce the risk of a clinical condition.

[0396] In some embodiments, the clinical condition is cancer, a myeloproliferative disorder, or a cardiovascular disease.

[0397] In some embodiments, the clinical condition is a cancer selected from lung cancer, lymphatic cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, plasma cell myeloma, biliary tract cancer, bladder cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, gastric cancer, colorectal cancer, colon cancer, urinary tract cancer, central nervous system cancer, neuroblastoma, kidney cancer, breast cancer, cervical cancer, testicular cancer, and soft tissue cancer.

[0398] In some embodiments, the clinical condition is a myeloproliferative disorder selected from primary myelofibrosis (MPN), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PFM), secondary myelofibrosis, acute myeloid leukemia (AML), secondary AML, chronic myelogenous leukemia (CML), clonal hematopoiesis of indeterminate potential (CHIP), and chronic myelomonocytic leukemia (CMML).

[0399] In some embodiments, the cardiovascular disease is selected from acute coronary syndrome, ischemic cerebrovascular disease, ischemic heart disease, thrombosis, venous thromboembolism, deep vein thrombosis, pulmonary embolism, refractory intraperitoneal thrombosis, peripheral arterial disease, hypertension, heart failure, atrial fibrillation, coronary heart disease, atherosclerosis, or clonal hematopoiesis.

[0400] In some embodiments, the subject is Philadelphia chromosome negative. In some embodiments, the subject is treatment naive. In some embodiments, the subject is resistant or refractory to one or more anti-cancer therapies, or is suspected to be resistant or refractory. In some embodiments, the subject is not eligible for stem cell transplantation. In some embodiments, the subject has been treated with stem cell transplantation.

[0401] In some embodiments, a method of treating, preventing, reducing the risk of, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises administering any of the compositions disclosed herein to a subject in need of such a method, wherein the administration comprises one or more administrations of the composition. In some embodiments, the clinical condition is cancer, a myeloproliferative disorder, or a cardiovascular disease.

[0402] In some embodiments, the method for inducing an immune response in a subject harboring a JAK2V617F and / or CALR exon 9 mutation comprises administering any of the compositions disclosed herein to a subject in need of the method, wherein administering comprises one or more administrations of the composition.

[0403] In some embodiments, a method of treating or preventing a myeloproliferative disorder in a subject comprises administering to a subject in need of such a method any of the compositions disclosed herein, where administering comprises one or more administrations of the composition. In some embodiments, the myeloproliferative disorder is selected from primary myelofibrosis (MPN), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PFM), secondary myelofibrosis, acute myelogenous leukemia (AML), secondary AML, chronic myelogenous leukemia (CML), clonal hematopoiesis of undetermined potential (CHIP), and chronic myelomonocytic leukemia (CMML).

[0404] In some embodiments, the method of treating cancer in a subject comprises administering to a subject in need of such a method any of the compositions disclosed herein, wherein the administration comprises one or more doses of the composition. In some embodiments, the cancer is selected from lung cancer, lymphatic cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, plasma cell myeloma, biliary tract cancer, bladder cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, gastric cancer, colorectal cancer, colon cancer, urinary tract cancer, central nervous system cancer, neuroblastoma, kidney cancer, breast cancer, cervical cancer, testicular cancer, and soft tissue cancer.

[0405] In some embodiments, the method of treating cardiovascular disease in a subject comprises administering to a subject in need thereof any of the compositions disclosed herein, wherein administering comprises one or more doses of the composition. In some embodiments, the cardiovascular disease is selected from acute coronary syndrome, ischemic cerebrovascular disease, ischemic heart disease, thrombosis, venous thromboembolism, deep vein thrombosis, pulmonary embolism, refractory intraperitoneal thrombosis, peripheral arterial disease, hypertension, heart failure, atrial fibrillation, coronary heart disease, atherosclerosis, or clonal hematopoiesis.

[0406] In some embodiments, the methods disclosed herein comprise administering to a subject in need thereof a composition comprising a polypeptide comprising at least two or more epitope sequences, or a polynucleotide encoding a polypeptide comprising at least two or more epitope sequences, or a vector comprising a polynucleotide encoding a polypeptide comprising at least two or more epitope sequences, wherein the epitope sequences are the CALR epitope of SEQ ID NO: 1 (MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE) or having at least 90% sequence identity with SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2 (EEAEDNCRRMMRTK) or having at least 90% sequence identity with SEQ ID NO: 2, a JAK2 epitope of SEQ ID NO: 4 (KLSHKHLVLNYGVCFCGDENILVQEFVKFG) or having at least 90% sequence identity with SEQ ID NO: 4, a JAK2 epitope of SEQ ID NO: 5 (VLNYGVCFC) or having at least 90% sequence identity with SEQ ID NO: 5, · selected from the JAK2 epitope of SEQ ID NO: 6 (FCGDENILV) or having at least 90% sequence identity with SEQ ID NO: 6, and combinations thereof.

[0407] In any of the methods disclosed herein, the composition administered to the subject may comprise a vector selected from an adenovirus vector, an alphavirus vector, a poxvirus vector, an adeno-associated virus vector, a retrovirus vector, a self-replicating RNA molecule, and combinations thereof. In some embodiments, the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, a self-replicating RNA molecule, and combinations thereof.

[0408] In some embodiments, the methods disclosed herein include one or more administrations of the compositions provided herein. For example, the methods include a first administration, followed by a second administration with a period between the two administrations.

[0409] In some embodiments, the first administration and the second administration may comprise the same or different compositions. For example, the first administration may comprise a composition comprising a vector selected from Ad26 vector, GAd20 vector, MVA vector, or self-replicating RNA molecule encoding the epitope sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the second administration may comprise a composition comprising a vector selected from Ad26 vector, GAd20 vector, MVA vector, or self-replicating RNA molecule encoding the epitope sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0410] In some embodiments, the first and second administrations are administered once during the subject's life, hi some embodiments, the first and second administrations are administered two or more times during the subject's life.

[0411] In some embodiments, the period between the first administration and the second administration is from about 1 week to about 2 weeks, from about 1 week to about 4 weeks, from about 1 week to about 6 weeks, from about 1 week to about 8 weeks, from about 1 week to about 12 weeks, from about 1 week to about 20 weeks, from about 1 week to about 24 weeks, or from about 1 week to about 52 weeks.

[0412] In some embodiments, the period between the first and second administration is about 2 weeks, about 3 weeks, about 4 weeks, 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, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, or about 52 weeks.

[0413] In some embodiments, the period between the first and second administration is about two weeks.

[0414] In some embodiments, the period between the first and second administration is about 4 weeks.

[0415] In some embodiments, the first administration and the second administration constitute a cycle, and the treatment regimen can include two or more cycles, each cycle being separated by about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.

[0416] The following examples are provided to further illustrate some of the embodiments disclosed herein. These examples are intended to illustrate, but not to limit, the embodiments of the present disclosure. In some embodiments, the first administration and the second administration may include any combination of vectors provided in Table 1, or any combination of epitopes provided in Table 2 below.

[0417] [Table 1]

[0418] [Table 2]

[0419] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding a CALR epitope of SEQ ID NO:1, a CALR epitope of SEQ ID NO:2, and a JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule.

[0420] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding a CALR epitope of SEQ ID NO:1, a CALR epitope of SEQ ID NO:2, a JAK2 epitope of SEQ ID NO:5, and a JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule.

[0421] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is an Ad26 vector; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2, the JAK2 epitope of SEQ ID NO: 5, and the JAK2 epitope of SEQ ID NO: 6, wherein the vector is an Ad26 vector.

[0422] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a GAd20 vector; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2, the JAK2 epitope of SEQ ID NO: 5, and the JAK2 epitope of SEQ ID NO: 6, wherein the vector is a GAd20 vector.

[0423] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is an MVA vector; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2, the JAK2 epitope of SEQ ID NO: 5, and the JAK2 epitope of SEQ ID NO: 6, wherein the vector is an MVA vector.

[0424] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a GAd20 vector; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a self-replicating RNA molecule.

[0425] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding two or more tandem repeats of the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding two or more tandem repeats of the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule.

[0426] Third dose In some embodiments, any of the methods disclosed herein may further include a third administration. For example, the method may include a first administration, a second administration, followed by a third administration, with a period between each administration.

[0427] In some embodiments, the first administration, the second administration, and the third administration may comprise the same or different compositions. For example, the first administration may comprise a composition comprising a vector selected from Ad26 vectors, GAd20 vectors, MVA vectors, or self-replicating RNA molecules encoding the epitope sequences represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the second administration may comprise a composition comprising a vector selected from Ad26 vectors, GAd20 vectors, MVA vectors, or self-replicating RNA molecules encoding the epitope sequences represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the third administration may comprise a composition comprising a vector selected from Ad26 vectors, GAd20 vectors, MVA vectors, or self-replicating RNA molecules encoding the epitope sequences represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0428] In some embodiments, the first administration, the second administration, and the third administration are administered once during the subject's life, hi some embodiments, the first, second, and third administrations are administered two or more times during the subject's life.

[0429] In some embodiments, the period between the second and third administrations is from about 1 week to about 2 weeks, from about 1 week to about 4 weeks, from about 1 week to about 6 weeks, from about 1 week to about 8 weeks, from about 1 week to about 12 weeks, from about 1 week to about 20 weeks, from about 1 week to about 24 weeks, or from about 1 week to about 52 weeks.

[0430] In some embodiments, the period between the second and third administration is about 2 weeks, about 3 weeks, about 4 weeks, 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, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, or about 27 weeks. about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, or about 52 weeks.

[0431] In some embodiments, the period between the second and third administration is about six weeks.

[0432] In some embodiments, the period between the second and third administration is about 8 weeks.

[0433] In some embodiments, the first administration, the second administration, and the third administration together constitute a cycle, and the treatment regimen can include two or more cycles, each cycle being separated by about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.

[0434] The following examples are provided to further illustrate some of the embodiments disclosed herein. The first, second, and third doses used in the methods disclosed herein can include any combination of epitopes and compositions provided in Tables 3 and 4 below.

