Vaccines using macrophage suppression

Abstract

Provided herein are IL-10 variant sequences. In some embodiments, the IL-10 variants are immunosilent, and / or are capable of suppressing monocyte, macrophage, or pDC activity. Also disclosed herein are vaccines comprising the IL-10 variants, and methods of treatment for diseases and disorders that correspond with activating adaptive immunity.

Description

UCDAV.009WO PATENTVACCINES USING MACROPHAGE SUPPRESSIONRELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of U.S. Provisional Ser. No. 63 / 724202, filed November 22, 2024, and U.S. Provisional Ser. No. 63 / 884779, filed September 19, 2025, both of which are hereby incorporated by reference in their entirety.REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled UCDAV009WO.xml, which was created and last modified on November 13, 2025, and is 107,256 bytes in size. The information in the electronic Sequence Listing is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0003] This invention was made with government support under All 50554 awarded by the National Institutes of Health. The government has certain rights in the invention.RELATED FIELD

[0004] Disclosed herein are IL- 10 variant sequences and their use in suppressing macrophage activity. In some embodiments, the IL- 10 variants are immunosilent. Also disclosed herein are vaccines comprising the IL-10 variants, and methods of treatment for diseases and disorders that correspond with an activated immune system.BACKGROUND

[0005] Interleukin 10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is a class II cytokine with diverse functions. Mature IL-10 is a homodimer of two subunits that are each 160 amino acids long. It primarily signals through a receptor complex comprising two IL-10 receptor-1 and two IL-10 receptor-2 proteins, which are also known as IL-10 receptor-alpha and IL-10 receptor-beta. IL-10 has beenshown to have multiple effects in both immunoregulation and inflammation, including downregulating the expression of Th 1 cytokines, MHC class II antigens, and costimulatory molecules on macrophages, and enhancing B cell survival, proliferation, and antibody production. IL-10 can block NF-KB activity, and is involved in the regulation of the JAK-STAT signaling pathway.SUMMARY

[0006] Aspects of the present disclosure relate to protein variants of human IL- 10. In some embodiments, the protein variant comprises a deletion of an at least one amino acid residue contributing to interaction with IL-10R1 or IL-10R2, wherein the deletion of at least one amino acid is at or corresponding with an amino acid at position 14, 18, 21, 22, 24, 28, 74, 90, 92, 96, 100, 104, 108, or any combination thereof, of SEQ ID NO: 49; and wherein the variant retains signaling function. In some embodiments, there is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 deletions at positions 14, 18, 21, 22, 24, 28, 74, 90, 92, 96, 100, 104, 108, or any combination thereof. In some embodiments, a cysteine residue corresponding to the cysteine at position 12 of SEQ ID NO: 49 is instead placed at position 11 of SEQ ID NO: 49, such that there is an amino acid other than cysteine at position 12.

[0007] Also disclosed herein are fusion proteins. In some embodiments, the fusion protein comprises: (a) an IL-10 or a variant of IL-10 having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92. 93, 94, 95, 96, 97, 98, or 99% sequence identity to IL-10, and (b) a segment of IL-10R2. In some embodiments, the variant IL-10 is the protein variant of any one of the embodiments of the present disclosure. In some embodiments, the fusion protein comprises: (a) a segment of human serum albumin and / or a segment of an Fc domain, and (b) a fusion protein or protein variant of any one of the embodiments of the present disclosure.

[0008] In some embodiments of the present disclosure, the fusion protein and / or IL- 10 protein variant has a specificity for signaling to myeloid cells that is calculated as the ratio between signaling activity to myeloid cell types and signaling activity7to T cells, wherein the specificity is greater than the specificity of cellular IL- 10 (cIL-10) for signaling to myeloid cells. In some embodiments, the specificity is for signaling to at least one of: monocytes, macrophages, plasmacytoid dendritic cells (pDCs), or any combination thereof. In some embodiments, the fusion protein and / or IL-10 protein variant is immunosilent. In some embodiments, the fusion protein and / or IL- 10 protein variant comprises a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90. 91. 92, 93, 94,95, 96, 97, 98, or 99 or 100% identity to any one of the sequences of SEQ ID NOs.: 6-40. In some embodiments, the segment is on the C- and / or N-terminal end. In some embodiments, the segment of IL-10R2 comprises the extracellular domain of IL-10R2, loop 2 residues Y59-K65, loop 3 residues S80-Y87, loop 5 residues W143-N152, full- length IL-10R2, or any combination thereof. In some embodiments, the fusion protein further comprises a linker sequence, optionally wherein the linker sequence is operably linked between the first fusion partner and the second fusion partner. In some embodiments, the linker sequence comprises: glycine, serine, threonine, alanine, or any combination thereof; optionally wherein the linker sequence comprises: glycine and / or serine. In some embodiments, the linker sequence is about 3 to about 20 amino acids in length; optionally wherein the linker sequence is about 5 to about 15 amino acids in length; optionally wherein the linker sequence is about 5 to 9 amino acids in length. In some embodiments, at least one of the amino acids at or corresponding with positions H14 or N18 of SEQ ID NO: 49 are deleted or mutated. In some embodiments, the amino acid at or corresponding with position R104 of SEQ ID NO: 49 is deleted or mutated, and wherein at least one of the amino acids at or corresponding with positions H14 or N18 of SEQ ID NO: 49 are deleted or mutated.

[0009] Also disclosed herein is a nucleotide sequence encoding the protein variant or fusion protein of any one of the embodiments of the present disclosure. In some embodiments, the nucleotide sequence comprises DNA and / or RNA.

[0010] Also disclosed herein is a delivery vector comprising the nucleotide sequence of any one of the embodiments of the present disclosure. In some embodiments, the deliver^' vector is a viral vector or lipid nanoparticle. In some embodiments, the viral vector is a lentiviral vector, retroviral vector, adenoviral vector, or any combination thereof. In some embodiments, the delivery vector further comprises an at least one self-cleaving peptide.

[0011] Also disclosed herein is a vaccine comprising the delivery vector of any one of the embodiments of the present disclosure. Also disclosed herein is a vaccine encoding the protein variant or fusion protein of any one of the embodiments of the present disclosure, and / or the nucleotide of any one of the embodiments of the present disclosure. In some embodiments, the vaccine is an mRNA, adenovirus, alphavirus, or lentivirus platform, or any combination thereof.

[0012] Also disclosed herein is a cell comprising the protein variant or fusion protein of any one of the embodiments of the present disclosure, the nucleotide sequenceof any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, or any combination thereof.

[0013] Also disclosed herein is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the protein variant or fusion protein of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, the vaccine of any one of the embodiments of the present disclosure, the cell of any one of the embodiments of the present disclosure, or any combination thereof. In some embodiments, the disease or disorder is an infection, autoimmune disease, an allergic disease or reaction, an inflammatory disease, a cancer, a tumor, or any combination thereof. In some embodiments, the disease or disorder is a chronic infection, optionally wherein the chronic infection is HIV or hepatitis.

[0014] Also disclosed herein is a use for the protein variant or fusion protein of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, the vaccine of any one of the embodiments of the present disclosure, the cell of any one of the embodiments of the present disclosure, or any combination thereof, in the preparation of a medicament.

[0015] Also disclosed herein is a method of stimulating an immune cell. In some embodiments, the method comprises administering to the immune cell the protein variant or fusion protein of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, the vaccine of any one of the embodiments of the present disclosure, the cell of any one of the embodiments of the present disclosure, or any combination thereof. In some embodiments, stimulating the immune cell comprises increasing the total number of activated T cells, and / or increasing the production of IFN-gamma and / or TNF-alpha. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the immune cell is a T cell, a B cell, or an NK cell. In some embodiments, the T cell is a CD4+ or a CD8+T cell. In some embodiments, the T cell is an MHC class Ib-restricted T cell.

[0016] Also disclosed herein is a method of inhibiting IL-1 activity, NLRP1 inflammasome activation, NLRP3 inflammasome activation, type 1 interferon production (IFN-I) production, IL-6 production, or any combination thereof in vivo. In someembodiments, the method comprises administering the protein variant or fusion protein of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, the vaccine of any one of the embodiments of the present disclosure, the cell of any one of the embodiments of the present disclosure, or any combination thereof. In some embodiments, the method further comprises administering an at least one single-chain trimer and / or IL-15 at the same time as administering the protein variant or fusion protein of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, the vaccine of any one of the embodiments of the present disclosure, the cell of any one of the embodiments of the present disclosure, or any combination thereof. In some embodiments, the method further comprises administering an at least one booster vaccine. In some embodiments, the method further comprises administering an at least one single-chain trimer and / or IL- 15 at the same time as the booster vaccine. In some embodiments, the at least one single-chain trimer comprises a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity w ith the sequence of any one of SEQ ID NOs: 52-55.

[0017] Also disclosed herein is a method of vaccinating a subject in need thereof. In some embodiments, the method comprises administering to the subject the protein variant or fusion protein of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, the vaccine of any one of the embodiments of the present disclosure, the cell of any one of the embodiments of the present disclosure, or any combination thereof In some embodiments, the vaccination results in increasing the total number of activated T cells, and / or increasing the production of IFN-gamma and / or TNF-alpha in the subject. In some embodiments, the vaccination results in the reduced occurrence, duration, and / or severity of at least one side effect as compared to vaccination in absence of the protein variant of any one of the embodiments of the present disclosure. In some embodiments, the at least one side effect is selected from: fever, malaise, granulocytosis, circulating IL-6, circulating total cytokine concentration, circulating specific cytokine concentration, or any combination thereof. In some embodiments, the circulating specific cytokine is IL-6 and / or IP-10 (CXCL10). In some embodiments, the subject is mammalian and / or human. In some embodiments, thevaccination is a booster vaccination. In some embodiments, the vaccination is performed on the subject more than once.

