Synthetic RIG-i like receptor agonists

Abstract

To provide synthetic RIG-I like receptor agonists.SOLUTION: In some embodiments, the disclosure provides a composition comprising: (a) a virus-like particle; and (b) at least one synthetic RIG-I like receptor (RLR) agonist that specifically binds to a RLR and comprises a ribonucleic acid (RNA) of 10-100 nucleotides in length, where the 5' most nucleotide of the RNA comprises 5' diphosphate or triphosphate moiety or a derivative or analog thereof, where the at least one RLR agonist is packaged in the virus-like particle.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 925,120, filed October 23, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Exogenous nucleic acids, particularly viral nucleic acids, introduced into cells induce an innate immune response, resulting in interferon (IFN) production and cell death, among other events. Upon detection of viral RNA, RIG-I-like receptors induce type I interferon (IFN) secretion, leading to increased expression of antiviral IFN-inducible proteins in infected and neighboring cells, thereby inhibiting viral replication. Further downstream events recruit immune cells and elicit an adaptive immune response. Furthermore, RIG-I ligands have been reported to induce apoptosis in many different types of tumor cells, but not in normal cells.

[0003] Virus-like particles (VLPs) are supramolecular structures assembled in a symmetrical fashion from multiple protein molecules of one or more types. VLPs lack the viral genome and are therefore non-infectious. VLPs can often be produced in large quantities by heterologous expression and are easily purified.

[0004] VLPs are used in vaccinology, immunology, and medicine due to both their structural properties and non-infectious properties. VLPs have been shown to be efficiently processed and presented on MHC class I molecules, possibly after uptake by micropinocytosis or other cellular uptake pathways, followed by cross-priming onto MHC class I molecules.

[0005] There remains a need for further and improved compositions and methods for modulating the activity of immunomodulatory proteins. Such agents can be used in cancer immunotherapy and the treatment of other conditions, such as chronic infections. There is a need for the development of improved RIG-I-like receptor ligands, including improved delivery methods, for a variety of therapeutic immunomodulatory applications. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure is based, at least in part, on the discovery of synthetic RNA molecules that function as RIG-I-like receptor (RLR) agonists. The present disclosure also provides compositions and methods for improving the biological activity of immunostimulatory nucleic acids, particularly RLR agonists, by packaging them into VLPs (RIG-VLPs). The compositions described herein can be used to induce potent and sustained immune responses that are particularly useful in the treatment of tumors.

[0007] Thus, in some aspects, the present disclosure provides: (a) a virus-like particle; (b) at least one synthetic RIG-I-like receptor (RLR) agonist that specifically binds to an RIG-I-like receptor (RLR), wherein the RLR agonist comprises a ribonucleic acid (RNA) of 10 to 100 nucleotides in length, wherein the 5'-most nucleotide of the RNA comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and wherein the at least one RLR agonist is packaged into a virus-like particle.

[0008] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one synthetic RIG-I-like receptor (RLR) agonist that specifically binds to an RIG-I-like receptor (RLR), the at least one synthetic RLR agonist comprising a ribonucleic acid (RNA) of 10 to 100 nucleotides in length, wherein the 5'-most nucleotide of the RNA comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, wherein the at least one RLR agonist is packaged into a virus-like particle.

[0009] In any of the above or related embodiments, the RNA is single-stranded. In other embodiments, some or all of the RNA is double-stranded.

[0010] In any of the above or related embodiments, the RLR agonist RNA is 10-15, 15-20, 20-25, 25-30, or 30-35 nucleotides in length.

[0011] In any of the above or related embodiments, the RLR agonist comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex. In some embodiments, the duplex comprises a hairpin. In some embodiments, the duplex comprises 10-15, 15-20, 20-25, 25-30, or 30-35 base pairs. In some embodiments, the duplex comprises fewer than 19 base pairs. In some embodiments, the first polynucleotide is connected to the second polynucleotide by a linker.

[0012] In any of the above or related embodiments, the RLR agonist comprises a sequence motif that confers at least one biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif.

[0013] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one synthetic RIG-I-like receptor (RLR) agonist that specifically binds to an RIG-I-like receptor (RLR), the at least one synthetic RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif. In some embodiments, the first polynucleotide comprises the sequence motif, and the at least one RLR agonist is packaged in a virus-like particle.

[0014] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one synthetic RIG-I-like receptor (RLR) agonist that specifically binds to an RIG-I-like receptor (RLR), the at least one synthetic RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif. In some embodiments, the first polynucleotide comprises the sequence motif, and the at least one RLR agonist is packaged in a virus-like particle.