[0435] [Table 3]

[0436] [Table 4-1]

[0437] [Table 4-2]

[0438] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding a CALR epitope of SEQ ID NO:1, a CALR epitope of SEQ ID NO:2, a JAK2 epitope of SEQ ID NO:5, and a JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a third administration comprising a third composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule.

[0439] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is Ad26; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is Ad26; and and a third administration comprising a third composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2, the JAK2 epitope of SEQ ID NO: 5, and the JAK2 epitope of SEQ ID NO: 6, wherein the vector is MVA.

[0440] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is Ad26; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is MVA; and and a third administration comprising a third composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2, the JAK2 epitope of SEQ ID NO: 5, and the JAK2 epitope of SEQ ID NO: 6, wherein the vector is MVA.

[0441] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is GAd; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is GAd; and and a third administration comprising a third composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2, the JAK2 epitope of SEQ ID NO: 5, and the JAK2 epitope of SEQ ID NO: 6, wherein the vector is MVA.

[0442] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is GAd20; and a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a self-replicating RNA; and and a third administration comprising a third composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO: 1, the CALR epitope of SEQ ID NO: 2, the JAK2 epitope of SEQ ID NO: 5, and the JAK2 epitope of SEQ ID NO: 6, wherein the vector is MVA.

[0443] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding two or more tandem repeats of the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding two or more tandem repeats of the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a third administration comprising a third composition comprising a vector comprising a polynucleotide encoding two or more tandem repeats of the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule.

[0444] Fourth dose In some embodiments, any of the methods disclosed herein may further include a fourth administration. For example, the method may include a first administration, a second administration, a third administration, followed by a fourth administration, with a period between each administration. In some embodiments, the first administration, the second administration, the third administration, and the fourth administration may include the same or different compositions.

[0445] For example, the fourth administration may comprise a composition comprising a vector selected from an Ad26 vector, a GAd20 vector, an MVA vector, or a self-replicating RNA molecule encoding an epitope sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0446] In some embodiments, the first, second, third, and fourth administrations are administered once during the subject's life, hi some embodiments, the first, second, third, and fourth administrations are administered two or more times during the subject's life.

[0447] In some embodiments, the period between the third administration and the fourth administration is from about 1 week to about 2 weeks, from about 1 week to about 4 weeks, from about 1 week to about 6 weeks, from about 1 week to about 8 weeks, from about 1 week to about 12 weeks, from about 1 week to about 20 weeks, from about 1 week to about 24 weeks, or from about 1 week to about 52 weeks.

[0448] In some embodiments, the period between the third and fourth administrations is about 2 weeks, about 3 weeks, about 4 weeks, 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, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, or about 27 weeks. about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, or about 52 weeks.

[0449] In some embodiments, the period between the third and fourth administration is about four weeks.

[0450] In some embodiments, the period between the third and fourth administrations is about 8 weeks.

[0451] In some embodiments, the first administration, the second administration, the third administration, and the fourth administration together constitute a cycle, and the treatment regimen can include two or more cycles, each cycle being separated by about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.

[0452] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding a CALR epitope of SEQ ID NO:1, a CALR epitope of SEQ ID NO:2, a JAK2 epitope of SEQ ID NO:5, and a JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a third administration comprising a third composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule; a fourth administration comprising a fourth composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is selected from an Ad26 vector, an MVA vector, a GAd20 vector, or a self-replicating RNA molecule.

[0453] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a GAd20 vector; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a GAd20 vector; and a third administration comprising a third composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is an MVA vector; and a fourth administration comprising a fourth composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a self-replicating RNA molecule.

[0454] In some embodiments, the method of inducing an immune response or treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject comprises treatment cycles, each cycle comprising: a first administration comprising a first composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a GAd20 vector; a second administration comprising a second composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a GAd20 vector; and a third administration comprising a third composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a self-replicating RNA molecule; and a fourth administration comprising a fourth composition comprising a vector comprising a polynucleotide encoding the CALR epitope of SEQ ID NO:1, the CALR epitope of SEQ ID NO:2, the JAK2 epitope of SEQ ID NO:5, and the JAK2 epitope of SEQ ID NO:6, wherein the vector is a self-replicating RNA molecule.

[0455] Maintenance Dosing In some embodiments, the method further comprises administering the composition to the subject at regular intervals during the treatment cycle, and may continue after the treatment cycle is completed. For example, the composition may be administered to the subject monthly during the treatment regimen, and may continue for an additional six months. In some embodiments, the composition may be administered between two treatment cycles. In some embodiments, the composition may be any of the compositions disclosed herein, such as a composition comprising a vector selected from an Ad26 vector, a GAd20 vector, an MVA vector, or a self-replicating RNA molecule encoding an epitope sequence.

[0456] Dosage and route of administration In some embodiments, the composition comprising the adenoviral vector comprises about 1×10 4 IFU (Infectious Unit) ~ approx. 1 x 10 12 IFU / dose, approximately 1 x 10 4 IFU ~ approx. 1 x 10 11 IFU / dose, approximately 1 x 10 4 IFU ~ approx. 1 x 10 10IFU / dose, approximately 1 x 10 4 IFU ~ approx. 1 x 10 9 IFU / dose, approximately 1 x 10 4 IFU ~ approx. 1 x 10 8 IFU / dose, or approximately 1 x 10 4 IFU ~ approx. 1 x 10 6 It is administered at a dose of IFU / dose.

[0457] In some embodiments, the composition comprising the adenoviral vector comprises about 1×10 6 VP (virus particle) ~ approx. 1 x 10 14 VP / dose, approximately 1×10 6 VP~approx. 1×10 12 VP / dose, approximately 1×10 6 VP~approx. 1×10 10 VP / dose, approximately 1×10 6 VP~approx. 1×10 8 VP / dose, or approximately 1 x 10 6 VP~approx. 1×10 7 Administered at a dose of VP / dose.

[0458] In some embodiments, a composition comprising an Ad26 vector comprises about 1×10 10 In some embodiments, the composition comprising an Ad26 vector is administered at about 1×10 IFU / dose. 11 In some embodiments, the composition comprising an Ad26 vector is administered at about 1×10 IFU / dose. 10 In some embodiments, the composition comprising the Ad26 vector is administered in an amount of about 1×10 11 Administered in VP / dose.

[0459] In some embodiments, a composition comprising a GAd20 vector comprises about 1×10 8 In some embodiments, the composition comprising a GAd20 vector is administered at about 1×10 IFU / dose. 10 In some embodiments, the composition comprising a GAd20 vector is administered at about 1×10 IFU / dose. 10 In some embodiments, the composition comprising the GAd20 vector is administered in an amount of about 1×1011 Administered in VP / dose.

[0460] In some embodiments, the composition comprising a poxvirus vector comprises about 1×10 4 IFU (infectious unit) ~ approx. 1 x 10 12 IFU / dose, approximately 1 x 10 4 IFU ~ approx. 1 x 10 11 IFU / dose, approximately 1 x 10 4 IFU ~ approx. 1 x 10 10 IFU / dose, approximately 1 x 10 4 IFU ~ approx. 1 x 10 9 IFU / dose, approximately 1 x 10 4 IFU ~ approx. 1 x 10 8 IFU / dose, or approximately 1 x 10 4 IFU ~ approx. 1 x 10 6 It is administered at a dose of IFU / dose.

[0461] In some embodiments, the composition comprising the MVA vector comprises about 1×8 10 In some embodiments, the composition comprising the MVA vector is administered at about 1×10 IFU / dose. 10 Administered from IFU / dose.

[0462] In some embodiments, a composition comprising a self-replicating RNA molecule is administered at a dosage of about 1 microgram to about 100 micrograms, about 1 microgram to about 90 micrograms, about 1 microgram to about 80 micrograms, about 1 microgram to about 70 micrograms, about 1 microgram to about 60 micrograms, about 1 microgram to about 50 micrograms, about 1 microgram to about 40 micrograms, about 1 microgram to about 30 micrograms, about 1 microgram to about 20 micrograms, about 1 microgram to about 10 micrograms, or about 1 microgram to about 5 micrograms of the self-replicating RNA molecule.

[0463] In some embodiments, the compositions disclosed herein can be administered to a subject by various routes, such as subcutaneous, topical, oral, and intramuscular. Administration of the composition can be accomplished orally or parenterally. Methods of parenteral delivery include topical, intraarterial (directly into tissue), intramuscular, subcutaneous, intramedullary, intrathecal, intracerebroventricular, intravenous, intraperitoneal, or intranasal administration. The present disclosure also has the objective of providing suitable topical, oral, systemic, and parenteral formulations for use in prophylactic and therapeutic methods.

[0464] In some embodiments, intramuscular administration of the vaccine composition can be accomplished by using a needle. An alternative is to use a needleless injection device (e.g., using Biojector™) to administer the composition, or a lyophilized powder containing the vaccine composition. For intravenous, cutaneous or subcutaneous injection, or injection into an affected area, the composition can be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using isotonic vehicles such as, for example, Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as necessary. Sustained release formulations can also be used.

[0465] Typically, administration will have a prophylactic purpose, generating an immune response against mutant CALR and / or mutant JAK2 before the onset of clinical symptoms. The composition of the present disclosure is administered to a subject to elicit an immune response in the subject. The amount of the composition to induce a detectable immune response is defined as an "immunologically effective dose." The composition of the present disclosure may induce humoral and cellular immune responses. In typical embodiments, the immune response is a protective immune response.

[0466] The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g., dosage, etc., is within the responsibility of general practitioners and other physicians, and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the practitioner.

[0467] In one exemplary regimen, a composition comprising an adenoviral vector is administered in an amount of about 10 4 ~10 12 The adenoviral vector composition is administered (e.g., intramuscularly) in a volume ranging from about 100 μL to about 10 mL, containing a concentration of 10 viral particles / mL. The adenoviral vector composition may be administered in a volume ranging from 0.25 to 1.0 mL, for example, in a volume of 0.5 mL.