[0018] Aspects of the present disclosure are also as detailed in the below enumerated alternatives:1. A protein variant of human IL- 10, wherein the variant comprises a deletion of an at least one amino acid residue contributing to interaction with IL-10R1 or IL-10R2, wherein the deletion of at least one amino acid is at or corresponding with an amino acid at position 14, 18, 21, 22, 24, 28, 74, 90, 92, 96, 100, 104, 108, or any combination thereof, of SEQ ID NO: 49; and wherein the variant retains signaling function. In some embodiments, there is at least 2. 3, 4, 5. 6, 7, 8. 9, 10, 11, 12, or 13 deletions at positions 14, 18, 21, 22, 24, 28, 74, 90, 92, 96, 100, 104, 108, or any combination thereof of SEQ ID NO: 49; and wherein the variant retains signaling function.2. The protein variant of alternative 1, wherein a cysteine residue corresponding to the cysteine at position 12 of SEQ ID NO: 49 is instead placed at position 11 of SEQ ID NO: 49, such that there is an amino acid other than cysteine at position 12.3. A fusion protein comprising:(a) an IL-10 or a variant of IL-10 having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to IL- 10, and(b) a segment of IL-10R2.4. The fusion protein of alternative 3, wherein the variant IL-10 is the protein variant of alternative 1 or 2.5. A fusi on protein compri sing :(a) a segment of human serum albumin and / or a segment of an Fc domain, and(b) a fusion protein or protein variant of any one of alternatives 1 -4.6. The protein variant or fusion protein of any one of alternatives 1-5, wherein the protein has a specificity for signaling to myeloid cells that is calculated as the ratio between signaling activity to myeloid cell types and signaling activity to T cells, wherein the specificity is greater than the specificity of cellular IL-10 (cIL-10) for signaling to myeloid cells.7. The protein variant or fusion protein of alternative 6, wherein the specificity is for signaling to at least one of: monocytes, macrophages, plasmacytoid dendritic cells (pDCs), or any combination thereof.8. The protein variant or fusion protein of any one of alternatives 1-7, wherein the protein variant is immunosilent.9. The protein variant or fusion protein of any one of alternatives 1-8, wherein the variant comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any one of the sequences of SEQ ID NOs.: 6-40.10. The protein variant or fusion protein of any one of alternatives 3-9, wherein the segment is on the C- and / or N-terminal end.11. The protein variant or fusion protein of any one of alternatives 3-10, wherein the segment of IL-10R2 comprises the extracellular domain of IL-10R2, loop 2 residues Y59-K65, loop 3 residues S80-Y87, loop 5 residues W143-N152, full-length IL- 10R2, or any combination thereof.12. The protein variant or fusion protein of any one of alternatives 1-11, further comprising a linker sequence, optionally wherein the linker sequence is operably linked between the first fusion partner and the second fusion partner.13. The protein variant or fusion protein of alternative 12, wherein the linker sequence comprises: glycine, serine, threonine, alanine, or any combination thereof; optionally wherein the linker sequence comprises: glycine and / or serine.14. The protein variant or fusion protein of alternative 12 or 13, wherein the linker sequence is about 3 to about 20 amino acids in length; optionally wherein the linker sequence is about 5 to about 15 amino acids in length; optionally wherein the linker sequence is about 5 to 9 amino acids in length.15. The protein variant or fusion protein of any one of alternatives 1-14, wherein at least one of the amino acids at or corresponding with positions H14 or N18 of SEQ ID NO: 49 are deleted or mutated.16. The protein variant or fusion protein of any one of alternatives 1-15, wherein the amino acid at or corresponding with position R104 of SEQ ID NO: 49 is deleted or mutated, and wherein at least one of the amino acids at or corresponding with positions H14 or N18 of SEQ ID NO: 49 are deleted or mutated.17. A nucleotide sequence encoding the protein variant or fusion protein of any one of alternatives 1-16.18. The nucleotide sequence of alternative 17, wherein the nucleotide sequence comprises DNA and / or RNA.19. A delivery vector comprising the nucleotide sequence of any one of alternatives 17-18.20. The delivery7vector of alternative 19, wherein the delivery' vector is a viral vector or lipid nanoparticle.21. The delivery vector of alternative 20, wherein the viral vector is a lentiviral vector, retroviral vector, adenoviral vector, or any combination thereof.22. The delivery7vector of any one of alternatives 19-21, further comprising an at least one self-cleaving peptide.23. A vaccine comprising the delivery vector of any one of alternatives 19-22.24. A vaccine encoding the protein variant or fusion protein of any one of alternatives 1-16, and / or the nucleotide of any one of alternatives 17 or 18.25. The vaccine of alternative 23 or 24, wherein the vaccine is an mRNA, adenovirus, alphavirus, or lentivirus platform, or any combination thereof.26. A cell comprising the protein variant or fusion protein of any one of alternatives 1-16, the nucleotide sequence of any one of alternatives 17-18, the delivery vector of any one of alternatives 19-22, or any combination thereof.27. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the protein variant or fusion protein of any one of alternatives 1 -16, the nucleotide sequence of any one of alternatives 17-18, the delivery7vector of any one of alternatives 19-22, the vaccine of any one of alternatives 23-25, the cell of alternative 26, or any combination thereof.28. The method of alternative 27. wherein the disease or disorder is an infection, autoimmune disease, an allergic disease or reaction, an inflammatory disease, a cancer, a tumor, or any combination thereof.29. The method of alternative 28, wherein the disease or disorder is a chronic infection, optionally wherein the chronic infection is HIV or hepatitis.30. A use for the protein variant or fusion protein of any one of alternatives 1- 16, the nucleotide sequence of any one of alternatives 17-18, the delivery vector of any one of alternatives 19-22, the vaccine of any one of alternatives 23-25, or the cell of alternative26, in the preparation of a medicament.31. A method of stimulating an immune cell, the method comprising administering to the immune cell the protein variant or fusion protein of any one ofaltematives 1-16, the nucleotide sequence of any one of alternatives 17-18, the delivery vector of any one of alternatives 19-22, or the vaccine of any one of alternatives 23-25.32. The method of alternative 31, wherein stimulating the immune cell comprises increasing the total number of activated T cells, and / or increasing the production of IFN-gamma and / or TNF -alpha.33. The method of alternative 31 or 32, wherein the immune cell is a lymphocyte.34. The method of alternative 33, wherein the immune cell is a T cell, a B cell, or an NK cell.35. The method of alternative 34, wherein the T cell is a CD4+ or a CD8+T cell.36. The method of any one of alternatives 34 or 35, wherein the T cell is an MHC class Ib-restricted T cell.37. A method of inhibiting IL-1 activity, NLRP1 inflammasome activation, NLRP3 inflammasome activation, type 1 interferon production (IFN-I) production. IL-6 production, or any combination thereof in vivo, the method comprising administering the protein variant of any one of alternatives 1-16, the nucleotide sequence of any one of alternatives 17-18, the delivery vector of any one of alternatives 19-22, the vaccine of any one of alternatives 23-25, or the cell of alternative 26.38. The method of any one of alternatives 31-37, further comprising administering an at least one single-chain trimer and / or IL-15 at the same time as administering the protein variant or fusion protein of any one of alternatives 1-16, the nucleotide sequence of any one of alternatives 17-18, the delivery' vector of any one of alternatives 19-22, the vaccine of any one of alternatives 23-25, or the cell of alternative 26.39. The method of any one of alternatives 31-38, further comprising administering an at least one booster vaccine.40. The method of alternative 39, further comprising administering an at least one single-chain trimer and / or IL- 15 at the same time as the booster vaccine.41. The method of any one of alternatives 38 or 40, wherein the at least one single-chain trimer comprises a sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. or 99% identity' with the sequence of any one of SEQ ID NOs: 52-55.42. A method of vaccinating a subject in need thereof, the method comprising administering to the subject the protein variant of any one of alternatives 1-16, the nucleotide sequence of any one of alternatives 17-18, the delivery vector of any one of alternatives 19-22, the vaccine of any one of alternatives 23-25, or the cell of alternative 26.43. The method of alternative 42, wherein the vaccination results in increasing the total number of activated T cells, and / or increasing the production of IFN-gamma and / or TNF-alpha in the subject.44. The method of alternative 42 or 43, wherein the vaccination results in the reduced occurrence, duration, and / or severity of at least one side effect as compared to vaccination in absence of the protein variant of any one of alternatives 1-16.45. The method of alternative 44, wherein the at least one side effect is selected from: fever, malaise, granulocytosis, circulating IL-6, circulating total cytokine concentration, circulating specific cytokine concentration, or any combination thereof.46. The method of alternative 45, wherein the circulating specific cytokine is IL-6 and / or IP-10 (CXCL10),47. The method of any one of alternatives 42-46, wherein the subject is mammalian and / or human.48. The method of any one of alternatives 42-47, wherein the vaccination is a booster vaccination.49. The method of any one of alternatives 42-48, wherein the vaccination is performed on the subj ect more than once.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A depicts a non-limiting example timetable of a first experiment (“Experiment 1”), as outlined in Example 1 below, for assessing vaccination with adenovectored vaccines that expressed SIV Gag alone (“Ad / Gag”, SEQ ID NO: 1) or SIV Gag and conventional IL- 10 (“Ad / Gag-cIL10,’' wherein the Gag and cIL-10 proteins are expressed from a bicistronic construct using an IRES element). Vaccines were administered to macaques (all groups) intramuscularly at weeks 0 and 4 of the experiment, and blood was drawn at regular intervals (shown as dots in the figure). The adenovectored vaccines were formulated in PBS for administration.

[0020] Figure IB depicts a non-limiting example timetable of a second experiment ("‘Experiment 2"). as outlined in Example 1 below, for assessing vaccination with an adenovectored vaccine that expressed SIV Gag alone (‘‘Ad / Gag”) or an adenovectored vaccine that expressed SIV Gag and monocyte / macrophage-specific IL- 10DE (“Ad / Gag-ILIODE”, using a bicistronic construct). Vaccines were administered to macaques (all groups) intramuscularly at weeks 0 and 4 of the experiment, and blood was drawn at regular intervals (shown as dots in the figure). The adenovectored vaccines were formulated in PBS for administration.

[0021] Figure 2 depicts quantification of the change in number of circulating granulocytes after vaccinating with adenovector vaccine that expressed Gag alone (dashed line) or Gag together with IL- 1 ODE (solid line) following Experiment 2.

[0022] Figures 3A-3B depict quantification of IL-6 production 24 hours following vaccination with Ad / Gag (Figure 3A) or Ad / Gag-ILIODE (Figure 3B) under the Experiment 2 procedure, wherein each plotted line represents a different individual.

[0023] Figures 4A-4B depict non-limiting example quantifications of cytokine production by CD8+T cells after stimulation with overlapping peptides from the Gag vaccine antigen. Separate graphs show the fraction of effector-memory CD8+T cells (CD95+CCR7‘) that tested positive for producing IFN-gamma (left panel), TNF -alpha (center panel), or both IFN-gamma and TNF-alpha (right panel) under the Experiment 2 and Experiment 1 procedures. Quantification was performed following vaccination with either Ad / Gag-ILI ODE vs. Ad / Gag (Figure 4A, Experiment 2), or Ad / Gag-cILlO vs. Ad / Gag (Figure 4B, Experiment 1).

[0024] Figures 5A-5B depict quantifications of the percent of effector-memory CD8+T cells that tested positive for producing IFN-gamma. PBMC samples of subjects vaccinated with Ad / Gag-ILIODE vs. Ad / Gag (Figure 5A, Experiment 2), or Ad / Gag-cILlO vs. Ad / Gag (Figure 5B, Experiment 1), were incubated with a Gag69 supertope peptide that is known to be bound by Mamu-E. The peptide sequence used in the experiment was KCVRMYNPTNILDVK (SEQ ID NO: 42). which contains the core 9mer, RMYNPTNIL (SEQ ID NO: 43), that is presented to T cells on Mamu-E.

[0025] Figures 6A-6B depict quantifications of the percent of CD4+T cells that tested positive for producing IFN-gamma following vaccination with Ad / Gag-ILIODE vs. Ad / Gag (Figure 6A, Experiment 2), or Ad / Gag-cILlO vs. Ad / Gag (Figure 6B, Experiment 1), when those cells are incubated with the Gag69 peptide presented on Mamu-E.

[0026] Figure 7 depicts quantification of the fraction of IFN+ and / or TFN+ T cells after incubation with stimulatory peptides (either Gag pool covering the entire vaccine antigen or Gag69), as measured by flow cytometry, in the presence or absence of inhibitory peptides. The CLIP inhibitory peptide has affinity for MHC class II molecules and so blocks T-cell responses to peptides displayed on class-II molecules. Since both samples in the second and third columns are stimulated in the presence of CLIP, the responding T cells are responding to peptides displayed on class la or class lb (Mamu-E, HLA-E) molecules. In the second column class Ib-restricted (Mamu-E-restricted) responses are additionally blocked. The increase in fraction of responding cells from column 2 to column 3 is therefore a measurement of T cells responding to stimulatory peptides bound to the MHC class lb molecule, Mamu-E. Because the Gag69 peptide used for stimulation in columns 4 and 5 is presented on Mamu-E, the VL9 peptide is used for blocking in column 4 and the increase from column 4 to column 5 shows that T cells are responding to the Gag69 peptide displayed on the surface of Mamu-E. Macaques were vaccinated with Gag and macrophage / monocyte-suppressive IL-10DE in Experiment 2, then tested 12 weeks after vaccination. T cells either underwent no stimulation (first column), or stimulation with Gag-pool peptides (columns 2-3) or with the Gag69 peptide displayed on the surface of Mamu-E (columns 4-5). The top, middle, and bottom row each represent a different animal subject that underwent vaccination.

[0027] Figure 8 depicts quantification of IL- 10 receptor activation in the presence of varying amounts and ratios of native (endogenous) IL-10 and IL-1 ODE. Receptor activation and downstream signaling were read out using HEK-Blue IL- 10 cells, which secrete alkaline phosphatase in response to IL- 10 receptor activation. The data show that IL- 1 ODE did not inhibit native (endogenous) IL- 10 signaling even at the high concentrations tested (1.7 or 8.5 pg / mL).

[0028] Figure 9 depicts a non-limiting example timetable of a third experiment (Experiment 3), as outlined in Example 4 below, of vaccinating three groups of macaques (n=3 per group). Group A macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene (SEQ ID NO: 3) packaged into LNPs at weeks 0 and 4 of the experiment. Group B macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene and 25 mcg of mRNA expressing conventional macaque IL- 10 (“cIL-10’‘, SEQ ID NO: 4) at weeks 0 and 4. Group C macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene and 25 mcg of mRNA expressing the macrophage / monocyte-suppressive IL-10 variant, IL-10 DE (SEQ ID NO: 5) at weeks 0 and 4. Blood samples were thencollected and T-cell responses analyzed before immunization and on days 14, 28, 35, 42, 56, 70, and 84.

[0029] Figures 10A-10B depict quantification of TNF alpha production (Figure 10A) and the CD8+: CD4+ratio of T cells (Figure 10B) 14 days since macaques were first vaccinated following the protocol depicted in Figure 9. PBMC were stimulated with overlapping Gag peptides before assessment of cytokine production. Quantification was performed using a cytokine flow cytometry assay.

[0030] Figure 11 depicts quantification of the surface markers (CD69 and CD137) in CD8+T cells 14 days since macaques were first vaccinated following the protocol depicted in Figure 9. PBMC were stimulated with overlapping Gag peptides before testing the extent of CD69 and CD137 up-regulation on the cell surface, indicating activation.

[0031] Figure 12 depicts quantification of the IFN-positive CD8+splenic T cells in mice after stimulation with overlapping Gag peptides. Three groups of mice were immunized with (A) mRNA encoding the SIV Gag protein only (SEQ ID NO: 3) (2 mcg injected into the tibialis anterior muscle), (B) mRNA encoding SIV Gag and mRNA encoding conventional IL-10 (SEQ ID NO: 4) (2 mcg + 1 mcg), or (C) mRNA encoding SIV Gag and mRNA encoding the macrophage-specific suppressive variant, IL- 1 ODE (SEQ ID NO: 5). The mRNA / LNP vaccines were injected on days 0 and 28 of the experiment; the mice were sacrificed on day 56 and harvested splenocytes analyzed by cytokine flow cytometry.