[0015] In any of the above or related embodiments, the RLR agonist is (i) GT repeat motif, (ii) GA repeat motif; (iii) AUCG repeat motif, (iv) AU repeat motifs; (v) dipyrimidine motif, (vi) ziprine motif, (vii) pyrimidine triplet motif, (viii) Printed triplet motif, (ix) a palindromic sequence motif, and (x) Contains a sequence motif selected from the group consisting of any combination of (i) to (ix).

[0016] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to form a duplex with the second polynucleotide, the duplex comprising fewer than 19 base pairs, and the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (i) GT repeat motif, (ii) GA repeat motif; (iii) AUCG repeat motif, (iv) AU repeat motifs; (v) dipyrimidine motif, (vi) ziprine motif, (vii) pyrimidine triplet motif, (viii) Printed triplet motif, (ix) a palindromic sequence motif, and (x) at least one synthetic RLR agonist comprising a sequence motif selected from any combination of (i) to (ix), wherein the at least one RLR agonist is packaged in a virus-like particle.

[0017] In some embodiments, the RLR agonists of the present disclosure comprise a combination of sequence motifs. In some embodiments, the combination of sequence motifs is a GT repeat motif and a purine triplet motif. In some embodiments, the combination of sequence motifs is an AUCG repeat motif and a dipyrimidine motif. In some embodiments, the combination of sequence motifs is an AUCG repeat motif and a dipurine motif.

[0018] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the at least one improved biological activity being (i) Increased RLR-mediated cytokine production; (ii) increased RLR-mediated expression of interferon-stimulated genes; (iii) increased RLR-mediated intracellular signaling; (iv) increased binding affinity to RLRs, and (v) A combination of any one of (i) to (iv) is selected.

[0019] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise a sequence motif that increases RLR-mediated type I interferon (e.g., IFN-α, IFN-β) production relative to agonists that do not comprise the sequence motif. In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif that increases RLR-mediated IL-1β production relative to agonists that do not comprise the sequence motif. In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif that increases RLR-mediated IP-10 production relative to agonists that do not comprise the sequence motif. In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif that increases RLR-mediated IL-6, IL-12p70, MCP-1, and / or MIP-1β production relative to agonists that do not comprise the sequence motif.

[0020] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is a GT repeat motif (e.g., GTGTGT) comprising a sequence of fewer than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of fewer than 19 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 15-18 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 15 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 10-15 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 10 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 5-10 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 5 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of about 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the GT repeat motif provides improved biological activity of the RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0021] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is a GT repeat motif comprising a sequence of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 18 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 16 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 14 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 12 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of 10 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of eight guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of six guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT repeat motif comprising a sequence of four guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is a GT repeat motif, and the GT repeat motif is [GT] n (wherein n=2 to 9, 3 to 7, or 4 to 8). In some embodiments, the GT repeat motif provides an improved biological activity of the RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to an RLR, and any combination of the above.

[0022] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected (operably linked) to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being a GT repeat motif comprising a sequence of about 14 guanine and thymine nucleotides, wherein the at least one RLR agonist is packaged within a virus-like particle.

[0023] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected (operably linked) to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being a GT repeat motif comprising a sequence of about 14 guanine and thymine nucleotides, wherein the at least one RLR agonist is packaged within a virus-like particle.

[0024] In some embodiments, the sequence motif is a GT repeat motif, and the GT repeat motif is [GT]7. In some embodiments, the improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0025] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being a GT repeat motif comprising a sequence of six guanine and thymine nucleotides, wherein the at least one RLR agonist is packaged within a virus-like particle.

[0026] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being a GT repeat motif comprising a sequence of six guanine and thymine nucleotides, wherein the at least one RLR agonist is packaged within a virus-like particle.

[0027] In some embodiments, the sequence motif is a GT repeat motif, and the GT repeat motif is [GT]3. In some embodiments, the sequence motif is a GT repeat motif, and the GT repeat motif is [GT]3, and the GT repeat is followed by a purplish triplet and a UCG, respectively. In some embodiments, the purplish triplet is GGA. In some embodiments, the improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0028] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is a GA repeat motif (e.g., GAGAGA) comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of less than 19 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of about 15-18 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of about 15 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of about 10-15 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of about 10 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of about 5-10 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of about 5 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of about 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the GA repeat motif provides improved biological activity of the RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to an RLR, or any combination of the above.