[0468] In one exemplary regimen, a composition comprising an MVA vector is administered in an amount of about 1×10 7 TCID 50 ~l×10 9 TCID 50 The vector is administered (e.g., intramuscularly) in a volume of saline ranging from about 100 μL to about 10 mL, containing a dose of 100 mg (50% tissue culture infectious dose) or Inf.U (infectious unit). MVA vectors may be administered in volumes ranging from 0.25 to 1.0 mL.

[0469] Second treatment In some embodiments, the methods disclosed herein further comprise administering a second therapeutic agent, hi some embodiments, the second therapeutic agent is an immunostimulant, a chemotherapeutic agent, an antibiotic, a checkpoint inhibitor, or a cell therapy.

[0470] In some embodiments, the second therapeutic agent is selected from a CTLA-4 antibody, a CTLA4 ligand, a PD-1 axis inhibitor, a PD-L1 axis inhibitor, a TLR agonist, a CD40 agonist, an OX40 agonist, hydroxyurea, ruxolitinib, fedratinib, a 41BB agonist, a CD28 agonist, a STING antagonist, a RIG-1 antagonist, a TCR-T therapy, a CAR-T therapy, an FLT3 ligand, aluminum sulfate, a BTK inhibitor, a JAK inhibitor, a CD38 antibody, a CDK inhibitor, a CD33 antibody, a CD37 antibody, a CD25 antibody, a GM-CSF inhibitor, IL-2, IL-15, IL-7, a CD3 redirection molecule, pomalimib, IFNγ, IFNα, TNFα, a VEGF antibody, a CD70 antibody, a CD27 antibody, a BCMA antibody, or a GPRC5D antibody, and any combination thereof.

[0471] In some embodiments, the second therapeutic agent is a checkpoint inhibitor selected from pilimumab, cetrelimab, pembrolizumab, nivolumab, sintilimab. cemiplimab, toripalimab, camrelizumab, tislelizumab, dostralimab, spartalizumab, prorugolimab, balstilimab, budigalimab, sasanlimab, avelumab, atezolizumab, durvalumab, embafolimab, or yodapolimab, or any combination thereof.

[0472] In some embodiments, the second therapeutic agent is a JAK inhibitor.

[0473] In some embodiments, the second therapeutic agent may be administered in combination with the first composition in a first administration, or the second composition in a second administration, or the third composition in a third administration, or the fourth composition in a fourth administration.

[0474] In some embodiments, the anti-CTLA-4 antibody is combined with either the first, or second, or third, or fourth administration of a composition of the present disclosure.

[0475] In some embodiments, an anti-PD-1 or anti-PD-L1 antibody is combined with any of the first, or second, or third, or fourth administrations of a composition of the disclosure.

[0476] In some embodiments, the checkpoint inhibitor is administered at a dose of about 0.5 to about 5 mg / kg, about 5 to about 10 mg / kg, about 10 to about 15 mg / kg, about 15 to about 20 mg / kg, about 20 to about 25 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 50 to about 75 mg / kg, about 50 to about 100 mg / kg, about 75 to about 100 mg / kg, about 100 to about 125 mg / kg, about 125 to about 150 mg / kg, about 150 to about 175 mg / kg, about 175 to about 200 mg / kg, about 200 to about 225 mg / kg, about 225 to about 250 mg / kg, or about 250 to about 300 mg / kg. EXAMPLES

[0477] Example 1: Identification of JAK2V617F epitope 2 and in-silico immunogenicity evaluation of the JAK2V617F epitope A 17-mer sequence of JAK2 protein with the V617F mutation as the central residue and all 9-mers of consecutive amino acids contained in this 17-mer sequence were generated. In-silico predictions of the binding affinity of these 9-mers to common HLA-A and HLA-B alleles were predicted using netMHCpan4.0. The binding predictions were classified into three categories: weak binding (predicted affinity >500 nM), medium binding (predicted affinity between 50 nM and 500 nM), and strong binding (predicted affinity <50 nM) to commonly occurring HLA-A alleles. The 9-mer JAK2 epitope 1 of SEQ ID NO:5 (VLNYGVCFC) and the novel 9-mer JAK2 epitope 2 of SEQ ID NO:6 (FCGDENILV) were selected from this analysis for further experimental follow-up. The peptide of SEQ ID NO:6 has not been previously described as an immunogenic JAK2 fragment. The predicted binding affinity of JAK2 epitope 2 shown in Table 5 is lower than the binding of JAK2 epitope 1.

[0478] [Table 5]

[0479] Example 2: Mutant JAK2 vs. wild-type JAK2 epitope immunogenicity in disease patient samples PBMCs were isolated from essential thrombocythemia (ET) or primary myelofibrosis (PMF) patients with a confirmed JAK617F mutation. Individual class I-associated mutated JAK2 or wild-type JAK2 peptides were exogenously administered at a concentration of 5 μg / mL to 250,000 PBMCs on day 0. Cells were cultured for 10 days in the presence of 10 IU / mL human IL-2 and 10 ng / mL human IL-15, and then the frequency of mutant or wild-type JAK2 antigen-specific T cells was assessed by peptide pulse recall and intracellular staining flow cytometric analysis of IFNγ- and TNFα-producing CD8+ T cells. Antigen-specific responses were considered positive if the frequency of IFNγ / TNFα+CD8+T cells was 3-fold or higher and 0.1% or higher compared to cells treated with DMSO alone on day 0. The results are summarized in Table 6.

[0480] [Table 6] "Yes" indicates a 3-fold increase in frequency of IFNγ / TNFα+CD8+ T cells compared to DMSO-only stimulator cells for each donor. Brackets indicate actual frequency of IFNγ / TNFα+CD8+ T cells.

[0481] Figure 1 shows the immunogenicity of JAK2 epitope 1 and JAK2 epitope 2 in disease patient samples compared to wild-type sequences of the same epitope regions. The corresponding wild-type peptide sequences of mutJAK2 epitopes I and II were also evaluated and shown to have a similar response as the DMSO control.

[0482] Example 3: Design and generation of CALR.JAK2 polynucleotides and polypeptides for Ad26 and MVA expression To select vaccine compositions that provided good antigen expression and T cell activation, a series of CALR.JAK2 polynucleotides and polypeptides were designed and tested.

[0483] CALR type 1 and type 2 mutations were included in the polynucleotide. Based on in silico T cell epitope prediction and HLA binding experiments, a 54-mer peptide of CALR mutant type 1 (SEQ ID NO: 1) and a truncated 14-mer peptide of CALR mutant type 2 protein (SEQ ID NO: 2) were included in the polynucleotide. The CARL portion of the polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 3. To ensure good intracellular processing of all proteins, the individual peptides were connected by AAY (ala-ala-tyr) linkers that facilitate proteasomal cleavage.

[0484] Also included were two separate JAK2 peptides, a 30-mer peptide (SEQ ID NO:4) or two 9-mer peptides (SEQ ID NO:5 or SEQ ID NO:6), each containing the V617F mutation, based on in silico T cell epitope predictions and HLA binding experiments. The peptide of SEQ ID NO:6 has not been previously described as an immunogenic JAK2 fragment.

[0485] To ensure good intracellular processing of all proteins, the individual peptides were also connected by AAY (ala-ala-tyr) linkers, which facilitate proteasomal cleavage. SEQ ID NO: 7 represents the amino acid sequence of two 9-mers of JAK2 separated by the AAY linker.

[0486] Two different constructs were designed in which the CALR portion was identical, but the JAK2 portion was either a 30-mer peptide, or two 9-mer peptides. These two different transgenes were designed to either contain a leader sequence (LS), HAVT20LS (MACPGFLWALVISTCLEFSMA, SEQ ID NO: 8), or an N-terminal ubiquitin signal (Ubiq) (MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGV、 The constructs were designed without either the HAVT20LS or ubiquitin sequences (SEQ ID NO: 54). The constructs are listed in Table 3. The HAVT20LS should direct the transgene into the ER, while the ubiquitin sequence targets the proteasome. This may result in different levels of expression of TG between the constructs. In addition, constructs were generated that only encoded the sequences of the two mutant CALR proteins and were used as controls for the expression of CALR proteins. The protein and polynucleotide sequences of the transgenes are listed in Table 7. The polynucleotide sequences shown were optimized for adenoviral expression.

[0487] [Table 7]

[0488] [Table 8]

[0489] The CALR.JAK2 construct was cloned into a pUC57 shuttle plasmid using standard methods for adenovirus expression. The expression cassette consisted of a cytomegalovirus (CMV) promoter containing a tetracycline operator (TetO), a Kozak sequence, the CALR.JAK2 polynucleotide, and a Simian Virus 40 (SV40) polyadenylation signal flanked by Ad26-specific sequences for use in combination with the PER.C6TetR cell line. These flanking Ad26-specific sequences were homologous to the Ad26 backbone plasmid sequence to ensure plasmid generation by homologous recombination. The sequence was a human gene optimized for enhanced transgene expression.

[0490] To test CALR.JAK2 expression by these different constructs, HEK293 cells were transfected with the respective DNA plasmids and expression was examined in cell lysates by Western blot 72 hours after transfection. In addition, the proteasome inhibitor MG132 was added for 4 hours before collecting samples to inhibit proteasome activity and examine the effect on transgene expression. All constructs expressed the CALR.JAK2 epitope, with the highest expression observed for the construct carrying HAVT20LS. Proteasome inhibition moderately promoted transgene expression by constructs carrying ubiquitin signals, but this difference may not be quantified by Western blot. Based on the CALR.JAK2 expression data, two constructs with the HATV20 leader sequence (LS) were selected for generation of adenoviruses, namely Ad26.LS_CALR_JAK2-30mer (Ad26HEME001) and Ad26.LS_CALR_JAK2-2x9mer (Ad26HEME002). In addition, a potential control, Ad26-LS-CALR (AD26HEME003), was generated. The polynucleotide sequence of Ad26HEME001, containing the tetracycline operator (TetO)-containing cytomegalovirus (CMV) promoter, Kozak sequence, CALR.JAK2, and Simian Virus 40 (SV40) polyadenylation signal, is shown in SEQ ID NO:23. The polynucleotide sequence of Ad26HEME002, which contains the tetracycline operator (TetO)-containing cytomegalovirus (CMV) promoter, a Kozak sequence, CALR.JAK2, and a Simian Virus 40 (SV40) polyadenylation signal, is shown in SEQ ID NO:24.