[0032] Figure 13 depicts quantification of alkaline phosphatase production by the HEK-Blue IL-10 cell line, as a measurement of proper B-macaqueIL-10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD (SEQ ID NO: 28) folding and secretion by Expi 293 cells that were used to produce the protein.

[0033] Figures 14A and 14B depict the selectively of B-macIL-10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD (SEQ ID NO: 28) for monocy tes over CD3+, CD4+, or CD8+T cells. Macaque peripheral blood mononuclear cells (PBMC) were treated with purified cellular IL-10 or its variants (i.e. conventional IL-10 [cIL-10], IL-10DE, or B- macIL- 10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD) for 10 minutes. Cells were then stained with fluorescent antibodies specific for phospho-STAT3 (Figure 14A) or phospho- STAT1 (Figure 14B). The amount of anti-phospho-STAT3 or -STAT1 binding as revealed in the mean fluorescence intensity (MFI) then indicated the amount of signaling occurring in the cells. The MFIs were normalized to the MFIs seen after stimulation with cIL-10.IL-10DE demonstrated minor specificity for HLA-DR+Monocytes as judged based on STAT3 phosphorylation (Figure 14A; higher bar for HLA-DR+Monocytes than for T cells) but substantial specificity based on STAT1 phosphorylation (Figure 14B). B-macIL-10- (GGGGS (SEQ ID NO: 44))3-IL-10R2ECD demonstrated superior specificity for monocytes as judged either by STAT3 (Figure 14A) or STAT1 (Figure 14B). In both cases, the ratio of monocyte-to-T-cell signaling is greater for B-macIL-10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD than for IL-10DE. B-macIL-10-(GGGGS (SEQ ID NO: 44))3-IL- 10R2ECD demonstrated a maximum selectivity of 4.36X (pSTATl normalized MFI for HLA-DR+Monocytes is 4.36X higher than for CDS T cells) while IL-10DE demonstrated a maximum selectivity of only 1.18X (pSTATl normalized MFI for HLA-DR+Monocytes vs. CD4+T cells).

[0034] Figures 15A, 15B, and 15C depict the assessment of IL-10 variants having reduced ability to associate with IL-10R2 due to deletion of key amino-acid residues involved in the interaction: cIL-10ndell3, cIL-10ndel!4, cIL-10del!4, cIL-10dell8, cIL- 10del25, IL-10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD, cIL-1 OshiftCdel 14, cIL- 1 OshiftCdel 18, and cIL-10shiftCdel25. Optimized expression constructs were designed that code for these variants, often in fusion with six histidine residues (6xHis) to facilitate purification. The resulting plasmids were transfected into Expi293F producer cells and the secreted proteins tested for the ability to signal to HEK IL-10 Blue cells (which respond to IL- 10 by producing alkaline phosphatase). Figure 15A shows that cIL-10dell4 and cIL- 1 Odel 18 variants, which carry deletions of the single amino acids Hl 4 or N18, respectively, both retain the ability to signal despite the fact that these residues are both involved in crucial interactions with IL-10R2. These deletion variants can therefore be used to optimally signal to monocytes and macrophages. cIL-10ndel!3. cIL-10ndel!4, and cIL- 10del25 all failed to signal to HEK IL- 10 Blue cells, however, indicating severe compromise and lack of utility. Figure 15B depicts repeat testing of the novel variants cIL- 10ndell4, cIL-10dell4, cIL-10dell8, and cIL-10del25, using the same methods as in Figure 15A. This testing confirmed the signaling ability of cIL-10dell4 and cIL-10dell8, as well as the failure of cIL-10ndell4 and cIL-10del25. Figure 15C shows repeat testing of some of the above variants while adding evaluation of '‘shiftC” variants that move the cysteine at position 12 backward in the IL-10 sequence. The results demonstrate that cIL- 1 OshiftCdel 14 and cIL-1 OshiftCdel 18 both retain capacity for signaling, which appears similar in magnitude to that of the parental variants, cIL-10dell4 and cIL-10dell8. cIL- 10shiftCdel25 fails to signal HEK IL-10 Blue cells.

[0035] Figure 16 depicts quantification of the fraction of IFN+ and TFN+ T cells after stimulation with the Gag69 peptide that is presented by Mamu-E. as measured by flow cytometry, following booster vaccination with type-5 adenovirus expressing Gag69 fused to the beta-2-microglobulin and Mamu-E heavy chain sequences from macaques (“single-chain trimer’" or SCT; SEQ ID NOs.: 47-48), as described in Example 7. The results show that exposure to MHC class Ib-based SCTs can expand T cells responding to MHC class Ib-restricted peptides such as Gag69. The top and bottom row each represent a different animal subject that underwent vaccination.

[0036] Figure 17 depicts a non-limiting example timetable of a fifth experiment (Experiment 5), as outlined in Example 7 below, of vaccinating six groups of macaques (n=5 per group) with combinations of IL-10 and IL-15 as adjuvants. Group A macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene packaged into LNPs at weeks 0 and 4 of the experiment. Group B macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene and 25 mcg of mRNA expressing conventional macaque IL- 10 (“cIL-10”) at weeks 0 and 4. Group C macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene and 25 mcg of mRNA expressing the macrophage / monocyte-suppressive IL-10 variant, IL-10 DE at weeks 0 and 4. Group D macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene and 25 mcg of mRNA expressing receptor-linker IL- 15 (“rlIL-15”) at weeks 0 and 4. rlIL-15 is a fusion protein of IL- 15 to the IL- 15 alpha receptor, which facilitates appropriate deli ven- of the IL-15 signal. Group E macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene, 12.5 mcg of mRNA expressing cIL-10 variant, and 12.5 mcg of mRNA expressing rlIL-15 at weeks 0 and 4. Group F macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene; 12.5 mcg of mRNA expressing the macrophage / monocyte-suppressive IL-10 variant, IL-10 DE; and 12.5 mcg of mRNA expressing rlIL-15 at weeks 0 and 4. Blood samples were then collected and T-cell responses analyzed before immunization and on days 14, 28, 35, 42, 56, 70, and 84.

[0037] Figure 18 presents the annotated protein sequence of IL-10R2 and its domains.

[0038] Figures 19A-19D depict nonlimiting example schematics of singlechain trimer sequences. Figure 19A shows the sequences and corresponding domains for EVDPIGHLYmagea3.hlaa0101.SCT (SEQ ID NO: 52), Figure 19B shows the sequences and corresponding domains for LAMPFATPMnyesol.hlacw0304.SCT (SEQ ID NO: 53), Figure 19C shows the sequences and corresponding domains forMPFATPMEAELnyesol.hlab3501.SCT (SEQ ID NO: 54), and Figure 19D shows the sequences and corresponding domains for FATPMEAELnyesol.hlacw0304.SCT (SEQ ID NO: 55).DETAILED DESCRIPTION

[0039] In the Summar\' Section above, the Detailed Description Section, and the claims below, reference is made to particular features of the invention. It is to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments of the invention, and in the invention generally.

[0040] Some embodiments of the present disclosure relate to IL- 10 variants, and their use in regulating activity' in monocytes, macrophages, and the immune system generally. Several features of the present disclosure include: (i) use of vaccine adjuvants that drive macrophage-suppressive STAT3 signaling to expand inflammatory T-cell responses, (ii) novel IL- 10 variants that provide macrophage / monocyte-specific suppression, (ii) use of such adjuvants in combination with IL-15 or with single-chain trimers, and (iii) expansion of T cells that are restricted by class-Ib MHC molecules.

[0041] Several advantages of the use of the variants described herein may include one or more of: (i) active reduction in vaccine reactogenicity / toxicity in association with greater adaptive immune responses, (ii) development of a greater quantity of antigen- or cancer-specific T cells that secrete inflammatory cytokines such as IFN-gamma, and (iii) development of T cells capable of IFN-gamma secretion. In some embodiments, the T cells are restricted by MHC class-Ib molecules such as HLA-E.

[0042] Previous approaches to expanding inflammatory T cells generally activate inflammatory pathways in the immune system, but can also be associated with toxicity. For example, monophosphoryl lipid A (MPL) is a detoxified LPS derivative that activates TLR4 and thereby increases vaccine responses, including T-cell responses. However, MPL works via activation of multiple immune cells (predominantly those expressing TLR4) and thus can cause temperature elevation or cytokine secretion. Similarly, co-expression of IL- 12 works as an adjuvant by directly activating T cells and causing IFN-gamma secretion. Suppression of macrophages via STAT3 signaling, incontrast, augments pathogen-specific or cancer-specific immune responses without nonspecific inflammatory effects — thus reducing the toxicity that can be associated with T-cell activation.

[0043] Some embodiments of the present disclosure relate to a fusion protein comprising: an IL- 10 or a variant IL- 10, and a segment of IL-10R2. In some embodiments, a variant of IL-10 is a sequence having at least 80. 85, 90, 95, 99, or 100% identity to SEQ ID NO: 49. Also disclosed herein is a protein variant of human IL-10, wherein the variant comprises a deletion of an amino acid residue contributing to interaction with IL-10R1 or IL- 10R2, werein the deletion of at least one amino acid is at or corresponding with an amino acid at position 14, 18, 21, 22, 24, 28, 74, 90, 92, 96, 100, 104, 108, or any combination thereof, of SEQ ID NO: 49; and wherein the variant retains signaling function. In some embodiments, there is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 deletions at positions 14, 18, 21, 22, 24, 28, 74, 90, 92, 96, 100, 104, 108, or any combination thereof of SEQ ID NO: 49. In some embodiments, the variant IL- 10 is the protein variant of any one of the embodiments of the present disclosure. Also disclosed herein is a fusion protein comprising any one of the fusion proteins and / or protein variants of the present disclosure, and a segment of human serum albumin and / or a segment of an Fc domain. In some embodiments, the protein has a specificity for signaling to myeloid cells versus for signaling to T cells, calculated as the ratio between signaling activity to myeloid cell types and signaling activity to T cells, wherein the specificity for signaling to myeloid cells is greater than the specificity of cellular IL-10 (cIL-10) for signaling to myeloid cells. In some embodiments, the myeloid cells receiving IL- 10 signal are at least one of: monocytes, macrophages, plasmacytoid dendritic cells (pDCs), or any combination thereof. In some embodiments, the protein variant is immunosilent. In some embodiments, the variant comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any one of the sequences of SEQ ID NOs. : 1 -48. In some embodiments, the protein comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO. 49. In some embodiments, the segment is on the C-terminal end. In some embodiments, the segment is on the N-terminal end. In some embodiments, the IL-10R2 segment comprises the extracellular domain of IL-10R2, the signal sequence of IL-10R2, loop 2 residues Y59- K65, loop 3 residues S80-Y87, loop 5 residues W143-N152, full-length IL-10R2, or any combination thereof. In some embodiments, the protein variant or fusion protein further comprises a linker sequence, optionally wherein the linker sequence is operably located between the first fusion partner and the second fusion partner. In some embodiments, thelinker sequence is comprised of glycine and serine residues. In some embodiments, at least one of the amino acids at or corresponding with positions H14 or N18 of SEQ ID NO: 49 are deleted or mutated. In some embodiments, the amino acid at or corresponding with position R104 of SEQ ID NO: 49 is deleted or mutated, and wherein at least one of the amino acids at or corresponding with positions H14 or N18 of SEQ ID NO: 49 are deleted or mutated.

[0044] Also disclosed herein is a nucleotide sequence encoding the fusion protein or protein variant of any one of the embodiments of the present disclosure. In some embodiments, the nucleotide sequence comprises DNA and / or RNA.

[0045] Also disclosed herein is a nucleotide sequence encoding a protein variant of human IL- 10. wherein the nucleotide sequence comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any one of the sequences of SEQ ID NOs.: 6- 40.

[0046] Also disclosed herein is a delivery vector comprising the nucleotide sequence of any one of the embodiments of the present disclosure. In some embodiments, the delivery vector is a viral vector or nanoparticle. In some embodiments, the viral vector is a lentiviral vector, retroviral vector, adenoviral vector, or any combination thereof. In some embodiments, the delivery vector further comprises at least one internal ribosomal entry site (IRES) or at least one sequence coding for a self-cleaving peptide.

[0047] Also disclosed herein is a vaccine comprising the delivery vector of any one of the embodiments of the present disclosure. Also disclosed herein is a vaccine encoding the protein variant of any one of the embodiments of the present disclosure, and / or the nucleotide sequence of any one of the embodiments of the present disclosure. In some embodiments, the vaccine is an mRNA. adenovirus, alphavirus, or lentivirus platform, or any combination thereof.

[0048] Also disclosed herein is a cell comprising the protein variant of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, or any combination thereof.