[0029] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is a GA repeat motif comprising a sequence of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of 18 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of 16 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of 14 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of 12 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of 8 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of six guanine and adenine nucleotides, or derivatives or analogs thereof, hi some embodiments, the sequence motif is a GA repeat motif comprising a sequence of four guanine and adenine nucleotides, or derivatives or analogs thereof.

[0030] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises a sequence motif, wherein the sequence motif is a GA repeat motif, wherein the GA repeat motif is [GT] n (wherein n=2 to 9, 3 to 7, or 4 to 8). In some embodiments, the GA repeat motif provides an improved biological activity of the RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to an RLR, and any combination of the above.

[0031] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being a GA repeat motif comprising a sequence of about 14 guanine and adenine nucleotides, wherein the at least one RLR agonist is packaged within a virus-like particle.

[0032] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being a GA repeat motif comprising a sequence of about 14 guanine and adenine nucleotides, wherein the at least one RLR agonist is packaged within a virus-like particle.

[0033] In some embodiments, the sequence motif is a GA repeat motif, and the GA repeat motif is [GT]. In some embodiments, the GA repeat motif provides an improved biological activity of the RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to an RLR, and any combination of the above.

[0034] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is an AUCG repeat motif (e.g., AUCGAUCG) comprising a sequence of fewer than 19, about 16, about 12-16, about 12, about 8-12, or about 6, 16, 12, or 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of fewer than 19 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of about 16 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of about 12-16 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of about 12 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of about 8-12 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of about 6 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of 16 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of 12 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is an AUCG repeat motif comprising a sequence of 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof.In some aspects, the AUCG repeat motif provides an improved biological activity of the RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to an RLR, and any combination of the above.

[0035] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises a sequence motif, wherein the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG] n (wherein n=2-4, or 2, 3, or 4). In some embodiments, the AUCG repeat motif provides an improved biological activity of the RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to an RLR, and any combination of the above.

[0036] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being an AUCG repeat motif comprising a sequence of approximately 12 guanine and adenine nucleotides. In some embodiments, the AUCG repeat motif is [AUCG]3, and the at least one RLR agonist is packaged within a virus-like particle.

[0037] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide comprising the sequence motif, the sequence motif being an AUCG repeat motif comprising a sequence of approximately 12 guanine and adenine nucleotides. In some embodiments, the AUCG repeat motif is [AUCG]3, and the at least one RLR agonist is packaged within a virus-like particle.

[0038] In some aspects, the AUCG repeat motif provides an improved biological activity of the RLR agonist, the improved biological activity being increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to an RLR, and any combination of the above.

[0039] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat motif, and the motif is preceded by a CG or dipyrimidine motif.In some embodiments, the AUCG repeat motif is preceded by a CG.In some embodiments, the AUCG repeat motif is [AUCG]3, and preceded by a CG.In some embodiments, the AUCG repeat motif is [AUCG]3, and preceded by a dipyrimidine motif CC.

[0040] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat motif, wherein the motif is preceded by a dipurine motif. In some embodiments, the dipurine motif is GA. In some embodiments, the AUCG repeat motif is [AUCG]3, wherein the dipurine motif GA is preceded. In some embodiments, the AUCG repeat motif is preceded by a dipurine motif II.

[0041] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise an AUGC repeat motif, in which one or more uridine nucleosides (U) are replaced with modified nucleosides. In some embodiments, the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, in which one or more uridine nucleosides (U) comprising the AUCG repeat motif are replaced with modified nucleosides, which are ribothymidine (T). In some embodiments, the AUCG repeat motif is [AUCG]3, in which one or more uridine nucleosides (U) comprising the AUCG repeat motif are replaced with modified nucleosides, which are ribothymidine (T), and in which the AUGC repeat motif is preceded by GG.

[0042] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise an AUCG repeat motif, in which one or more guanosine nucleosides (G) are replaced with modified nucleosides. In some embodiments, the modified nucleosides are inosine (I). In some embodiments, the AUCG repeat motif is [AUCG]3, in which one or more guanosine nucleosides (G) constituting the AUCG repeat motif are replaced with modified nucleosides, in which the modified nucleoside is ribothymidine (T), and in which GG precedes the AUGC repeat motif.

[0043] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise an AUCG repeat motif, where the motif is preceded by IG. In some embodiments, the AUCG repeat motif is [AUCG]3, where the AUCG repeat motif is preceded by IG.

[0044] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat, wherein one or more guanosine nucleosides (G) are substituted with inosine (I), and the AUCG repeat is preceded by an inosine (I). In some embodiments, the guanosine nucleosides (G) comprising the AUCG repeat are substituted with inosine (I), the AUCG repeat is preceded by an inosine (I), and the 5'-most nucleotide of the first polynucleotide comprises an inosine (I).