[0491] To select cell lines producing Ad26HEME001 and Ad26HEME002, the rescue ability of the virus was measured by transduction of viral genomic DNA in PER.C6® compared to PER.C6TetR cells. Cytopathic effect (CPE) and plaque formation (together indicating rescue ability) were comparable between Ad26HEME001 and Ad26HEME002 constructs (Table 9). In summary, both constructs showed complete CPE within 8 days in PER.C6TetR but not in PER.C6® cells. Ad26HEME001 and Ad26HEME002 showed about 13 and about 11 plaques on PER.C6® cells, respectively, and more than 100 plaques in PER.C6 TetR cells for co-transduction on day 8. This indicated that rescue ability was inhibited in PER.C6® cells. Adenoviral vectors that are difficult to rescue have limited productivity and a high risk of deletions and mutations in the transgene expression cassette. Based on these results, PER.C6 TetR was selected as the cell line for virus production of Ad26HEME001 and Ad26HEME002. Ad26HEME003 was also produced in PER.C6TetR cells.

[0492] [Table 9]

[0493] Example 4: Generation of Ad26HEME001 and Ad26HEME002 Research batches were generated from cells transfected with the single genomic pAd26HEME001 (pAd26.LS_CALR_JAK2-30mer), pAd26HEME002 (pAd26.LS_CALR_JAK2-2x9mer), and pAd26HEME003 (pAd26.LS_CALR) plasmids.

[0494] Using pUC57 plasmid DNA, the Ad26 early region 1 (E1) TG expression cassette was cloned into the pAd26 backbone to generate the pAd26HEME001, pAd26HEME002, and pAd26HEME003 plasmids. The E1 region of the pAd26 backbone was deleted to render the vector incapable of replication in non-complementing cells, such as human cells. In addition, a portion of the Ad26 early region 3 (E3) region was removed (ΔE3) to create sufficient space in the viral genome for the insertion of foreign antigens, and the Ad26 early region 4 (E4) open reading frame (orf) 6 was replaced with the adenovirus type 5 (Ad 5) homolog, allowing the generation of Ad26 vectors incapable of replication in Ad5-E1-complementing cell lines, such as HEK293, PER.C6®, and HER96 cells. The pAd26 backbone was linearized by using a unique restriction site (AsiSI) in the region where the TG cassette was later introduced. Both ends of this linearized plasmid contained overlapping sequences homologous to Ad26-specific sequences present outside the E1 transgene expression cassette, which allowed the generation of a plasmid by cloning the transgene cassette into the pAd26 backbone using homologous recombination techniques. The complete plasmid sequence was verified.

[0495] The plasmids were transfected into PER.C6TetR cells to generate Ad26HEME001, Ad26HEME002, and Ad26HEME003 vectors. Viruses were amplified in PER.C6TetR cells, purified, and tested for the integrity and identity of the adenoviral genome and for correct expression of TG.

[0496] Batch generation and characterization One Ad26HEME001, two Ad26HEME002, and one Ad26HEME003 research batches were generated and characterized for infectivity, transgene expression, batch genetic stability, and relative productivity assessment in suspension PER.C6 (sPER.C6)TetR cells. All research batches were generated on adherent PER.C6TetR cells. Batch quality was characterized by expression of the encoded antigen under non-replicating conditions, number of virus particles (VP), and infectious units (IU).

[0497] All produced research batches showed expression of the encoded antigen as shown by Western blot, and all batches had low VP / IU ratios. The research batch of Ad26HEME001 had a VP / IU ratio of 29. Two research batches of Ad26HEME002 had a VP / IU ratio of 5, and Ad26HEME003 had a VP / IU ratio of 8. Both genetic stability and productivity are important for the possibility of scaling up the production of adenoviral vectors to clinical trial material (CTM) or commercial scale. The risk of genetic instability, defined by the vector genome changing during growth in a production cell line and the emergence of populations with undesirable properties, can be evaluated by growing several virus populations on a small scale. For all research batches, identity polymerase chain reaction (ID PCR) for the transgene region, E3 gene region, and E4 gene region was performed to evaluate genetic stability in the final batch material. In addition, the polymerase chain reaction (PCR) products of the transgene region were sequenced. All study batches were confirmed to be genetically stable.

[0498] Productivity (as defined by titer in VP / mL) was assessed in small-scale experiments by comparing vaccine candidate vectors to their respective benchmark controls with known performance in 10 L process intensification (PIN)-bioreactors. Briefly, sPER.C6TetR cells were transduced with purified research batch material at 70, 300, and 900 VP / cell, as well as two Ad26 benchmark controls. Ad26HEME001, Ad26HEME002, and Ad26HEME003 showed similar productivity to the standard controls at 70, 300, and 900 VP / cell, and all three vectors showed good productivity (the controls performed as expected), reaching ~10 VP / mL over 2-3 days of culture. 11 VP / mL were produced.

[0499] The polynucleotide sequence of the Ad26HEME002 vector containing the various elements is shown in SEQ ID NO:24, and the amino acid sequence encoded thereby is SEQ ID NO:10.

[0500] Example 5: Generation of MVA.CALR.JAK2 and GAd20.CALR.JAK2 constructs The amino acid sequence of the CALR.JAK 2 transgene was converted to a nucleotide sequence based on human codon usage. The terminal motif (TTTTTnT) of the MVA vector was avoided. A BamH1 restriction site and a Kozak sequence were then added upstream of the nucleotide sequence. Two stop codons followed by an Ascl restriction site were added downstream of the nucleotide sequence. A T cell enhancer (TCE) polynucleotide (SEQ ID NO: 29) encoding a 28 aa long small peptide fragment from the Mandarin fish invariant chain was placed at the N-terminus of the transgene. Preclinical data showed that this sequence increases the immune response of the viral vector. Synthesis of the transgene was performed using known methods.

[0501] The MVA transgene, cloned under the control of the vaccinia P7.5 early / late promoter (SEQ ID NO: 32), was inserted into the deletion III locus of MVA (FlankIII-1 and -2 regions) by homologous recombination and recombinant viral particles were generated using known methods.

[0502] The GAd20 transgene was subcloned into a shuttle plasmid between the CMV promoter and BGH polyA with two TetO repeats via EcoRI-Not1 restriction sites. The resulting expression cassette was transferred into the GAd20 genome by homologous recombination in a suitable E. coli strain and transformed with the construct carrying the CMV-transgene-BGH DNA fragment and the GAd20 genome. The recombination included CMV and BGH as homology arms already present in the GAd20 construct, in place of the E1 deletion (the site of insertion of the transgene). The recombinant GAd20 vector was then rescued by transfection of E1-complemented, TetR-expressing M9 cells and amplified by subsequent reinfection of fresh M9 cells.

[0503] MVA and GAd20 vectors expressing the polypeptide TCE_CALR_JAK2-2x9mer (SEQ ID NO: 31) (HCalJ-9.9) were generated using known methods.

[0504] Example 6: The Ad26.CALR.JAK2 vector expresses, processes, and presents antigens to autologous T cells. The purpose of this experiment was to test the ability of human antigen-presenting cells (APCs) to process and present neo-antigens from a vaccine and to mount a T cell response.

[0505] Self CD1c + DC and autologous CD4 + / CD8 + Frozen vials of T cells were isolated by magnetic bead negative selection. +DCs were infected overnight with Ad26HEME002 or Ad5HEME002 (Ad5 vector engineered to express the LS_CALR_JAK2-2x9mer transgene) at a multiplicity of infection (MOI) of 50,000VP. 18–24 h postinfection, DCs were transfected with the same donor autologous T cells and CD1c + DCs were mixed at a ratio of 10:1. Cultures were supplemented with 10 ng / mL human IL-15 on day 0 and half-medium changes were performed with 2×IL-15 and IL-2 (10 IU / mL) every 2–3 days until day 11. On day 11, peptides corresponding to CALRmut, JAK2V617F, or adenohexon protein (positive control) were added overnight to DC / T cell cultures along with a protein transport inhibitor. The resulting cultures were stained for T cell markers (CD4, CD8, CD3) and markers of activated T cells (IFN-γ, TNF-α) and analyzed by flow cytometry. Increased IFN-γ and TNF-α staining after overnight stimulation with peptides on day 11 specifically indicated proliferation of CALRmut and / or JAK2V617F antigen-specific T cells due to Ad26 / Ad5-HEME002-driven transgene expression. A three-fold increase in IFN-γ and TNF-α double positive CD8 T cells over Ad26 / Ad5 empty vector was considered a positive response. Exclusion criteria were (i) adenohexon responses less than 0.5% of IFN-γ and TNF-α double positive T cells or (ii) viral antigen (adeno-CEF construct) IFN-γ and TNF-α double positive T cells less than 1%. In this case, the experiment was not considered suboptimal and negative responses were considered likely to be technical failure of the donor or experimental setup.

[0506] A total of 24 unique healthy donor cells were screened. Figure 2 shows a representative result of screening one donor for CALR mutation-specific T cell responses. In the experiments, cells were gated on live / CD3 / CD8 cells and assessed by IFN-γ and TNF-α staining. Both CD8 (class I) and CD4 (class II) responses were observed after infection. The experiments were performed in three independent assays, and the frequency of responses was comparable between experiments, about 35% with either Ad26 or Ad5. Figure 3 shows that both JAK2 epitopes included in the vaccine (SEQ ID NO: 5 (VLNYGVCFC), epitope 1 in Figure 3, and SEQ ID NO: 6, FCGDENILV epitope 2 (Figure 3)) are immunogenic. Figure 4 shows a representative result of screening one donor for JAK2 mutation-specific T cell responses. The data showed that both JAK2 mutant neo-epitopes were processed from the vaccine transgene sequence and these epitopes could be presented by cancer cells. The HLA types of all donors tested are shown in Table 10.