[0049] Also disclosed herein is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the protein variant of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, the vaccine of any one of the embodiments ofthe present disclosure, the cell of any one of the embodiments of the present disclosure, or any combination thereof. In some embodiments, the disease or disorder is an infection, an autoimmune disease, an allergic disease or reaction, an inflammatory disease, a cancer, a tumor, or any combination thereof. In some embodiments, the disease or disorder is a chronic infection, optionally wherein the chronic infection is HIV or hepatitis.

[0050] Also disclosed herein is a use for the protein variant of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, the vaccine of any one of the embodiments of the present disclosure, or the cell of any one of the embodiments of the present disclosure, in the preparation of a medicament.

[0051] Also disclosed herein is a method of stimulating an immune cell, the method comprising administering to the immune cell the protein variant of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, or the vaccine of any one of the embodiments of the present disclosure. In some embodiments, stimulating the immune cell comprises increasing the total number of activated T cells, and / or increasing the production of IFN-gamma and / or TNF -alpha. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the immune cell is a T cell, a B cell, or an NK cell. In some embodiments, the T cell is a CD4+ or a CD8+T cell. In some embodiments, the T cell is an MHC class Ib-restricted T cell.

[0052] Also disclosed herein is a method of inhibiting IL-1 activity, NLRP1 inflammasome activation, NLRP3 inflammasome activation, type 1 interferon production (IFN-I) production, IL-6 production, or any combination thereof in vivo, the method comprising administering the protein variant of any one of the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, or the vaccine of any one of the embodiments of the present disclosure. In some embodiments, the method further comprises administering an at least one booster vaccine. In some embodiments, the at least one booster vaccine encodes or comprises single-chain trimers and / or IL-15.

[0053] Also disclosed herein is a method of vaccinating a subject in need thereof, the method comprising administering to the subject the protein variant of any oneof the embodiments of the present disclosure, the nucleotide sequence of any one of the embodiments of the present disclosure, the delivery vector of any one of the embodiments of the present disclosure, or the vaccine of any one of the embodiments of the present disclosure. In some embodiments, the vaccination results in: (1) increasing the total number of activated T cells, (2) increasing the production of IFN-gamma and / or TNF-alpha in the subject, (3) increasing the frequency of cells in the subject that can produce IFN-gamma and / or TNF-alpha after stimulation with an antigen, or (4) any combination of (l)-(3) thereof. In some embodiments, the vaccination results in reduced side effects as compared to vaccination in absence of the protein variant of any one of the embodiments of the present disclosure. In some embodiments, the subject is mammalian and / or human. In some embodiments, the vaccination is a booster vaccination. In some embodiments, the vaccination is performed on the subject more than once.

[0054] Disclosed herein are vaccines that express monocyte- and / or macrophage-suppressing molecules to promote inflammatory T-cell functions, which may include one or more of (i) development of a greater quantity of antigen- or cancer-specific T cells that secrete inflammatory cytokines such as IFN-gamma or TNF-alpha, and (ii) development of T cells capable of IFN-gamma secretion that respond to MHC class-Ib- restricted “supertopes.” Also disclosed herein are IL- 10 variants. In some embodiments, the IL-10 variants are macrophage-suppressive and / or immunosilent. In some embodiments, the vaccine comprises an IL- 10 variant. In some embodiments, the IL- 10 variant comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any one of the sequences of SEQ ID NOs.: 1-48. In some embodiments, the vaccine comprises at least one of: IL- 10, an IL- 10 variant, an antigen, or any combination thereof. In some embodiments, the vaccine comprises a nucleic acid construct that encodes an IL- 10 variant of any one of the embodiments of the present disclosure. In some embodiments, the nucleic acid construct is an mRNA sequence. In some embodiments, the nucleic acid construct is a DNA sequence. In some embodiments, the vaccine comprises an adjuvant that comprises or expresses monocyte- or macrophage-suppressing molecules. In some embodiments, the vaccine comprises an IL-10 variant that has function as an adjuvant (i.e. boosts the effectiveness of the vaccine).

[0055] In some embodiments IL-10 variants are provided. In some embodiments, the IL-10 variant is derived from wild type IL-10, such that it has 1, 2, or 3 of: (1) immunosilence. (2) increased suppression of monocytes or macrophages; and / or (3) increased immune system activation compared to wildtype IL-10. In some embodiments,“immunosilence'’ comprises an incapability to elicit a host immune response due to inclusion of only (i) host sequences and / or (ii) linker sequences that comprise exclusively smaller amino acids and exclude MHC-binding subsequences. In some embodiments, the IL-10 variant comprises 1, 2, 3, or 4 of: (1) an N-terminal deletion, wherein the deletion is at least 5, 10, 13, 15, 18, 21, 22, or 25 amino acids; (2) a first fusion partner, wherein the first fusion partner comprises a segment of human serum albumin and / or a segment of an Fc domain; (3) a second fusion partner, wherein the second fusion partner comprises a segment of IL-10R2; and / or (4) at least one deletion or mutation of an amino acid residue capable of interacting with IL-10R2. In some embodiments, the amino acid deletion or mutation is at or corresponds with position 12, 14, 18, 21, 22, 24, 90, 92, 96. 104, 108, or any combination thereof of SEQ ID NO: 49. In some embodiments, the variant comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any one of the sequences of SEQ ID NOs. : 1 -48. In some embodiments, the variant comprises a mutation to at least one residue selected from the group consisting of: C12, H14, N18, N21, M22, Cl 08, or any combination thereof. In some embodiments, the variant comprises a C- or N-terminal fusion partner. In some embodiments, the fusion partner comprises a fragment, domain, or full-length sequence of: human serum albumin, an Fc region of an antibody, IL-10R2, or any combination thereof. In some embodiments, the variant comprises the extracellular domain of IL-10R2, the signal sequence of IL-10R2, loop 2 (Y59-K65). loop 3 (S80-Y87), loop 5 (W143-N152), full-length IL-10R2, or any combination thereof. In some embodiments, the variant comprises a linker sequence. In some embodiments, the linker sequence is between the first fusion partner and the second fusion partner. In some embodiments, the linker sequence is GGGGS (SEQ ID NO: 44), GGGGSGGGGS (SEQ ID NO: 45), or GGGGS GGGGS GGGGS (SEQ ID NO: 46). In some embodiments, the variant comprises a fragment, domain, or full-length sequence of an antibody. In some embodiments, the variant comprises a fragment, domain, or full-length sequence of the Fc region of an antibody. In some embodiments, the variant comprises a fragment, domain, or full-length sequence of a single-domain antibody.

[0056] In some embodiments, constructs are provided that encode one or more variants. In some embodiments, the construct encodes an IL-10 variant of any one of the embodiments of the present disclosure. In some embodiments, the construct comprises a bicistronic construct, the bicistronic construct further comprising coding sequences for an IL- 10 variant and an antigen. In some embodiments, the construct comprises a bicistronic construct, the bicistronic construct further comprising coding sequences for an IL-10variant and an immunogen. Non-limiting examples of such immunogens are HIV Gag; SIV Gag; composite HIV T-cell immunogens that comprise multiple HIV proteins, which are optionally fused into a single protein; CMV pp65; other CMV proteins; EBV proteins; KSHV proteins; other herpesvirus proteins; SARS-CoV-2 proteins; tumor-associated antigens or epitopes derived therefrom; and tumor neoantigen or epitopes derived therefrom. In some embodiments, the Gag-ILIO bicistronic construct comprises monocyte / macrophage-specific IL- 1 ODE or IL-10-(GGGGS)3-IL-10R2ECD. In some embodiments, the Gag-ILIO bicistronic construct comprises conventional IL-10 (i.e., normal macaque or human IL-10). In some embodiments, the Gag-ILIO bicistronic construct comprises any one of the IL- 10 variants of the present disclosure. In some embodiments, the Gag-ILIO bicistronic construct comprises an IL- 10 variant comprising 1, 2, 3, or 4 of (1) an N-terminal deletion, wherein the deletion is at least 5, 10, 13, 15, 18, 21, 22, or 25 amino acids; (2) a first fusion partner, wherein the first fusion partner comprises a segment of human serum albumin and / or a segment of an Fc domain; (3) a second fusion partner, wherein the second fusion partner comprises a segment of IL-10R2; and / or (4) at least one deletion or mutation of an amino acid residue capable of interacting with IL-10R2, wherein the amino acid is at or corresponds with position 12, 14, 18, 21, 22, 24, 25, 90, 92, 96, 104, 108, or any combination thereof of SEQ ID NO: 49.

[0057] In some embodiments, a composition is provided. In some embodiments, the composition comprises an isolated antigen and / or immunogen. In some embodiments, the composition comprises an IL-10 variant. In some embodiments, the composition comprises the IL-10 variant, and an isolated antigen and / or immunogen.

[0058] In some embodiments, a vaccine in provided. In some embodiments, the vaccine is an inactivated vaccine, live-attenuated vaccine, messenger RNA (mRNA) vaccine, subunit, recombinant, polysaccharide, or conjugate vaccine; toxoid vaccine, or viral vector vaccine. In some embodiments, the vaccine is an adenovector vaccine. In some embodiments, the vaccine comprises at least one protein or nucleotide construct. In some embodiments, the vaccine comprises a protein or nucleotide construct that encodes an IL- 10 or an IL- 10 variant. In some embodiments, the IL- 10 or IL- 10 variant acts as an adjuvant. In some embodiments the adjuvant is a construct encoding an IL- 10 variant. In some embodiments, the vaccine further comprises an at least one further adjuvant. In some embodiments, the adjuvant is selected from: (1) a cytokine, such as IL-15 or IL-12, or a cytokine variant having at least 80% identity with the native cytokine; (2) a lipid organic or inorganic nanoparticle; (3) a salt comprising aluminum; (4) an oil-in-water emulsionsuch as 1MF59. AS03, or Montanide ISA; (5) a saponin-based adjuvant such as Matrix-M or QS-21; (6) 3M-052, AS01, Freund’s adjuvant, or GLA-LSQ; (7) a TLR agonist, such as CpG, Poly I:C, imiquimod, resiquimod, or flagellin; (8) or any combination thereof.

[0059] In some embodiments, the adjuvant is capable of driving macrophagesuppressive STAT3 signaling to expand inflammatory T-cell responses in a subject. In some embodiments, the subject is mammal and / or human. In some embodiments, the adjuvant is combined with IL- 15 or with at least one single-chain trimer. Nonlimiting examples of single-chain trimers include any one of SEQ ID NOs: 52-55, or a sequence having at least 80% identity with any one of SEQ ID NOs: 52-55.

[0060] Also disclosed herein are methods of administering a vaccine. In some embodiments, the vaccine is administered through injecting a mammal and / or human subject. Also disclosed herein are methods of immunizing a subject by administering the vaccine. In some embodiments, the vaccine comprises an antigen, a wild type IL 10, an IL10 variant of any one of the embodiments of the present disclosure, or any combination thereof. In some embodiments, the vaccine comprises a nucleotide construct encoding the wild type or variant IL 10. In some embodiments, the antigen is encoded by a nucleic acid construct. In some embodiments, the antigen and the IL 10 and / or IL 10 variant is encoded in the same construct. In some embodiments, the antigen and the IL 10 and / or IL 10 variant are encoded in different constructs. In some embodiments, following administration, the subject expresses the antigen, IL 10. variant IL- 10, or any combination thereof in vivo. In some embodiments, an immune cell of the subject expresses the antigen, IL10, variant IL- 10, or any combination thereof following vaccination. In some embodiments, the vaccine is administered intravenously, intramuscularly, intradermally, or subcutaneously. In some embodiments, the vaccine is administered orally, intranasally, or by inhalation. In some embodiments, the vaccine is combined with a pharmaceutical carrier. In some embodiments, the vaccine comprises a composition that is formulated for its method of administration to the subject. In some embodiments, the vaccine is administered to the subject at least once. In some embodiments, the vaccine is administered to the subject at least 2, 3, or 5 times. In some embodiments, the vaccine is administered as a booster vaccine. In some embodiments, the vaccination results in increasing the total number of activated T cells, and / or increasing the production of IFN-gamma and / or TNF-alpha in the subject. In some embodiments, T cell and / or immune activity are monitored in a subject, and the vaccine is administered when the T cell and / or immune activity is below a threshold.

[0061] The previous known approach to reliably expanding T cells capable of IFN-gamma secretion that respond to supertopes and are restricted by MHC class-Ib molecules such as HLA-E is vaccination with certain cytomegalovirus vectors. These vaccines have myriad off-target effects due to the expression of hundreds of cytomegalovirus genes. The approach of the present disclosure is simpler and also adaptable to multiple vaccine vector platforms including mRNA, adenovirus, alphavirus vectors, lentivectors, etc.