[0507] [Table 10]

[0508] Example 7: Ad26.CALR.JAK2 vector induces cellular responses in mice The objectives of the experiment were to test whether the T cell responses induced by Ad26-containing Ad26HEME001 or A26HEME002 were higher than those induced by the empty Ad26 vector using IFN-γ enzyme-linked immunospot assay (ELISpot) and to select the most suitable mouse strain for further immunogenicity studies.

[0509] C57BL / 6 and Balb / c mouse strains were tested for this immunogenicity study because epitope prediction analysis indicated that potential CD8 T cell epitopes were present in these mouse strains. 10VP / mouse doses of Ad26HEME001 or Ad26HEME002, or Ad26 encoding no transgene (Ad26.Empty) were injected. Overlapping 15mer peptide pools spanning the Ad26HEME001 or Ad26HEME002 inserts were used to assess cellular immune responses (IFN-γ ELISpot) 14 days after prime vaccination. Experimental groups are shown in Table 11.

[0510] [Table 11]

[0511] Insert-specific T cell responses were elicited in C57BL / 6 and Balb / c mice 14 days after priming, as measured by IFN-γ ELISpot, and the immune responses induced in animals immunized with Ad26HEME001 (Figure 5) or Ad26HEME002 (Figure 6) were significantly higher than those induced in animals immunized with Ad26.Empty. No cellular immune responses were detected against the LS peptide (data not shown). There were no significant differences between the immune responses elicited by the two vectors across mouse strains. Similarly, there were no significant differences in the immune responses induced in Balb / c and C57BL / 6 to the HEME001 peptide pool across vaccine candidates. There were significant differences in the immune responses induced in Balb / c and C57BL / 6 to the HEME002 peptide pool across vaccine candidates.

[0512] Example 8: Ad26.CALR.JAK2 and MVA.CALR.JAK2 vectors induce cellular responses in mice. The objective of these experiments was to determine whether a modified vaccinia Ankara (MVA) vector encoding HCalJ-9.9 (i.e., MVA-HCalJ-9.9, MVAHEME002) would enhance the immune response induced by priming Balb / c mice with Ad26HEME002. The Balb / c mouse strain was chosen based on the data described in Example 7 showing less variability in the Balb / c mouse strain compared to the C57BL / 6 mouse strain.

[0513] In the first experiment, Ad26HEME002 was 10 10 VP, MVA-HCalJ-9.9 is 10 7 IU. At week 0, mice were immunized by IM injection with Ad26HEME002, half of the animals were not boosted (group 2), and half were boosted with MVA-HCalJ-9.9 at week 3 (group 3). Another group of mice was primed with MVA-HCalJ-9.9 at week 3 (group 1). Control mice were primed at week 0 with Ad26.Empty (group 4). At week 4, all animals were sacrificed and splenocytes were stimulated with a 15mer overlapping peptide pool spanning the Ad26HEME002 insert, or a peptide pool covering the CALR sequence in the insert, or a 9mer covering two JAK2 9mer sequences. Induction of IFN-γ producing cells was measured by IFN-γ ELISpot. Table 12 shows the different experimental groups.

[0514] [Table 12]

[0515] All animals immunized with Ad26HEME002 alone or in combination with the MVA-HCalJ-9.9 boost developed IFN-γ-producing cells upon stimulation with a peptide pool of 15-mer peptides overlapping by 11 amino acids and covering the entire HEME002 (LS_CALR.JAK2.2x9mer) (Figure 7) or a mutCALR peptide pool of 15-mer peptides overlapping by 11 amino acids and covering only the mutCALR portion of HEME002 (Figure 8), whereas stimulation with the two 9-mer JAK2 peptides did not induce detectable induction of cytokine-producing cells (data not shown). Importantly, priming with Ad26HEME002 followed by boosting with MVA-HCalJ-9.9 (group 3) resulted in significantly higher responses compared to animals only primed with Ad26HEME002 at week 0 (group 2) (HEME002 peptide pool: p=0.038 CALRmut peptide pool: p=0.00025; ANOVA). In conclusion, the ELISpot data showed that MVA was able to enhance insert-specific cellular immune responses induced by priming immunization with Ad26HEME002.

[0516] In the second experiment, Ad26HEME002 was 10 9 VP or 10 10 VP, MVA-HCalJ-9.9 is 10 7 Mice were immunized at week 0 with Ad26HEME002 IM followed by a boost with MVA-HCalJ-9.9 at week 3. At week 4, all animals were sacrificed and splenocytes were stimulated with a 15mer overlapping peptide pool spanning the Ad26HEME002 insert, or a peptide pool covering the CALR sequence in the insert, or a 9mer covering two JAK2 9mer sequences. Induction of IFN-γ producing cells was measured by IFN-γ ELISpot. Ad26HEME002 was administered at 1 × 10 10 Prime with VP, then MVA-HCalJ-9.9 at 1 × 10 7 Boosting with plaque-forming units (pfu) yielded 1 × 10 10There was a statistically significant increase in IFN-γ (3.2-fold increase, p = 0.0042) compared to Ad26.Empty vector alone with VP. In contrast, priming with Ad26HEME002 at 1 × 10 9 VP, followed by 1 × 10 MVA-HCalJ-9.9 7 No significant change in IFN-γ was observed at the low dose boosted with pfu (p=0.090). 10 VP Ad26HEME002, 1×10 9 An increase in IFNγ-producing splenocytes was observed compared to VP administration. Figure 9 shows the results of the experiment.

[0517] Example 9: Immunogenicity of Ad26HEME002 and MVA-HCALJ9.9 in Non-Human Primates (NHPs) The primary objective of the study was to determine whether vaccination with Ad26HEME002 and MVA-HCalJ-9.9 induces CALR- and / or JAK2-specific T cell responses in NHPs that are greater in magnitude and longer lasting than vaccination with Ad26HEME002 alone. A secondary objective was to evaluate whether the combination of the anti-CTLA-4 monoclonal antibody YERVOY® (ipilimumab) ([Ipi]) with Ad26HEME002 and MVA-HCalJ-9.9 could enhance immune responses. In addition, a pilot objective was to evaluate whether the combination of an anti-CTLA-4 antibody and a homologue could enhance insertion-specific T cell responses with two regimens of Ad26HEME002 compared to one regimen of Ad26HEME002.

[0518] Cynomolgus macaques were immunized with Ad26HEME002 and / or MVA-HCalJ-9.9 IM alone or in combination with Ipi (10 mg / kg intravenously [IV]) according to the schedule shown in Table 13. Briefly, NHPs were immunized with Ad26HEME002 (5×10 10Animals were immunized with MVA-HCalJ-9.9 alone (10 VP, IM) (groups 1 and 2) or in combination with Ipi 10 mg / kg IV (groups 3 and 4). 8 Animals were boosted with Ad26HEME002 IU, IM, group 2), or MVA-HCalJ-9.9 combined with Ipi 10 mg / kg IV (group 3), or animals were boosted with Ad26HEME002 combined with Ipi at week 4 and MVA-HCalJ-9.9 combined with Ipi 10 mg / kg IV at week 14 (group 4), or animals did not receive any boost (group 1). Animals were bled at different time points and PBMCs and serum were isolated for immunological assays. Induction of immune responses against CALRmut or JAK2 was assessed in PBMCs at different time points during the experiment by IFN-γ ELISpot using peptide pools containing CALRmut or JAK2 2x9-mer inserts, leader sequences, or whole inserts (HEME002; CALR_JAK2-2x9mer).

[0519] [Table 13]

[0520] High non-specific background responses were seen in some animals at various time points during the study, which may obscure the interpretation of particularly low responses. All animals were included in the data set.

[0521] No immune responses to JAK2 or leader sequence peptide pools were detected at any of the time points measured (data not shown). The magnitudes as well as response rates measured to CALR and CALR_JAK2-2x9mer(HEME002) peptide pools were very similar, so only the CALR_JAK2-2x9mer(HEME002) data set is shown. Figure 10 shows the kinetics of induction of IFN-γ producing human HEME002 specific T cells across the four groups. A single Ad26HEME002 immunization did not induce detectable IFN-γ producing cells to the HEME002 peptide pool in any of the animals (Figure 10). In contrast, low but detectable HEME002-specific IFN-γ producing cells were detected in 5 of 12 animals (groups 3 and 4 combinations) immunized with Ad26HEME002 in combination with Ipi at weeks 2 (Figure 12) and 4 (data not shown), and this immune response was significantly higher than that detected in animals receiving Ad26HEME002 alone (p<0.001).

[0522] In animals treated with Ad26HEME002 and MVA-HCalJ-9.9, immune responses to CALR_JAK2-2x9mer (HEME002) were observed from week 6 onwards. There was only a small change in response rates (3-4 of 7 animals) whether animals received one (Figure 13) or two (Figure 14) doses of MVA-HCalJ-9.9. Among the animals that responded, the average magnitude of the HEME002 immune response ranged from 129 to 453 SFU / 10 6 The immune responses ranged from 0.1 to 1.5 cells (Figures 13-15). Figure 11 shows the immune response at -2 weeks of the study (i.e., 2 weeks before the first dose). Figure 12 shows the immune response at week 2. Figure 13 shows the immune response at week 6. Figure 14 shows the immune response at week 10. Figure 15 shows the immune response at week 16. At weeks 4 and 8, animals receiving MVA-HCalJ-9.9 in combination with Ipi (group 3) showed an increased magnitude of HEME002-specific immune response compared to following Ad26HEME002 immunization (mean response of responding animals: 93 SFU / 10 at week 2). 6 Cells, week 6 1945SFU / 10 6Cells, and 983SFU / 10 at 10 weeks 6 cells), but there was no change in the overall response rate compared to before and after MVA administration.

[0523] Two doses of Ad26HEME002 plus Ipi increased the amount of IFN-γ producing cells compared to a single dose of Ad26HEME002 plus Ipi (group 4). The mean HEME002 response in responding animals was 93 SFU / 10 at week 2 (after the first dose). 6 Cells, 580SFU / 10 at week 6 (after the second dose) 6 At week 14, group 4 received a combination of MVA-HCalJ-9.9 and Ipi, which increased the mean response in responding animals by 4.6-fold (400 SFU / 10 at week 12). 6 cells, 1,827 SFU / 10 at 16 weeks 6 No changes in response rates were observed (cells, Tobit p<0.001 for change scores) or in response rates. Among responders, only a slight reduction in immune response was observed, with the mean response to HEME002 at week 20 (6 weeks after MVA-HCalJ-9.9+Ipi administration) being 1,698 SFU / 10 6 cells (group 4).

[0524] Analysis of area under the curve (AUC; Tobit with Bonferroni correction) was performed for the HEME002 immune response for the following time periods: (i) weeks 2-20, (ii) weeks 2-12, and (iii) weeks 16-20. Due to the high variability and amount of censored values, no statistically significant differences were found when comparing the AUC of groups 1 and 2, or groups 2 and 3 (Figure 10). In contrast, there was a significant difference in the AUC for HEME002 response when comparing groups 3 and 4 for the time period 16-20 weeks (p=0.029). Animal #3002 (sample #14) was euthanized on day 43. Animal #4003 (sample #22) at week 16 had too many wells to count, and this sample had a 2,000 SFU / 10 6 The cells were also used for statistical analysis.

[0525] Preclinical studies have demonstrated that Ad26 and MVA vectors expressing CALRmut and JAK2 2x9-mer fusion proteins were able to induce T cell activation in vitro and cellular immune responses in mice and NHPs.

[0526] Example 10: MutCALR / MutJAK2 Construct Experiments, Administration, and Scheduling The primary objective of the study was to compare the subcutaneous (SC) and intravenous (IV) administration of anti-CTLA4 antibodies in combination with Ad / Ad / MVA vaccine. The objective was also to evaluate multiple doses of anti-CTLA4 antibodies versus a single dose in combination with Ad / Ad / MVA vaccine. The secondary objective was to evaluate whether the combination of anti-CTLA-4 monoclonal antibody YERVOY® (ipilimumab) (hereinafter "Ipi") and anti-PD-1 antibody (nivolumab) could further enhance specific T cell responses.

[0527] Anti-CTLA4 antibodies were administered SC at the same time as Ad26 vaccination placed in the Ad26 injection. Alternatively, anti-CTLA4 antibodies were infused IV over time immediately following Ad26 administration. SC administration of anti-CTLA4 antibodies was compared to IV administration of 3 mg / kg per animal. In the week 2 analysis, 16 animals received anti-CTLA4 antibodies (3 mg / kg) intravenously and 31 animals received anti-CTLA4 antibodies (3 mg / kg) SC. Similar responding animals were observed in the anti-CTLA4 IV group (7 / 16 (44%)) and the anti-CTLA4 SC group (13 / 31 (42%), respectively. Furthermore, the mean magnitude of responses was not significantly different as determined by TobitLRT analysis with Bonferroni correction p<0.05 (Figure 16, Table 14).

[0528] [Table 14]

[0529] Inclusion of anti-αPD-1 antibody (nivolumab 10 mg / kg IV) in Ad / Ad / MVA+ipilimumab starting at week 4 improves the magnitude of mutCALR-specific T cell responses (Figure 17). MutCALR peptide responses were calculated based on mutCALR-specific responses minus background. Preliminary ELISpot analysis at week 6 also demonstrated that multiple dosing with anti-αCTLA4 antibody and Ad / Ad / MVA dosing regimen was superior to single dosing (Figure 17).

[0530] Example 11: Generation of a self-replicating RNA CALR_JAK2-2x9 construct The LS_CALR.JAK2-2x9mer polynucleotide was cloned into a self-replicating RNA.

[0531] The TC-83 strain of the Venezuelan Equine Encephalitis Virus (VEEV) genomic sequence served as the nucleotide sequence used to construct the replicon of the present invention. This sequence was modified by placing the downstream LooP (DLP) from Sindbis virus upstream of the nonstructural protein 1 (nsP1) and joining the two with a 2A ribosomal skipping element from Porcine Teschovirus-1. The first 193 nucleotides of nsP1 were duplicated downstream of the 5'UTR and upstream of the DLP, except for the start codon, which was mutated to TAG. This ensured that all regulatory and secondary structures required for replication were maintained and translation of this partial nsp1 sequence was prevented. The structural genes were removed and EcoRV and AscI restriction sites were placed downstream of the subgenomic promoter as multiple cloning sites (MCS) to allow easy insertion of genes of interest (Figure 19). LS_CALR_JAK2-2x9mer was inserted into the cloning site. The transgene was synthesized by IDT as a dsDNA fragment with 40 bp of homology to the MCS at its 5′ and 3′ ends and cloned into a VEEV-derived self-replicating RNA vector digested with EcoRV and AscI using NEB HiFi DNA assembly master mix (catalog no. E2621S).

[0532] The polynucleotide sequence of the complete self-replicating RNA plasmid is shown in SEQ ID NO:33. The polynucleotide sequence of the T7 terminator is shown in SEQ ID NO:34. The polynucleotide sequence of the AmpR promoter is shown in SEQ ID NO:35. The polynucleotide sequence of the minimal 26S promoter is shown in SEQ ID NO:36. The polynucleotide sequence of the T7 promoter is shown in SEQ ID NO:37. The polynucleotide sequence of the polyA site is shown in SEQ ID NO:38. The polynucleotide sequence of the alpha 5' replicative sequence from nsP1 is shown in SEQ ID NO:39. The polynucleotide sequence of the DLP SEQ is shown in SEQ ID NO:40. The polynucleotide sequence of P2A is shown in SEQ ID NO:41. The polynucleotide sequence of Bom is shown in SEQ ID NO:42. The polynucleotide sequence of the DLPnspORF is shown in SEQ ID NO:43. The polynucleotide sequence of nsP2 is shown in SEQ ID NO:44. The polynucleotide sequence of nsP4 is shown in SEQ ID NO:45. The polynucleotide sequence of nsP3 is shown in SEQ ID NO:46. The polynucleotide sequence of nsP1 is shown in SEQ ID NO:47. The polynucleotide sequence of KanR is set forth in SEQ ID NO: 48. The polynucleotide sequence of Rop is set forth in SEQ ID NO: 49. The polynucleotide sequence of the 5'UTR is set forth in SEQ ID NO: 50. The polynucleotide sequence of the 3'UTR is set forth in SEQ ID NO: 51.

[0533] Example 12: srRNA.CALR / JAK2 replicon RNA platform induces cellular responses in mice The purpose of these experiments was to determine whether a self-replicating RNA molecule encoding CALR_JAK2-2x9mer would promote an immune response in Balb / c mice. The Balb / c mouse strain was chosen based on the data described in Example 7 showing less variability in the Balb / c mouse strain compared to the C57BL / 6 mouse strain.

[0534] In the first experiment, self-replicating RNA encoding LS_CALR_JAK2-2x9mer (srRNA.CALR / JAK2) was administered at 3, 10 and 30 μg. At week 0, Balb / c mice were immunized by IM injection of srRNA.CALR / JAK2 at the indicated doses (3, 10 and 30 μg), while the control group was injected with saline. At week 2, all animals were sacrificed and splenocytes were stimulated with a 15-mer overlapping peptide (SEQ ID NO: 3) pool covering the CALR sequence of the insert, or a 9-mer covering two JAK2 9-mer sequences (SEQ ID NO: 7). Induction of IFN-γ producing cells was measured by IFN-γ ELISpot. CD8 and CD4 polyfunctional T cell responses were determined by measuring the production of IFN-γ, TNFα and IL-2 by flow cytometry. Table 15 shows the different experimental groups.

[0535] [Table 15]

[0536] All animals immunized with srRNA.CALR / JAK2 expressed IFNγ-producing cells when stimulated with a peptide covering the CALRmut sequence (FIG. 21A), whereas no induction of cytokine-producing cells was detected when stimulated with two 9mer JAK2 peptides (SEQ ID NOs: 5 and 6) (data not shown). Intracellular flow cytometry demonstrated that the anti-CALR response was CD4-driven and polyfunctional at all doses tested (FIG. 21B).

[0537] The self-replicating RNA molecules from the first experiment were injected naked into animals. In the second experiment, the self-replicating RNA molecules were formulated in lipid nanoparticles (LNPs) and similar experiments were performed as outlined in Table 16. As in the first experiment, all animals immunized with srRNA.CALR / JAK2 expressed IFNγ-producing cells when stimulated with a peptide covering the CALRmut sequence (Figure 22A), whereas no induction of cytokine-producing cells was detected when stimulated with two 9mer JAK2 peptides (SEQ ID NOs: 5 and 6) (data not shown). Intracellular flow cytometry demonstrated that the anti-CALR response was CD4-driven and polyfunctional at all doses tested, both unformulated and LNP-formulated (Figure 22B).

[0538] [Table 16]

[0539] In a third experiment, srRNA.CALR / JAK2 was tested in combination with two other vaccine platforms, Ad26 and MVA, to determine whether heterologous prime / boost regimens would increase anti-CALR T cell function. All platforms encoded the CALR_JAK2-2x9mer construct. At week 0, Balb / c mice were immunized with saline, Ad26HEME002 (10 10 At week 4, mice were injected with Ad26HEME002, MVA-HCalJ-9.9 (10 PFU), or srRNA / CALR.JAK2 (20 μg). 7 Mice were injected with either srRNA.CALR / JAK2 (PFU) or srRNA.CALR / JAK2 and spleens were analyzed one week after the boost (see experimental design below, Table 17). A control group was added to bridge to previous data using Ad26 / MVA with a prime / boost interval of 3 weeks and spleen analysis 2 weeks later. Spleens were analyzed by ELISpot and ICS as above using overlapping CALR peptides.

[0540] [Table 17]

[0541] Adding Ad26 or MVA boost to self-replicating RNA prime resulted in a statistically significant boost by IFNγ ELISpot (p<0.001, 5.1-fold and 6.5-fold increase, respectively). Ad26 prime followed by self-replicating RNA as boosting agent resulted in a 1.7-fold increase (not statistically significant) compared to Ad26 prime alone (Figure 23A). Intracellular cytokine staining showed that self-replicating RNA prime followed by Ad26 and / or MVA boost resulted in a statistically significant increase in polyfunctional IFNγ+TNFα+IL-2+CD4 T cells (p<0.001, 4.9-fold and 3.6-fold increase, respectively). Ad26 prime followed by boost with self-replicating RNA resulted in a 2-fold increase in polyfunctional 3 cytokine positive T cells compared to Ad26 prime alone (Figure 23B).