[0062] In some embodiments, the IL- 10 variant, construct, nucleotide, and / or vaccine have use in the preparation of a medicament, and / or in the prevention or treatment of a disease or disorder. In some embodiments, the medicament has use in boosting a vaccine's efficacy. In some embodiments, the disease or disorder is an autoimmune or inflammatory disease or disorder. In some embodiments, the disease or disorder is an allergic disease or reaction. In some embodiments, the disease or disorder is a cancer and / or a tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the disease or disorder is at least one of an infectious disease, cancer, a tumor, or allergic reaction. Non-limiting examples of infectious diseases or disorders include: HIV and the resulting AIDS, CMV, EBV, KSHV, HSV-1, HSV-2, malaria, tuberculosis, dengue, Zika, Chikungunya, Lassa, Nipah, and Marburg. Non-limiting examples of cancers or tumors include those originating from any cell, tissue, or system type, such as prostate, breast, lung, and leukemia. Non-limiting example of antigens causing an allergic reaction include pollen, dust mite antigens, pet dander, and insect-sting antigens.

[0063] Some embodiments of the present disclosure present a method of treating a subject in need thereof, the method comprising administering the IL- 10 variant, construct comprising the IL- 10 variant, cell comprising the IL- 10 variant, and / or vaccine comprising the IL-10 variant, to the subject. Also disclosed herein are formulations comprising IL-10, such as a composition formulated as part of a vaccine.

[0064] Also disclosed herein is a method of activating an immune cell by contacting the immune cell with the IL- 10 variant of any one of the embodiments of the present disclosure. In some embodiments, the immune cell is a myeloid cell or a T cell. In some embodiments, the immune cell is a CD4+ or CD8+ T cell. In some embodiments, contacting the immune cell with the IL- 10 variant of any one of the embodiments of the present disclosure results in an increase in IFN-gamma and / or TNF-alpha production. In some embodiments, contacting the immune cell with the IL-10 variant of any one of the embodiments of the present disclosure results in increasing the total number of activatedimmune cells. In some embodiments, contacting the immune cell with the IL- 10 variant of any one of the embodiments of the present disclosure results in increasing the total number of activated T cells.

[0065] In the present disclosure, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in this disclosure, including the drawings and claims, are not meant to be limiting. Some embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.Terms

[0066] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0067] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0068] The articles “a’' and “an’" are used herein to refer to one or to more than one (for example, at least one) of the grammatical object of the article, unless the context dictates otherwise. By way of example, ‘‘an element” means one element or more than one element.

[0069] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is approximately the recited value. Where it is not clear from the context what is encompassed by “about,” it will mean the value recited + / - 10%.

[0070] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises.” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meantincluding, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0071] The terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and mean a human or a non-human mammal, e.g.. a dog, a cat, a mouse, a rat, a cow, a sheep, a pig. a goat, a non-human primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. The term “mammal” is used in its usual biological sense. Thus, it includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0072] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and / or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%. about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, a substance that is “isolated” may be “pure” (e g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multi-cellular organism or tissue.

[0073] As used herein, “in vivo” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method insideliving organisms, usually plants, animals, and mammals, including humans, mice, and monkeys, as opposed to a tissue extract or dead organism.

[0074] As used herein, “ex vivo” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside a living organism with little alteration of natural conditions.

[0075] As used herein, “in vitro” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.

[0076] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, earner, vehicle, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzy me-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry’, infrared spectrometry, mass spectrometry', nuclear magnetic resonance, gravimetry, or titration, or any7combination thereof.

[0077] The terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to that amount of a recited composition or compound that results in an observable effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity’ of the composition, formulation, route ofadministration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0078] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to a biological, enzymatic, or therapeutic function.

[0079] The term “inhibit” as used herein has its plain and ordinary meaning as understood in light of the specification, and may refer to the reduction or prevention of a biological activity. The reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected. The delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. 100%, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay may not necessarily indicate a 100% inhibition or delay. A partial inhibition or delay may be realized.

[0080] As used herein, the terms “treating” or “treatment” have their plain and ordinary meaning as understood in light of the specification, and refer to an approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (e.g., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the recurrence of disease, and remission, whether partial or total and whether detectable or undetectable. “Treating” and “treatment” as used herein also include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may include a single administration or may include a series ofadministrations. The compositions are administered to the subject in an amount and for a duration sufficient to treat the subject. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the subject, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.

[0081] “Tumor,"’ as used herein, has its plain and ordinary meaning as understood in light of the specification, and refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. The term “neoplasia” encompasses the term tumor.

[0082] The terms “cancer” and “cancerous” have their plain and ordinary meanings as understood in light of the specification, and refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include lung cancer including small-cell lung cancer, non-small cell lung cancer and lung adenocarcinomas with neuroendocrine features; neuroendocrine prostate cancer, melanoma, gliomas, low-grade gliomas and glioblastoma, medullary' thyroid cancer, carcinoid tumors, neuroendocrine tumors in the pancreas, bladder cancer, testicular cancer squamous cell cancer (e.g. epithelial squamous cell cancer), neuroendocrine neoplasms, such as neuroendocrine tumors of unknown primary, neuroendocrine neoplasms of the small bowel, carotid body, adrenal gland, colorectal gy necological organ, abdomen, esophagus, GI tract, bile duct, ner ous system, appendix, liver, anal, thymus, ileocecal junction, head and neck, breast, peritoneum and retroperitoneum, kidney, thyroid, stomach, bone,; adenocarcinomas, such as adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma,sali vary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, bone cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases. The term cancer includes adult and pediatric solid cancers. In some embodiments, the cancer can be a solid tumor. In some embodiments, the cancer is a highly fibrotic tumor or cancer. In some embodiments, the cancer is a desmoplasia.

[0083] As used herein, the term "therapeutic target" has its plain and ordinary meaning as understood in light of the specification and refers to a gene or gene product that, upon modulation of its activity (e.g., by modulation of expression, biological activity, and the like), can provide for modulation of the disease phenotype. As used throughout, "modulation" is meant to refer to an increase or a decrease in the indicated phenomenon (e.g., modulation of a biological activity refers to an increase in a biological activity or a decrease in a biological activity).

[0084] The term “immunosilent’' has its plain and ordinary meaning as understood in light of the specification and refers to a substance such as a molecule, nucleotide, peptide, or cell, that does not induce an immune response. In some embodiments, an immunosilent compound does not elicit a host immune response. In some embodiments, “immunosilence” comprises an incapability to elicit a host immune response due to inclusion of only (i) host sequences and / or (ii) linker sequences that comprise exclusively smaller amino acids and exclude MHC-binding subsequences. A non-limiting example of an immunosilent construct includes one consisting of host sequences and linker sequences, where the latter are not presented on MHC molecules.

[0085] The term “antigen” has its plain and ordinary meaning as understood in light of the specification, and refers to a foreign substance recognized by the immune system. The term “immunogen” has its plain and ordinary meaning as understood in light of the specification, and refers to an antigen capable of inducing an immune response.

[0086] The terms “variant” or “derivative” have their plain and ordinary meaning as understood in light of the specification, and refer to a substance that is altered compared to a parent substance. In some embodiments, it retains the same properties as the parent. In some embodiments, it has different properties compared to the parent. In some embodiments, the derivative or variant comprises non-naturally occurring amino acid residues. A non-limiting example of a variant or derivative is an IL-10 derivative, in which at least one amino acid residue and / or chemical group are modified, mutated, inserted, and / or deleted. In some embodiments, the IL- 10 derivative is a non-natural derivative. Insome embodiments, a variant or derivative has at least 70, 80, 85, 90, 95, or 99% identity with the parent. In some embodiments, a variant or derivative has less than 99, 95, 90, 85, 80, or 70% identify with the parent.

[0087] The term “administering” includes oral administration, inhaled administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intra-tumoral, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini- osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intra-tumoral, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, nebulized formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein.

[0088] As used herein, “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meanings as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory7agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or carrier can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical formulation is administered. Such pharmaceutical diluent, excipient, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers,particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include sugars, starch, glucose, fructose, lactose, sucrose, maltose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, salts, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also include one or more of the following: antioxidants, such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids; carbohydrates such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as glycerol, erythritol, threitol. arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol. fucitol, iditol, inositol, isomalt, maltitoL or lactitol; salt-forming counterions such as sodium; and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The formulation, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These formulations can take the form of solutions, suspensions, emulsions, sustained release formulations and the like. The formulation should suit the mode of administration.

[0089] The terms “first,” “second,” and “third” used in combination with substances are intended to designate distinguishable features to similar substances and do not imply any particular order unless otherwise specified.

[0090] The term “antibody” is used in the broadest sense and includes various antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies, antibody fragments, and any other constructs that retain antigen-binding activity. In some embodiments, antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. The VH region and / or the VL region are responsible for binding the antigen recognized by the antibody. The term antibody includes intact immunoglobulins, as well the variants and portions thereof, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as,bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology. 3. sup. rd Ed.. W.H. Freeman & Co., New York, 1997.

[0091] As used herein, the term “gene” means nucleic acid in the genome of a subject capable of being expressed to produce a mRNA and / or protein in addition to intervening intronic sequences and in addition to regulatory regions that control the expression of the gene, e.g., a promoter or fragment thereof.

[0092] As used herein, the term “diagnosis”, and variants thereof, such as, but not limited to “diagnose” or “diagnosing” shall include, but not be limited to, a primary diagnosis of a clinical state or any primary diagnosis of a clinical state. A diagnostic assay described herein is also useful for assessing the remission of a subject, or monitoring disease recurrence, or tumor recurrence, such as following surgery, radiation therapy, adjuvant therapy or chemotherapy, or determining the appearance of metastases of a primary' tumor.

[0093] The term “control” refers to a sample or standard used for comparison with a sample which is being examined, processed, characterized, analyzed, etc. In some embodiments, the control is a sample obtained from a healthy patient or anon-tumor tissue sample obtained from a patient diagnosed with a cancer. In some embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of people with an infectious disease, or group of samples that represent baseline or normal values, such as the level or activation of white blood cells in circulation).

[0094] Although the present disclosure has been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of embodiments of the disclosure as defined in the appended claims.

[0095] The present disclosure will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the disclosure in any way.Table 1: Non-limiting Example SequencesEXAMPLES

[0096] The non-limiting example methodologies as disclosed herein are used in the following working Examples.Example 1 : Vaccines that co-express a target molecule from a pathogen with macrophagesuppressive and -selective interleukin (IL-10).

[0097] The IL- 10 molecule has previously been modified in order to produce variants that selectively induce STAT3 and STAT1 signaling in monocytes and macrophages (while minimally affecting T and / or B cells). These variants were predicted to suppress macrophage activation without stimulating inflammatory CD8+T cells (e.g., without stimulating the inflammatory T cells that are intentionally expanded by most vaccine strategies). Herein, it was reasoned that macrophage suppression by vaccines in vivo might instead promote inflammatory' T-cell functions by inhibiting IL-1 signaling effects and inhibiting NLRP1 and NLRP3 inflammasome activation, which are both associated with suppression of gene expression from genetic vaccines. Furthermore, it was speculated that monocyte- and macrophage-specific STAT3 stimulation in absence of B- cell STAT3 stimulation would prevent development of regulatory' B cells (B regs) that express TGF-beta and endogenous (host) IL- 10, which are both molecules capable of suppressing T-cell activation and cytokine secretion.

[0098] It was therefore hypothesized that, despite the established expectation that monocyte and macrophage suppression via STAT3 and STAT1 would be associated with a reduction in inflammatory T-cell functions, STAT3-driven monocyte / macrophage suppression could sustain gene expression and lead to stimulation of inflammatory CD8+T cells — reflected in superior immune responses to genetic vaccines that express the macrophage-suppressive molecules. To test the possibility', two experiments were conducted that assessed vaccination with adenovector vaccine that expressed SIV Gag alone (control, SEQ ID NO: 1), and (1) adenovector vaccine that expressed SIV Gag andmonocyte / macrophage-specific IL-1 ODE (SEQ ID NO: 2), or (2) SIV Gag and conventional IL-10 (SEQ ID NO: 58) (i.e., normal macaque IL-10). The SIV Gag gene alone ('‘Ad / Gag”) or Gag-ILIO bicistronic constructs (“Ad / Gag-cILlO” or ‘'Ad / Gag- IL10DE”) were engineered into the El region of adenoviral vectors, which were administered at weeks 0 and 4 of the experiment (Figures 1A-1B). This latter form of IL- 10, IL- 1 ODE, was selected based its specific stimulation of STAT3 signaling in monocytes / macrophages, such as THP-1 cells, and reduced STAT3 signaling in B cells, such as Daudi cells. Experiment 1 included one group receiving Ad / Gag vaccination (adenovectors expressing the Gag gene only) and another receiving Ad / Gag-cILlO vaccination (adenovectors simultaneously expressing Gag and conventional [wild-type host] IL-10). Experiment 2 included animals receiving Ad / Gag vaccination and those receiving Ad / Gag-ILIODE vaccination (adenovectors simultaneously expressing Gag and macrophage / monocyte-specific IL-1 ODE).