[0542] Example 13. Immunogenicity of srRNA.CALR / JAK2 and MVA-HCalJ-9.9 in Non-Human Primates (NHP) The primary objective of this study is to determine whether vaccination with srRNA.CALR / JAK2 and MVA-HCalJ-9.9 induces antigen-specific T cell responses in NHPs that are greater in magnitude and longer lasting than vaccination with srRNA.CALR / JAK2 alone. A secondary objective is to evaluate whether the combination of the anti-CTLA-4 monoclonal antibody YERVOY® (ipilimumab) ([Ipi]) with srRNA.CALR / JAK2 and MVA-HCalJ-9.9 can enhance vaccine-induced immune responses. In addition, a pilot objective is to evaluate whether the anti-PD-1 monoclonal antibody OPDIVO® (nivolumab), when combined with the vaccine regimen and anti-CTLA-4 antibody, induces similar or increased insertion site-specific T cell responses compared to animals not receiving anti-PD-1. Cynomolgus monkeys were immunized IM with srRNA.CALR / JAK2 and / or MVA-HCalJ-9.9 alone or in combination with Ipi (3 mg / kg subcutaneous [SC]) or in combination with Nivolumab (10 mg / kg intravenous [IV]) according to the schedule shown in Table 18. Briefly, NHPs are immunized with srRNA.CALR / JAK2 and MVA-HCalJ-9.9 in combination with Ipi 3 mg / kg SC (group 2) or with Ipi 3 mg / kg SC and Nivolumab IV 10 mg / kg (group 4). Animals are bled at various time points and PBMCs and serum are isolated for immunological assays. The induction of immune responses specific to the CALR antigen is evaluated in PBMCs at various time points during the study by IFNγ ELISpot using peptide pools consisting of 15mer overlapping peptides corresponding to the entire CALR insert sequence.

[0543] The srRNA.CALR / JAK2 vaccine is expected to induce antigen-specific T cell responses that can be further enhanced when administered as a regimen in combination with MVA-HCALJ-9.9. The use of immune checkpoint inhibitor monoclonal antibodies, anti-CTLA-4 antibodies and / or anti-PD-1 antibodies in combination with srRNA.CALR / JAK2 and MVA-HCALJ-9.9 will result in larger, higher quality and more durable antigen-specific T cell responses.

[0544] [Table 18]

[0545] Example 14. Immunogenicity of GAd.CALR.JAK2 and sequential presentation of srRNA.CALR / JAK2 in non-human primates (NHPs) The primary objective of this study is to confirm that vaccination with GAd20.CALR.JAK2 induces antigen-specific T cell responses in combination with anti-CTL-4 antibodies and MVA or sRNA. This study is also designed to determine whether vaccination with srRNA.CALR / JAK2 increases T cells when combined with GAd20 or GAD20 / MVA and whether srRNA can be used in place of MVA. The study also aims to evaluate whether GAD20 / MVA / srRNA in combination with anti-CTLA4 antibodies can induce greater antigen-specific T cell responses than GAD20 / MVA / srRNA alone. Adding the srRNA.CALR / JAK2 vector to the GAd20 / MVA vaccine in a triple combination is expected to elicit greater and more sustained antigen-specific T cell responses in cancer patients.

[0546] The second objective of this experiment is to evaluate a multi-dose regimen dose of srRNA.CALR.JAK2, without the need for anti-CTL-4 antibodies, after MVA administration or completely. Additionally, the experiment will be constructed to determine whether srRNA can be administered multiple times (monthly) to continuously increase or maintain antigen-specific T cell responses. The highest and most durable levels of anti-mutCALR / mutJAK2 targeting T cells generated require continuous neoantigen presentation by a vector that is not susceptible to Ab-mediated neutralization, resulting in clearance of the malignant clone and clinical benefit in MPN patients. One potential advantage of self-replicating RNA-based vaccines is the lack of developed vector-specific immunity. The absence of a vector-specific immune response may allow repeated administration of self-replicating RNA without diminishing antigen presentation, as self-replicating RNA-based vaccines cannot generate vector-specific neutralizing antibodies. A multi-dose regimen will be tested to evaluate whether srRNA.CALR / JAK2 can maintain antigen-specific T cell responses on a monthly intramuscular administration schedule.

[0547] In this experiment, cynomolgus monkeys are immunized according to the schedule shown in Table 19. Animals are bled at various time points to isolate PBMCs and serum for immunological assays. Induction of immune responses specific to the CALR antigen is assessed in PBMCs at various time points during the study by IFNγ ELISpot using peptide pools consisting of 15mer overlapping peptides corresponding to the entire CALR insert sequence.

[0548] Administration of srRNA.CALR / JAK2 after GAd20 / MVA immunization allows for a continuous boost to prolong antigen-specific T cell responses, either independently or in combination with CPI administration. High-dose srRNA.CALR / JAK2 self-replicating RNA-based regimens are expected to elicit larger T cell responses for longer periods of time.

[0549] [Table 19]

[0550] Embodiment The following list of embodiments is intended to complement, not replace or supplant, the foregoing description. Embodiment 1. MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE (SEQ ID NO: 1), or a sequence at least 90% identical to SEQ ID NO: 1; EEAEDNCRRMMRTK (SEQ ID NO: 2), or a sequence at least 90% identical to SEQ ID NO: 2; KLSHKHLVLNYGVCFCGDENILVQEFVKFG (SEQ ID NO: 4), or a sequence at least 90% identical to SEQ ID NO: 4; VLNYGVCFC (SEQ ID NO:5), or a sequence at least 90% identical to SEQ ID NO:5; A polypeptide comprising at least two or more epitope sequences selected from the group consisting of FCGDENILV (SEQ ID NO: 6), or a sequence at least 90% identical to SEQ ID NO: 6, and combinations thereof.

[0551] Embodiment 2. The polypeptide comprises: MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE (SEQ ID NO: 1), or a sequence at least 90% identical to SEQ ID NO: 1; EEAEDNCRRMMRTK (SEQ ID NO: 2), or a sequence at least 90% identical to SEQ ID NO: 2; VLNYGVCFC (SEQ ID NO:5), or a sequence at least 90% identical to SEQ ID NO:5, and 2. The polypeptide of embodiment 1, comprising an epitope sequence that is FCGDENILV (SEQ ID NO:6), or a sequence that is at least 90% identical to SEQ ID NO:6.

[0552] Embodiment 3. The polypeptide comprises: MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE (SEQ ID NO: 1), or a sequence at least 90% identical to SEQ ID NO: 1; EEAEDNCRRMMRTK (SEQ ID NO: 2), or a sequence at least 90% identical to SEQ ID NO: 2, and 2. The polypeptide of embodiment 1, comprising an epitope sequence that is KLSHKHLVLNYGVCFCGDENILVQEFVKFG (SEQ ID NO: 4), or a sequence that is at least 90% identical to SEQ ID NO: 4.

[0553] Embodiment 4. The polypeptide comprises: MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE (SEQ ID NO: 1), or a sequence at least 90% identical to SEQ ID NO: 1, and 2. The polypeptide of embodiment 1, comprising an epitope sequence that is EEAEDNCRRMMRTK (SEQ ID NO:2), or a sequence that is at least 90% identical to SEQ ID NO:2.

[0554] Embodiment 5. MACPGFLWALVISTCLEFSMA (SEQ ID NO: 8), MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGV (SEQ ID NO:54), and 5. The polypeptide according to any one of embodiments 1 to 4, further comprising a leader sequence at the N-terminus selected from MGQKEQIHTLQKNSERMSKQLTRSSQAV (SEQ ID NO: 29).

[0555] Embodiment 6. A polypeptide according to any one of embodiments 1 to 5, wherein the epitope sequences are linked to each other by a linker sequence.

[0556] Embodiment 7. The linker sequence is AAY, RR, DPP, HHAA (SEQ ID NO:56) , HHA, HHL, RKSYL (SEQ ID NO:57) , R.K.S.Y. (SEQ ID NO:58), SSL, or REKR (SEQ ID NO:59) 7. The polypeptide of embodiment 6, selected from:

[0557] Embodiment 8. The polypeptide of embodiment 6, wherein the linker sequence comprises a protease cleavage site.

[0558] Embodiment 9. The polypeptide is an amino acid sequence of SEQ ID NO:3 or having at least 90% sequence identity with SEQ ID NO:3; an amino acid sequence of SEQ ID NO:7 or having at least 90% sequence identity with SEQ ID NO:7; an amino acid sequence of SEQ ID NO:9 or having at least 90% sequence identity with SEQ ID NO:9; an amino acid sequence of SEQ ID NO:10 or having at least 90% sequence identity with SEQ ID NO:10; an amino acid sequence of SEQ ID NO:11 or having at least 90% sequence identity with SEQ ID NO:11; an amino acid sequence of SEQ ID NO:12 or having at least 90% sequence identity with SEQ ID NO:12; an amino acid sequence of SEQ ID NO:13 or having at least 90% sequence identity with SEQ ID NO:13; an amino acid sequence of SEQ ID NO: 14 or having at least 90% sequence identity with SEQ ID NO: 14; an amino acid sequence of SEQ ID NO: 15 or having at least 90% sequence identity with SEQ ID NO: 15; and 2. The polypeptide of embodiment 1, selected from the amino acid sequence of SEQ ID NO: 31 or having at least 90% sequence identity with SEQ ID NO: 31.

[0559] Embodiment 10. A polynucleotide encoding the polypeptide according to any one of embodiments 1 to 9.