[0099] In Experiment 2, the number of circulating granulocytes was seen to clearly increase in blood after the priming vaccination with adenovirus (Figure 2, dashed lines). Such neutrophilia is evidence of non-specific inflammation due to stimulation of the innate immune system, which is associated with vaccine side effects (malaise and fever). Impressively, inclusion of the myeloid / macrophage-suppressive molecule in the vector eliminated these increases completely, thus demonstrating reduced toxicity (Figure 2, solid lines). Similarly, macaques receiving conventional vaccination (Ad / Gag only) manifested spikes in production of the inflammatory pyrogenic cytokine, IL-6, one day after each vaccination in the experiment (Figure 3A). Inclusion of myeloid / macrophage- suppressive IL- 10 in the vaccination (Ad / Gag-ILIODE group) lowered or eliminated the IL-6 spike in all macaques tested (Figure 3B).

[0100] Furthermore, the vaccine expressing myeloid / macrophage-suppressive IL- 10, but not conventional unmodified IL- 10, had increased capacity to promote inflammatory CD8+T-cell functions (Figures 4A-4B). Recipients of the vaccine expressing this molecule produced a higher frequency of CD8+T cells that responded to peptides from the vaccine antigen (SIV Gag) with production of the pro-inflammatory cytokines, IFN-gamma and TNF-alpha (Figure 4A). Conventional IL- 10, however, did not have a significant effect on the frequency of CD8+T cells responding to vaccination (Figure 4B).Example 2: Macrophage suppression promotes MHC class Ib-restricted T-cell functions in vivo.

[0101] Unexpectedly, the pro-inflammatory T-cell functions promoted by myeloid / macrophage-suppressive IL- 10 included the expansion of T cells recognizing “supertopes” restricted by the oligomorphic MHC molecule, Mamu-E. To determine the frequency of these cells, PBMC samples were incubated with the Gag69 supertope peptide that is known to be bound by Mamu-E (KCVRMYNPTNILDVK (SEQ ID NO: 42)). Recipients of the vaccine expressing this myeloid-biased IL-10 variant produced a higher frequency of CD8+T cells that responded to Gag69 supertope peptide with production of the pro-inflammatory cytokine, IFN-gamma (Figure 5A). Co-expression of conventional IL- 10 had no effect on Gag69 responses (Figure 5B). Similarly, the vaccine expressing macrophage-suppressive IL-10 was associated with an increased frequency of CD4+T cells that responded to Gag69 supertope peptide with production of IFN-gamma, while coexpression of conventional IL-10 had no effect (Figures 6A-6B).

[0102] To test if these responses were authentically restricted by Mamu-E, T- cell assays were performed in the presence or absence of the “VL9” peptide inhibitor, which binds to Mamu-E, thus preventing the binding of a Mamu-E binding peptide such as Gag69. The result is that VL9 addition prevents signaling to T cells that recognize other peptides in the context of Mamu-E. The following combinations of stimulatory peptides and inhibitors were employed:

[0103] (i) Gag pool (covering the whole protein) in the presence ofVL9 and CLIP, which inhibit TCR engagement with vaccine-derived peptide in the context of Mamu-E (VL9) or MHC class II (CLIP)

[0104] (ii) Gag pool (covering the whole protein) in the presence of CLIP only, inhibiting TCR engagement with vaccine-derived peptide in the context of MHC class II. Increased T-cell responses in this sample compared to one that includes VL9+CLIP allows estimation of the Mamu-E restricted response to peptides in the complete Gag pool.

[0105] (iii) Gag69 in the presence of VL9 alone, inhibiting TCR engagement with Gag69 peptide in the context of Mamu-E.

[0106] (iv) Gag69 stimulus only. Increased T-cell responses in this sample compared to one that includes VL9 allows estimation of the Mamu-E restricted response to Gag69.

[0107] The results showed that the macaques that were vaccinated with Gag and macrophage / monocyte-suppressive IL-10DE in Experiment 2, then tested 12 weeks after vaccination, had T-cells that were responsive to either Gag-pool peptides or to Gag69 in the context of Mamu-E (Figure 7). Note in this figure the increased cytokine production in absence of inhibitory VL9 peptide (column 3 vs. column 2, or column 5 vs. column 4). Samples from Experiment 1 never showed evidence of Mamu-E restriction despite repeated testing.

[0108] Reliably eliciting inflammatory T-cell responses to peptides that are restricted by class-Ib MHC molecules is a hitherto unsuspected outcome of expressing macrophage-suppressive molecules with vaccination. The only previously known vaccine modalities that reliably elicit such responses are certain rhesus cytomegalovirus-vectored vaccines. These have multiple drawbacks including greater complexity, replication in the vaccinated host, expression of multiple undesired gene products, and difficulties in manufacturing.Example 3: The macrophage / monocyte-suppressive IL-10 variant. IL-10DE. is not an IL- 10 inhibitor or functional antagonist.

[0109] The finding that the macrophage- and monocyte-specific molecule, IL- 10DE, potentiates inflammatory vaccine responses was surprising because IL- 10 activities on myeloid cells are predominantly anti-inflammatory, and because this variant was designed for specific anti-inflammatory signaling to those cells. It remained possible, however, that IL- 1 ODE was acting as a "functional antagonist’" or inhibitor of IL- 10 signaling to T cells, thus relieving them of suppressive (counter-regulatory) IL- 10 signaling that might occur normally after vaccination. Partial agonists can sometimes act as functional antagonists (inhibitors) by competing with the full agonist for receptor occupancy and producing a net decrease in the receptor activation observed with the full agonist alone.

[0110] This possibility for IL-10DE as a functional antagonist was tested by measuring IL- 10 receptor activation in the presence of varying amounts and ratios of native (endogenous) IL- 10 and IL- 1 ODE (Figure 8). Receptor activation and downstream signaling were read out using HEK-Blue IL- 10 cells, which secrete alkaline phosphatase in response to IL- 10 receptor activation. The results showed no competitive antagonism by IL- 1 ODE, regardless of the concentration of either IL- 10 or IL- 1 ODE. No submaximalsignaling from IL- 10 was produced in the presence of IL- 1 ODE at any point in the response curve. Thus, post-vaccination promotion of inflammatory T-cell functions by IL-10DE was mediated by the suppressive effects on macrophages for which it was designed, not by functional antagonism or competitive inhibition.Example 4: mRNA / LNP vaccines that co-express a target molecule from a pathogen with macrophage-suppressive and -selective interleukin (IL- 10),

[0111] Though co-expression of macrophage-suppressive IL-10 with a vaccine immunogen from adenovectors had proven advantageous (Figures 1A-7), it was unclear if similar success could be achieved using mRNA packaged into lipid nanoparticles. Two important differences in this situation include (1) the innate immune response to adenovectors creates an environment that favors adaptive immunity, including T-cell responses, so it was not clear if macrophage / monocyte suppression could have a similar effect in the setting of mRNA vaccination; and (2) vaccinating with two transcripts (one each for the immunogen and for macrophage / monocyte specific cytokine) may produce too much variant cytokine, resulting in absence of the desired effect, namely, promotion of inflammatory T-cell functions that are responding to the immunogen. The experimental design comprised three groups (n=3 macaques per group; Figure 9). Group A macaques were vaccinated with 25 mcg of mRNA expressing the S1V gag gene packaged into LNPs at weeks 0 and 4 of the experiment. Group B macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene and 25 mcg of mRNA expressing conventional macaque IL-10 ("cIL- I O") at weeks 0 and 4. Group C macaques were vaccinated with 25 mcg of mRNA expressing the SIV gag gene and 25 mcg of mRNA expressing the macrophage / monocyte-suppressive IL-10 variant, IL-10 DE, at weeks 0 and 4. Blood samples were then collected and T-cell responses analyzed before immunization and on days 14, 28, 35, 42, 56, 70, and 84.

[0112] The first results obtained (from day 14) again demonstrated the superior capacity of macrophage-suppressive and -selective interleukin (IL- 10) variants to stimulate inflammatory CD8+T cells. In the cytokine flow cytometry assay - which measured production of TNF alpha and other cy tokines in response to stimulation with Gag-derived peptides - macrophage-suppressive IL-10 DE (Group C) caused significantly greater TNF- a production than achieved either in of Groups A or B (Figure 10A). There was also significantly greater concentration of the TNF response in CD8+vs. CD4+T cells (Figure10B), demonstrating the particular utility of macrophage / monocyte suppression for stimulating inflammatory’ CD8+T cells. A second assay was also performed, that measured surface markers of T-cell activation in response to peptide stimulation, the ‘'activation- induced marker” or “AIM” assay. In agreement with the results obtained by cytokine flow cytometry’, macrophage suppression using IL-1 ODE allowed for production of significantly more inflammatory CD8+T cells responding to the vaccine immunogen in the AIM assay (Figure 11).

[0113] In a parallel study, mRNA vaccination was conducted in mice to determine the advantages of IL- 10 variants that suppress macrophages for stimulating inflammatory CD8+T-cell responses. As had been done in macaques, three groups were immunized with (A) mRNA encoding the SIV Gag protein only (2 mcg injected into the tibialis anterior muscle), (B) mRNA encoding SIV Gag and mRNA encoding conventional IL- 10 (2 mcg + 1 mcg), or (C) mRNA encoding SIV Gag and mRNA encoding the macrophage-specific suppressive variant, IL- 1 ODE (2 mcg + 1 mcg). The mRNA / LNP vaccines were injected on days 0 and 28 of the experiment; the mice were sacrificed on day 56 and harvested splenocytes analyzed by cytokine flow cytometry (Figure 12). The results showed that recipients of mRNA / Gag and mRNA / IL-lODE developed superior inflammatory CD8+T-cell responses to the Gag immunogen, as demonstrated by development of more CD8+T cells capable of producing IFN-y in response to stimulation with overlapping peptides from the immunogen (Figure 12).Example 5: Novel IL- 10 variants that are macrophage-suppressive and immunosilent.

[0114] Previously created macrophage-suppressive IL- 10 variants such as human and macaque IL-10D (D25A mutant from Yoon, JBC 281:35088

[2006] ; SEQ ID NOs 56-57) or IL-10DE carry missense mutations that affect binding of the molecule to the IL-10R2 molecule, which results in the macrophage / monocyte-specific suppressive activity. Such missense mutations, however, can elicit adaptive immune responses against the changed amino-acid sequence, particularly in the context of the unexpected promotion of inflammatory functions uncovered. In this example, three approaches were designed to create IL- 10 variant proteins more likely to be immunosilent: (1) fusing IL- 10 to IL-10R2, (2) deleting key residues that interact with IL-10R2, and (3) adding N-terminal fusion partners.Fusing IL-10 to IL-10R2

[0115] The first approach undertaken was fusion of segments of the IL- 10R2 to IL- 10 or its variants. The basis for macrophage selectively of the IL- 10 variants mentioned above, e.g., IL-10D or IL- 1 ODE, is poor interaction with and recruitment of IL-10R2 to the receptor complex. This interaction and recruitment occur partly on the basis of interactions occurring near the N-terminus of IL- 10 with portions of the IL- 10R2 protein. Novel fusions of IL-10R2 segments to intact IL- 10 or IL- 10 variants were therefore created. Such fusionprotein designs approximate IL-10 or variant IL- 10 to its binding-partner amino acids of IL-2, leading to their preferential intra-molecular association and exclusion of native IL- 10R2 protein from the complex. These novel proteins are immunosilent because they comprise only sequences found in the host, possibly along with short, non-immunogenic linker sequences.

[0116] In some embodiments, variable segments of the IL-10R2 protein sequence may be included, e.g., loop 5 including residues W143-N152 (L5; numbering includes the native IL-10R2 signal sequence; Figure 18), loop 3 including residues S80- Y87 (L3), loop 2 including residues Y59-K65 (L2), combinations of these, or the complete or near-complete IL-10R2 extracellular domain. For example, fusions of IL-10R2 L5 to IL-lOndell 1 (IL-lOndell 1 is the mature IL-10 protein lacking its first 11 amino acids) are as shown in the below Table 2:Table 2: IL-10R2 protein sequences with IL-l Ondel fusion partners

[0117] Similar fusion proteins can be made, e.g., with L3 or with the combination of L2 and L3. as shown in the below Table 3:Table 3: IL-10 fusion sequences

[0118] IL-10 was also fused to the full-length or near full-length IL-10R2 extracellular domain (ECD). This protein operates by a similar mechanism to those above: the fused IL-10R2 associates with IL- 10 and thereby prevents association with the native transmembrane IL-10R2, preventing signaling through that receptor to the inside of the cell. In this case it was predicted that N-terminal placement of the IL- 10 protein may be favorable, as shown in the below Table 4:Table 4: IL-10 fusion sequences with native secretion signals

[0119] The IL-10-IL-10R2 fusion may also be constructed with the IL-10R2 sequence preceding that of IL-10, as shown in the below Table 5:Table 5: IL-10R2 sequences fused to IL-10

[0120] To demonstrate that B-macaqueIL-10-(GGGGS (SEQ ID NO: 44))3-IL- 10R2ECD is properly folded and secreted, and retains the ability to signal through IL-10R1 , an optimized coding sequence was synthesized for the protein and cloned into a plasmid vector under control of a cytomegalovirus (CMV) promoter. The purified plasmid was transfected into Expi 293 cells; supernatants were harv ested three days later and filtered through a 0.2-micron filter. The supernatants were then serially diluted before incubation with HEK-Blue IL- 10 cells (InVivoGen), which respond to IL- 10 signaling by secreting alkaline phosphatase. The results showed that each of two supernatants produced strong signaling at dilutions of 1: 10, 1 :20, or 1:200 (Figure 13). Thus B-macaqueIL-10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD is properly folded and secreted, and it retains the ability to interact with IL-10R1.

[0121] The inventors next evaluated the specificity of B-macaqueIL-10- (GGGGS (SEQ ID NO: 44)) -IL-10R2ECD for monocyte / macrophage suppression. Macaque peripheral blood mononuclear cells (PBMC) were isolated and then treated with cIL-10, IL-10DE, or B-macaqueIL-10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD. After incubating for ten minutes, the treated cells were placed on ice and then stained extracellularly with fluorescent antibodies against surface markers (CD3, CD4, CD8, and CD20) and intracellularly with antibodies specific for the phosphorylated form of STAT3 (Figure 14A) or STAT1 (Figure 14B). Because the phosphory lated forms of these STAT proteins represent the activated (signaling) form, detection of the phosphoproteins indicates signaling from the added IL- 10 to the interior of the cell. The mean fluorescence intensity of anti-STAT3 or anti-STATl staining was normalized to the intensity achieved by cIL-10 protein. Monocyte / macrophage specificity in this assay is indicated by more signaling to those cells (more STAT3 and STAT1 phosphory lation in those cells) than to CD3+, CD4+, or CD8+T cells.

[0122] The results indicated that the action of IL- 1 ODE is partially monocyte specific, as expected (Figures 14A-14B, unfilled bars). With respect to STAT3 signaling, marginally more phosphorylation was achieved in HLA-DR+monocytes than in CD3+, CD4+, or CD8 T cells, indicating very modest specificity (Figure 14A). Better results were obtained when considering STAT 1 signaling, where the normalized MFI for IL- 1 ODEsignaling to HLA-DR+monocytes was 1.18 times higher than the MFI for signaling to CD4+T cells.

[0123] The STAT3 and STAT1 phosphorylation results for B-macIL-10- (GGGGS (SEQ ID NO: 44))3-IL-10R2ECD indicated substantially greater specificity. Considering STAT3 phosphorylation (Figure 14A), B-macIL-10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD demonstrated 1.6-2.5X more signaling to HLA-DR+ monocytes than to the T-cell subsets examined. With respect to STAT1 signaling instead (Figure 14B), specificity was even better, with 4.36X higher STAT1 phosphorylation seen in monocytes vs. CD8+T cells. Thus, using either signaling measure, B-macIL-10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD manifested more specific signaling to monocytes than did IL- 10DE.

[0124] All of the macrophage / monocyte-specific IL- 10 variants above will retain their ability to suppress these myeloid cells and promote inflammatory CD8+T cells when administered as Fc fusions, whether encoded in a genetic deliver}' vehicle such as mRNA or delivered as protein. The Fc region may be placed C-terminally or N-terminally to the IL-10 variants, and expression of such Fc fusions is predicted to be higher when a monomeric variant of IL-10 is employed. Two examples of this approach as shown in the below Table 6:Table 6: Monomeric variant sequences of IL- 10

[0125] These molecules are predicted to have superior macrophage / monocyte suppressive activity’ due to longer half-life and greater ability to cluster IL- 10 receptors on the macrophage surface. They will accordingly have greater ability to promote the inflammatory functions of CD8+ T cells.Deletion of Key Residues

[0126] The second approach to creating macrophage / monocyte specific IL- 10 proteins that are more likely to be immunosilent is deletion from the amino acid chain of key residues that interact with IL-10R2, with optional shifting of Cys 12 in the protein sequence to retain its proper position relative to Cys 108. Preferred amino acids for deletion include residues 14, 18, 24, 90, 92, 96, and 104. The sequences as shown in the below Table 7 (which retain the native IL-10 signal sequence) show deletion of residues H14,N18, or D25, with or without a shift in the position of Cys 12, as well as combined deletions of residues R104 and H14; or residues R104 and N18:Table 7: IL-10 variant sequences with specific deletions

[0127] These deletions can optionally be combined with missense mutations that also reduce binding to IL-10R2, e.g. D25A and / or E96A; or with tags for purification such as polyhistidine.

[0128] To test which of the above variants would be best folded and secreted, and would retain the ability to signal through IL-10R1 despite sub-optimal association with IL-10R2, optimized coding sequences were synthesized for Monomeric cIL-10-6xHis, Monomeric cIL-10, cIL-10ndell3-6xHis, cIL-10ndell4-6xHis, cIL-10dell4-6xHis. cIL- 10dell8-6xHis, cIL-10del25-6xHis, macaquelL- 10-(GGGGS (SEQ ID NO: 44))3-IL- 10R2ECD, cIL-10shiftCdell4-6xHis, cIL-10shiftCdell8-6xHis, and cIL-10shiftCdel25- 6xHis. These sequences were cloned into a plasmid vector under control of a cytomegalovirus (CMV) promoter. The purified plasmid was transfected into Expi 293 cells; supernatants were harvested three days later and filtered through a 0.2-micron filter. The supernatants were then serially diluted before incubation with HEK-Blue IL- 10 cells (InVivoGen), which respond to IL- 10 signaling by secreting alkaline phosphatase. The results showed that cIL-10“nderi3 and cIL- I O"ndel'’ l 4 variants were inactive, doubtless due to removal of structurally important residues such as Cl 2; cIL-10del25 was also inactive (Figures 15A and 15B). However, two mutants with deletions of residuesinteracting with IL-10R2 were active, cIL-10dell4 and cIL-10dell8, as were their “shiftC” variants (Figures 15A, 15B, and 15C). Furthermore, B-macaqueIL-10-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD was again shown to retain the ability to interact with IL-10R1 and signal via the receptor to HEK IL- 10 Blue cells (Figure 15A).N-terminal fusion partners

[0129] The third approach is to add N-terminal fusion partners to IL-lO’s deleted variants, e.g., cIL-10dell4 and cIL-10dell8, in order to provide steric interference with IL-10R2 binding, due to the presence of the N-terminal fusion protein, resulting in further selective preference for suppression of macrophage functions. The two N-terminal fusion partners initially explored were human serum albumin and the crystallizable fragment of antibody (Fc). Exemplary protein sequences are as shown in the below Table 8:Table 8: IL-10 protein sequences with N-terminal fusion partners

[0130] ** The specific IL- 10 deletion variant chosen for these examples wasIL-10dell4, but any of the deletions that retain signaling capacity above could be used, e.g., IL- 1 Odel 18.

[0131] The ability of all these variant IL-lOs to specifically suppress macrophages, monocytes, and other myeloid cells, but to suppress B and T cells to a lesser degree or not at all, is assessed by comparing activities of wild-type IL-10 and the candidate variant IL- 10 across a panel of four cell lines representing different classes of immune cells. Daudi cells are B lymphoblasts; Jurkat cells are immortalized T lymphocytes; YT-1 cells have a surface phenotype similar to human NK cells and have NK-like killing activity; and THP-1 cells are human monocytic cells that can be differentiated into macrophages with PMA. By testing the ability of wild-type IL- 10 or variant IL-10 (selected from those above) to induce STAT3 phosphorylation, an estimate was derived for the percent activity' of the variant molecule in reference to wild-type IL- 10. The valuable variants for evoking pro-inflammatory T-cell functions via macrophage / monocyte suppression are those that retain >50% activity in myeloid THP-1 cells and <25% activity in either Daudi or Jurkat cells.

[0132] Alternatively, the ability of these variant IL-lOs to specifically suppress macrophages, monocytes, and other myeloid cells, but to suppress T cells to a lesser degree, was assessed by stimulating peripheral blood mononuclear cells with varying concentrations of the proteins, then using antibodies specific for phospho-STATl or phospho-STAT3 to examine the extent of increased phosphorylation in monocytes vs. T cells. This was the approach taken in Figures 14A-14B to demonstrate the monocyte specificity of B-macIL-10-(GGGGS)3-IL-10R2ECD.Example 6: IL-10R2 fusion protein for cell type-specific signaling through the IL-22 pathway.

[0133] The IL-10R1 is a high-affinity, private receptor while IL-10R2 is a lower-affinity subunit shared with certain other IL- 10 family cytokines including IL-22, IL-26, IL-28A, IL-28B, and IL-29. The approach used above to restrict cytokine signaling to macrophage / monocyte-lineage cells works in part because those cells express sufficient IL-10R2 to overcome the IL-10R2-specific inhibition imposed by the fusion protein. IL- 22 acts on macrophages via interactions with its high-affinity receptor, IL-22R, and with IL-10R2. The effect of IL-22 on macrophages is M2 polarization, which is associated with IL-10 and TGF-beta production. Furthermore, M2-type macrophages cause T cells to produce molecules like IL-4 and TGF-0 that promote antibody production by B cells. Thus, the cell type-specific effects of IL-22-IL-10R2ECD fusion protein may overlap with those of IL-10-IL-10R2ECD fusion proteins. The IL-22 fusion protein that is analogous to IL- 10-(GGGGS (SEQ ID NO: 44))?-IL-10R2ECD is IL-22-(GGGGS (SEQ ID NO: 44)>IL- 10R2ECD:

[0134] AALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKS NFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFT LEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKK LGESGEIKAIGELDLLFMSLRNACIGGGGSGGGGSGGGGSMVPPPENVRMNSVNF KNILQWESPAFAKGNLTFTAQYLSYRIFQDKCMNTTLTECDFSSLSKYGDHTLRV RAEFADEHSDWVNITFCPVDDTIIGPPGMQVEVLADSLHMRFLAPKIENEYETWTMKNVYNSWTYNVQYWKNGTDEKFQITPQYDFEVLRNLEPWTTYCVQVRGFLP DRNKAGEWSEPVCEQTTHDETVPS (SEQ ID NO: 41).

[0135] Multiple sclerosis (MS), a chronic inflammatory disease of the CNS, is characterized by increased serum levels of IL-2, IL-4, IL-6, IL-13, IL-17, IL-21, IL-22 and IL-33. Despite being usually associated with proinflammatory activity, several studies have additionally recognized a neuroprotective role of IL-22. Thus, MS pathogenesis is apparently associated with dysregulated IL-22 signaling. Indeed, there is a genetic MS risk variant (SNP rsl7066096) located downstream of IL22RA2, encoding the IL-22 binding protein (IL-22BP), an antagonist molecule that regulates the actions of IL-22. This susceptibility variant is associated with higher IL22RA2 expression in differentiated monocytes, suggesting that a reduction in IL-22 signaling is associated with increased risk. Patients with high lesion loads were found to have significantly higher levels of IL-22BP in the CSF.

[0136] To show the benefit of IL-22-(GGGGS (SEQ ID NO: 44))3-IL- 10R2ECD in an animal model, experimental autoimmune encephalomyelitis (EAE) was induced by injection of myelin oligodendrocyte protein (MOG) with adjuvant into rodents. Immune cell activation has been found to often found to peak at approximately seven days after immunization. IL-22-(GGGGS (SEQ ID NO: 44))3-IL- 10R2ECD administration was started either (i) before MOG immunization or (ii) beginning approximately 7-9 days after MOG immunization (EAE induction), which is after the pathogenic priming events have taken place. During the 30 days after MOG administration, the EAE score was followed, which is a numerical marker of disease severity (0, no paralysis; 1, tail paralysis; 2, hind limb paresis 3, hind limb paralysis; 4, hind and front limb paralysis; 5, dead). The beneficial effect of IL-22-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD protein was significantly lower EAE scores in the intervention group. Other beneficial effects observed included smaller lymph-node size (indicating a lesser response) and / or fewer antigen-specific T cells that produce interferon gamma.