[0560] Embodiment 11. The polynucleotide comprises: A nucleic acid sequence of SEQ ID NO: 16 or having at least 90% sequence identity with SEQ ID NO: 16; A nucleic acid sequence of SEQ ID NO: 17 or having at least 90% sequence identity with SEQ ID NO: 17; A nucleic acid sequence of SEQ ID NO: 18 or having at least 90% sequence identity with SEQ ID NO: 18; A nucleic acid sequence of SEQ ID NO:19 or having at least 90% sequence identity with SEQ ID NO:19; A nucleic acid sequence of SEQ ID NO:20 or having at least 90% sequence identity with SEQ ID NO:20; A nucleic acid sequence of SEQ ID NO:21 or having at least 90% sequence identity with SEQ ID NO:21; A nucleic acid sequence of SEQ ID NO:22 or having at least 90% sequence identity with SEQ ID NO:22; A nucleic acid sequence of SEQ ID NO:26 or having at least 90% sequence identity with SEQ ID NO:26, and 11. The polynucleotide of embodiment 10, selected from the group consisting of a nucleic acid sequence of SEQ ID NO:27 or having at least 90% sequence identity to SEQ ID NO:27.

[0561] Embodiment 12. A vector comprising the polynucleotide of embodiment 10 or embodiment 11.

[0562] Embodiment 13. The vector of embodiment 12, wherein the vector is selected from an adenovirus vector, an alphavirus vector, a poxvirus vector, an adeno-associated virus vector, a retrovirus vector, a self-replicating RNA molecule, and combinations thereof.

[0563] Embodiment 14. The adenoviral vector is selected from the group consisting of hAd5, hAd7, hAd11, hAd26, hAd34, hAd35, hAd48, hAd49, hAd50, GAd20, Gad19, GAd21, GAd25, GAd26, GAd27,...

Claims

1. MKDKQDEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE (SEQ ID NO: 1), EEAEDNCRRMMRTK (SEQ ID NO:2), VLNYGVCFC (SEQ ID NO:5), and FCGDENILV (SEQ ID NO: 6) A polypeptide comprising an epitope sequence of

2. MACPGFLWALVISTCLEFSMA (SEQ ID NO: 8), MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGV (SEQ ID NO:54), or 2. The polypeptide of claim 1, further comprising an N-terminal leader sequence comprising MGQKEQIHTLQKNSERMSKQLTRSSQAV (SEQ ID NO:29).

3. The polypeptide of claim 1 or 2, wherein the epitope sequences are linked to each other by a linker sequence.

4. 4. The polypeptide of claim 3, wherein the linker sequence is selected from AAY, RR, DPP, HHAA (SEQ ID NO:56), HHA, HHL, RKSYL (SEQ ID NO:57), RKSY (SEQ ID NO:58), SSL, and REKR (SEQ ID NO:59).

5. The polypeptide is The amino acid sequence of SEQ ID NO: 10, the amino acid sequence of SEQ ID NO: 12, or A polypeptide according to any one of claims 1 to 4, comprising the amino acid sequence of SEQ ID NO:

31.

6. The polypeptide of claim 5, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ ID NO:

31.

7. A polynucleotide encoding the polypeptide according to any one of claims 1 to 6.

8. The polynucleotide is The nucleic acid sequence of SEQ ID NO: 17, The nucleic acid sequence of SEQ ID NO: 19, The nucleic acid sequence of SEQ ID NO: 26, or The polynucleotide of claim 7 comprising the nucleic acid sequence of SEQ ID NO:

27.

9. The polynucleotide is The nucleic acid sequence of SEQ ID NO: 26, or The polynucleotide of claim 8 comprising the nucleic acid sequence of SEQ ID NO:

27.

10. A vector comprising the polynucleotide according to any one of claims 7 to 9.

11. 11. The vector of claim 10, wherein the vector is selected from an adenovirus vector, an alphavirus vector, a poxvirus vector, an adeno-associated virus vector, a retrovirus vector, a self-replicating RNA molecule, and combinations thereof.

12. The vector of claim 10 or 11, wherein the vector is an MVA vector comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:

31.

13. The vector of claim 10 or 11, wherein the vector is a GAd20 vector comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:

31.

14. The vector of claim 10 or 11, wherein the vector is a self-replicating RNA molecule comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:

12.

15. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 6, a polynucleotide according to any one of claims 7 to 9, or a vector according to any one of claims 10 to 14.

16. 16. The pharmaceutical composition of claim 15 for use in a method for treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject, inducing an immune response in a subject having a JAK2V617F and / or CALR exon 9 mutation, treating or preventing a myeloproliferative disorder in a subject, treating cancer in a subject, or treating a cardiovascular disease in a subject.

17. The pharmaceutical composition of claim 16, wherein the myeloproliferative disorder is selected from primary myelofibrosis (MPN), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PFM), secondary myelofibrosis, acute myeloid leukemia (AML), secondary AML, chronic myelogenous leukemia (CML), clonal hematopoiesis of undetermined potential (CHIP), and chronic myelomonocytic leukemia (CMML).

18. The pharmaceutical composition of claim 16, wherein the cancer is selected from lung cancer, lymphatic system cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, plasma cell myeloma, biliary tract cancer, bladder cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, gastric cancer, colorectal cancer, colon cancer, urinary tract cancer, central nervous system cancer, neuroblastoma, kidney cancer, breast cancer, cervical cancer, testicular cancer, and soft tissue cancer.

19. The pharmaceutical composition of claim 16, wherein the cardiovascular disease is selected from acute coronary syndrome, ischemic cerebrovascular disease, ischemic heart disease, thrombosis, venous thromboembolism, deep vein thrombosis, pulmonary embolism, refractory intraperitoneal thrombosis, peripheral arterial disease, hypertension, heart failure, atrial fibrillation, coronary heart disease, atherosclerosis, and clonal hematopoiesis.

20. 1. A pharmaceutical composition for use in a method of treating, preventing, reducing the risk of onset, or delaying the onset of a clinical condition characterized by expression of a JAK2V617F and / or CALR exon 9 mutation in a subject, inducing an immune response in a subject having a JAK2V617F and / or CALR exon 9 mutation, treating or preventing a myeloproliferative disorder in a subject, treating cancer in a subject, or treating a cardiovascular disease in a subject, comprising: The pharmaceutical composition comprises: the CALR epitope of SEQ ID NO: 1, CALR epitope of SEQ ID NO:2, The JAK2 epitope of SEQ ID NO:5, and JAK2 epitope of SEQ ID NO:6 The pharmaceutical composition comprises a vector comprising a polynucleotide encoding the

21. 21. The pharmaceutical composition of claim 20, wherein the myeloproliferative disorder is selected from primary myelofibrosis (MPN), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PFM), secondary myelofibrosis, acute myeloid leukemia (AML), secondary AML, chronic myelogenous leukemia (CML), clonal hematopoiesis of undetermined potential (CHIP), and chronic myelomonocytic leukemia (CMML).

22. 21. The pharmaceutical composition of claim 20, wherein the cancer is selected from lung cancer, lymphatic system cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, plasma cell myeloma, biliary tract cancer, bladder cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, gastric cancer, colorectal cancer, colon cancer, urinary tract cancer, central nervous system cancer, neuroblastoma, kidney cancer, breast cancer, cervical cancer, testicular cancer, and soft tissue cancer.

23. 21. The pharmaceutical composition of claim 20, wherein the cardiovascular disease is selected from acute coronary syndrome, ischemic cerebrovascular disease, ischemic heart disease, thrombosis, venous thromboembolism, deep vein thrombosis, pulmonary embolism, refractory intraperitoneal thrombosis, peripheral arterial disease, hypertension, heart failure, atrial fibrillation, coronary heart disease, atherosclerosis, and clonal hematopoiesis.

24. 24. The pharmaceutical composition of any one of claims 20 to 23, wherein the vector is selected from an adenoviral vector, an alphavirus vector, a poxvirus vector, an adeno-associated virus vector, a retroviral vector, a self-replicating RNA molecule, and combinations thereof.

25. The vector comprises: the CALR epitope of SEQ ID NO: 1, CALR epitope of SEQ ID NO:2, The JAK2 epitope of SEQ ID NO:5, and JAK2 epitope of SEQ ID NO:6 The pharmaceutical composition according to any one of claims 20 to 24, which is an Ad26 vector comprising a polynucleotide encoding a polypeptide comprising:

26. The vector comprises: the CALR epitope of SEQ ID NO: 1, CALR epitope of SEQ ID NO:2, The JAK2 epitope of SEQ ID NO:5, and JAK2 epitope of SEQ ID NO:6 The pharmaceutical composition of any one of claims 20 to 24, which is a GAd20 vector comprising a polynucleotide encoding a polypeptide comprising:

27. The vector comprises: the CALR epitope of SEQ ID NO: 1, CALR epitope of SEQ ID NO:2, The JAK2 epitope of SEQ ID NO:5, and JAK2 epitope of SEQ ID NO:6 The pharmaceutical composition according to any one of claims 20 to 24, which is an MVA vector comprising a polynucleotide encoding a polypeptide comprising:

28. The vector comprises: the CALR epitope of SEQ ID NO: 1, CALR epitope of SEQ ID NO:2, The JAK2 epitope of SEQ ID NO:5, and JAK2 epitope of SEQ ID NO:6 The pharmaceutical composition according to any one of claims 20 to 24, which is a self-replicating RNA molecule comprising a polynucleotide encoding a polypeptide comprising:

29. 29. The pharmaceutical composition of any one of claims 16-28, wherein the method further comprises administering a second therapeutic agent selected from a CTLA-4 antibody, a PD-1 antibody, a PD-L1 antibody, a TLR agonist, a CD40 agonist, an OX40 agonist, hydroxyurea, ruxolitinib, fedratinib, a 4-1BB agonist, a CD28 agonist, a FLT3 ligand, aluminum sulfate, a BTK inhibitor, a JAK inhibitor, a CD38 antibody, a CDK inhibitor, a CD33 antibody, a CD37 antibody, a CD25 antibody, a GM-CSF inhibitor, IL-2, IL-15, IL-7, IFNγ, IFNα, TNFα, a VEGF antibody, a CD70 antibody, a CD27 antibody, a BCMA antibody, a GPRC5D antibody, or a combination thereof.

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