[0137] Inflammatory skin disease including psoriasis and atopic dermatitis affect a large number of people worldwide. To demonstrate the benefit of IL-22-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD for such conditions, imiquimod was applied to the skin of C57 / BL6J mice to induce psoriasis-like lesions. Progression of the disease can be followed using PASI scoring (Psoriasis Area and Severity Index, based on the redness, thickness, and scaliness of lesions) and / or histological evaluation (epidermal thickness and microabscesses). IL-22-(GGGGS (SEQ ID NO: 44))3-IL-10R2ECD protein wasadministered at different timings relative to imiquimod, e.g., starting before imiquimod or on the same day or after imiquimod. The beneficial effects of this protein in psoriasis or dermatitis are evident based on reductions in the PASI scores, epidermal thicknesses, or lesional thicknesses of mice receiving the protein.Example 7: Boosting IL-10-elicited MHC class Ib-restricted T cells using single-chain trimers or vaccines that co-express IL- 15,

[0138] The elicitation of Mamu-E-restricted (i.e., MHC class Ib-restricted) inflammatory T cells to “supertope” peptides as described above is an unexpected result of expressing macrophage-suppressive IL-10 molecules in vaccines. Such T cells may be particularly useful effector cells for fighting infectious diseases and cancers. Infectious diseases such as HIV have evolved a form of evolutionary resistance to common human cytotoxic T-cell responses that are restricted by conventional MHC class-la molecules, as highly vulnerable epitopes that elicit strong conventional T-cell responses are gradually eliminated from circulating virus. Similarly, the human immune system helps to suppress the outgrowth of cancer cells in part by cytotoxic T-cell recognition of abnormal peptides presented on the cancer-cell surface. Such peptides may be abnormal because they are present in high abundance (tumor-associated antigens, TAAs) or due to altered sequence (tumor-specific antigens. TSAs). In many cases, people have limited repertoires of T cells that are capable of responding to peptides derived from TAAs or TSAs presented on MHC class la alleles (i.e., conventional peptide presentation) because many such T cells are eliminated by negative selection in the thymus. In addition, cancer cells are known to reduce or eliminate their expression of MHC class la molecules, undermining all class la- restricted responses. Eliciting MHC class Ib-restricted (e.g., HLA-E-restricted or Mamu- E-restricted) inflammatory T cells using macrophage-suppressive IL- 10 variants will allow the immune system to target cancer cells that have evaded the more common class la- restricted responses. Furthermore, eliciting such MHC class Ib-restricted T cells provides a source of novel TCRs that are expected to be particularly effective components of transgenic T-cell therapies.

[0139] Two approaches were used to boost the unique inflammatory T cells elicited in the presence of macrophage-suppressive IL- 10. First, having initially vaccinated in the presence of macrophage-suppressive IL- 10 to elicit class Ib-restricted T cells (Figures 5A, 5B, 6A, 6B, and 7), a second vaccination was conducted by delivery of mRNA or other genetic material (e.g., DNA delivered by adenovirus, vaccinia, or MV A)that expressed the target peptides that are capable of presentation on MHC class lb in fusion with the class-Ib molecules and with beta-2-microglobulin (single-chain trimers or SCTs). These SCTs may be conventional SCTs or modified to alter affinity for CD4 or CD8. In this experiment, two macaques received a single injection with type-5 adenovirus expressing Gag69 fused to the beta-2-microglobulin and Mamu-E heavy chain sequences from macaques (see nucleotide and protein sequences below). Two weeks after vaccination, PBMCs were isolated from the blood of each animal and exposed to the Gag69 supertope peptide, to determine if T cells having specificity for the peptide could be detected based on their ability to produce IFN-gamma and / or TNF-alpha. The result was clear detection of both cytokines in each vaccinated animal (Figure 16), which confirms that SCTs based on class-Ib molecules can expand the relevant cells.

[0140] Second, much of the ability of RhCMV-vectored vaccines to expand MHC class Ib-restricted cells is likely dependent on IL-15 signaling. Myeloid-specific IL- 10 variants can synergistically increase the responsiveness of memory cytotoxic T lymphocytes to IL- 15, resulting in more proliferation. Thus, the unique inflammatory T cells elicited in the presence of macrophage-suppressive IL- 10 can be boosted to an unexpected degree (more than additive) by simultaneously providing IL- 15 or IL- 15 fusion proteins. To demonstrate the unexpectedly synergistic adjuvant power of monocyte / macrophage-specific IL-10 administered with IL-15, an experiment was performed as diagrammed in Figure 17. An mRNA vaccine against the lentiviral Gag protein is delivered either alone (Group A) or with different forms of IL-10 (Groups B-C and E-F) and / or IL- 15 (Groups D-F, receiving mRNA coding for receptor-linker IL- 15 fusion protein). The results demonstrate that T-cell responses in all of Groups B-D (receiving either IL- 10 or IL- 15 in addition to the vaccine immunogen) are improved relative to T-cell responses in Group A. The results in Groups E and F, however (both IL- 10 and IL- 15), demonstrate an additional synergistic improvement over that expected due to the separate influences of the two cytokines. In particular, T-cell responses in Group F were improved by at least an additional 1.2 to 20 times more than expected. If Group C’s and Group D’s responses are each improved by 2X (on average) relative to those in Group A, for example, then the responses in Group F are improved by 4.8 to 80X.

[0141] The macaque Gag69-beta-2-microglobulin-Mamu-E sequence (singlechain trimer or SCT sequence) is as shown in the below Table 9: Table 9: Macaque Gag69SCT sequence

[0142] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently descnbing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as wall be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 markers refers to groups having 1, 2, or 3 markers. Similarly, a group having 1-5 markers refers to groups having 1, 2, 3. 4, or 5 markers, and so forth.

[0143] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. Any feature, step, element, embodiment, or aspect disclosed herein can be used in combination with any other unless specifically indicated otherwise.

[0144] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A protein variant of human IL- 10, wherein the variant comprises a deletion of an at least one amino acid residue contributing to interaction with IL-10R1 or IL-10R2, wherein the deletion of at least one amino acid is at or corresponding with an amino acid at position: 14, 18, 21, 22, 24, 28, 74, 90, 92, 96, 100, 104, 108, or any combination thereof, of SEQ ID NO: 49; and wherein the variant retains signaling function.

2. The protein variant of claim 1, wherein a cysteine residue corresponding to the cysteine at position 12 of SEQ ID NO: 49 is instead placed at position 11 of SEQ ID NO: 49, such that there is an amino acid other than cysteine at position 12.

3. A fusion protein comprising:(a) an IL-10 or a variant of IL-10 having at least 80.

81.

82.

83. 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to IL- 10, and(b) a segment of IL-10R2.

4. The fusion protein of claim 3, wherein the variant IL- 10 is the protein variant of claim 1 or 2.

5. A fusion protein comprising:(a) a segment of human serum albumin and / or a segment of an Fc domain, and(b) a fusion protein or protein variant of any one of claims 1-4.

6. The protein variant or fusion protein of any one of claims 1 -5, wherein the protein has a speci I'icity for signaling to myeloid cells that is calculated as the ratio between signaling activity' to myeloid cell types and signaling activity to T cells, wherein the specificity is greater than the specificity of cellular IL- 10 (cIL-10) for signaling to myeloid cells.

7. The protein variant or fusion protein of claim 6, wherein the specificity is for signaling to at least one of: monocytes, macrophages, plasmacytoid dendritic cells (pDCs), or any combination thereof.

8. The protein variant or fusion protein of any one of claims 1-7, wherein the protein variant is immunosilent9. The protein variant or fusion protein of any one of claims 1-8, wherein the variant comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any one of the sequences of SEQ ID NOs.: 6-40.

10. The protein variant or fusion protein of any one of claims 3-9, wherein the segment is on the C- and / or N-terminal end.

11. The protein variant or fusion protein of any one of claims 3-10, wherein the segment of IL-10R2 comprises the extracellular domain of IL-10R2, loop 2 residues Y59- K65, loop 3 residues S80-Y87, loop 5 residues W143-N152, full-length IL-10R2, or any combination thereof.

12. The protein variant or fusion protein of any one of claims 1-11, further comprising a linker sequence, optionally wherein the linker sequence is operably linked between the first fusion partner and the second fusion partner.

13. The protein variant or fusion protein of claim 12, wherein the linker sequence comprises: glycine, serine, threonine, alanine, or any combination thereof; optionally wherein the linker sequence comprises: glycine and / or serine.

14. The protein variant or fusion protein of claim 12 or 13, wherein the linker sequence is about 3 to about 20 amino acids in length; optionally wherein the linker sequence is about 5 to about 15 amino acids in length; optionally wherein the linker sequence is about 5 to 9 amino acids in length.

15. The protein variant or fusion protein of any one of claims 1-14, wherein at least one of the amino acids at or corresponding with positions H14 or N18 of SEQ ID NO: 49 are deleted or mutated.

16. The protein variant or fusion protein of any one of claims 1-15, wherein the amino acid at or corresponding with position R 104 of SEQ ID NO: 49 is deleted or mutated, and wherein at least one of the amino acids at or corresponding with positions H14 or N18 of SEQ ID NO: 49 are deleted or mutated.

17. A nucleotide sequence encoding the protein variant or fusion protein of any one of claims 1-16.

18. The nucleotide sequence of claim 17, wherein the nucleotide sequence comprises DNA and / or RNA.

19. A delivery’ vector comprising the nucleotide sequence of any one of claims 17-18.

20. The delivery vector of claim 19, wherein the delivery' vector is a viral vector or lipid nanoparticle.

21. The delivery vector of claim 20, wherein the viral vector is a lenti viral vector, retroviral vector, adenoviral vector, or any combination thereof.

22. The delivery vector of any one of claims 19-21, further comprising an at least one self-cleaving peptide.

23. A vaccine comprising the delivery vector of any one of claims 19-22.

24. A vaccine encoding the protein variant or fusion protein of any one of claims 1-16, and / or the nucleotide of any one of claims 17 or 18.

25. The vaccine of claim 23 or 24, wherein the vaccine is an mRNA, adenovirus, alphavirus, or lentivirus platform, or any combination thereof.

26. A cell comprising the protein variant or fusion protein of any one of claims 1-16, the nucleotide sequence of any one of claims 17-18, the delivery vector of any one of claims 19-22, or any combination thereof.

27. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the protein variant or fusion protein of any one of claims 1-16, the nucleotide sequence of any one of claims 17-18, the deli \ er\ vector of any one of claims 19-22, the vaccine of any one of claims 23-25, the cell of claim 26, or any combination thereof.

28. The method of claim 27, wherein the disease or disorder is an infection, autoimmune disease, an allergic disease or reaction, an inflammatory disease, a cancer, a tumor, or any combination thereof.

29. The method of claim 28, wherein the disease or disorder is a chronic infection, optionally wherein the chronic infection is HIV or hepatitis.

30. A use for the protein variant or fusion protein of any one of claims 1 -16, the nucleotide sequence of any one of claims 17-18, the delivery' vector of any one of claims 19-22, the vaccine of any one of claims 23-25, or the cell of claim 26, in the preparation of a medicament.

31. A method of stimulating an immune cell, the method comprising administering to the immune cell the protein variant or fusion protein of any one of claims 1-16, the nucleotide sequence of any one of claims 17-18, the delivery vector of any one of claims 19-22, or the vaccine of any one of claims 23-25.

32. The method of claim 31, wherein stimulating the immune cell comprises increasing the total number of activated T cells, and / or increasing the production of IFN- gamma and / or TNF-alpha.

33. The method of claim 31 or 32, wherein the immune cell is a lymphocyte.

34. The method of claim 33, wherein the immune cell is a T cell, a B cell, or anNK cell.

35. The method of claim 34, wherein the T cell is a CD4+ or a CD8+T cell.

36. The method of any one of claims 34 or 35, wherein the T cell is an MHC class Ib-restricted T cell.

37. A method of inhibiting IL-1 activity, NLRP1 inflammasome activation, NLRP3 inflammasome activation, type 1 interferon production (IFN-I) production, IL-6 production, or any combination thereof in vivo, the method comprising administering the protein variant of any one of claims 1-16, the nucleotide sequence of any one of claims 17- 18, the delivery vector of any one of claims 19-22, the vaccine of any one of claims 23-25, or the cell of claim 26.

38. The method of any one of claims 31-37, further comprising administering an at least one single-chain trimer and / or IL- 15 at the same time as administering the protein variant or fusion protein of any one of claims 1-16, the nucleotide sequence of any one of claims 17-18, the delivery vector of any one of claims 19-22, the vaccine of any one of claims 23-25, or the cell of claim 26.

39. The method of any one of claims 31-38, further comprising administering an at least one booster vaccine.

40. The method of claim 39, further comprising administering an at least one single-chain trimer and / or IL-15 at the same time as the booster vaccine.

41. The method of any one of claims 38 or 40, wherein the at least one singlechain trimer comprises a sequence having at least 80, 81, 82, 83, 84, 85, 86.

87.

88.

89. 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence of any one of SEQ ID NOs: 52-55.

42. A method of vaccinating a subject in need thereof, the method comprising administering to the subject the protein variant of any one of claims 1-16. the nucleotide sequence of any one of claims 17-18, the delivery vector of any one of claims 19-22, the vaccine of any one of claims 23-25, or the cell of claim 26.

43. The method of claim 42, wherein the vaccination results in increasing the total number of activated T cells, and / or increasing the production of IFN-gamma and / or TNF-alpha in the subject.

44. The method of claim 42 or 43, wherein the vaccination results in the reduced occurrence, duration, and / or severity of at least one side effect as compared to vaccination in absence of the protein variant of any one of claims 1-16.

45. The method of claim 44, wherein the at least one side effect is selected from: fever, malaise, granulocytosis, circulating IL-6, circulating total cytokine concentration, circulating specific cytokine concentration, or any combination thereof.

46. The method of claim 45, wherein the circulating specific cytokine is IL-6 and / or IP-10 (CXCL10),47. The method of any one of claims 42-46, wherein the subject is mammalian and / or human.

48. The method of any one of claims 42-47, wherein the vaccination is a booster vaccination.

49. The method of any one of claims 42-48, wherein the vaccination is performed on the subject more than once.

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