[0045] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2, and wherein the AUCG repeat motif is preceded by a dipurine motif. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2, and wherein the AUCG repeat motif is preceded by a dipurine motif, wherein the dipurine motif is GG.

[0046] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2 and is preceded by a print triplet motif. In some embodiments, the print triplet is GGG. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2 and is preceded by a print triplet motif, wherein the AUCG repeat motif is GGG. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2 and is preceded by CCCCCG. In some embodiments, the sequence motif is an AUCG repeat motif, wherein the AUCG repeat motif is [AUCG]2 and is preceded by TCGUCG.

[0047] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one synthetic RLR agonist that specifically binds to an RLR, the synthetic RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising an [AUCG]n repeat motif (where n=2-4), with GG, CG, or IG preceding the 5'-most AUCG repeat motif, wherein the at least one RLR agonist is packaged within a virus-like particle. In some embodiments, n=3. In some embodiments, each G in the AUCG motif is substituted with an inosine.

[0048] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one synthetic RLR agonist that specifically binds to an RLR, the synthetic RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to the second polynucleotide to form a duplex, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising an [AUCG]n repeat motif (where n=2-4), with GG, CG, or IG preceding the 5'-most AUCG repeat motif, wherein the at least one RLR agonist is packaged within a virus-like particle. In some embodiments, n=3. In some embodiments, each G in the AUCG motif is substituted with an inosine.

[0049] In any of the above or related embodiments, the RLR agonists of the present disclosure comprise a sequence motif, which is a palindromic sequence comprising less than 19, about 15-18, about 15, about 10-15, about 10, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising less than 19 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising about 15-18 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising about 15 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of about 10-15 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of about 10 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 18 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 17 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 16 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 15 nucleotides, or derivatives or analogs thereof, linked in any order to form a palindrome.In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 14 nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 13 nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 12 nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 11 nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 10 nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 9 nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of eight nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of seven nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of six nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of five nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of four nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome.

[0050] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises a linker, wherein the linker is flanked by AU. In some embodiments, the linker is flanked by AU repeat motifs, wherein the AU repeat motif is [AU] n (where n=2 to 3). In some embodiments, the AU repeat motif is [AU]2.

[0051] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; at least one of N1, N2, N3, and N4 is inosine, and / or at least one of X1 and / or X2 comprises at least one inosine nucleoside, which inosine nucleoside base pairs with a cytidine in the hairpin RNA; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0052] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (iv) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (v) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (vi) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; at least one of N1, N2, N3, and N4 is inosine, and / or at least one of X1 and / or X2 comprises at least one inosine nucleoside, which inosine nucleoside base pairs with a cytidine in the hairpin RNA; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0053] In some aspects, the RLR agonists of the present disclosure have improved biological activity, where the improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0054] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, and having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) at least one RNA agonist comprising: When inosine is present, it base pairs with cytidine, Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0055] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, and having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) at least one RNA agonist comprising: When inosine is present, it base pairs with cytidine, Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0056] In some aspects, the RLR agonists of the present disclosure have improved biological activity, where the improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0057] In some embodiments, N1 comprises inosine and N4 comprises cytidine. In some embodiments, N1 comprises inosine and N4 comprises cytidine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 comprises cytidine and N4 comprises inosine. In some embodiments, N2 comprises inosine and N3 comprises cytidine. In some embodiments, N2 comprises cytidine and N3 comprises inosine. In some embodiments, N1 comprises guanosine. In some embodiments, N2 comprises guanosine. In some embodiments, N1 comprises cytidine. In some embodiments, N2 comprises cytidine. In some embodiments, N1 and N2 comprise guanosine, and N3 and N4 comprise cytidine. In some embodiments, N1 and N2 comprise cytidine, and N3 and N4 comprise guanosine. In some embodiments, N1 and N2 comprise inosine, and N3 and N4 comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise inosine.

[0058] In any of the above or related embodiments, the RLR agonist of the present disclosure has the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; When inosine is present, it base pairs with cytidine, and N1 comprises an inosine, N4 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N2 comprises an inosine, N3 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise inosine and do not include a guanosine nucleoside. In some embodiments, N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise inosine and do not include a guanosine nucleoside. In some aspects, the RLR agonists of the present disclosure have improved biological activity, where the improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0059] In any of the above or related embodiments, the RLR agonist of the present disclosure has the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; When inosine is present, it base pairs with cytidine, and N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 comprise inosine and do not comprise guanosine nucleosides. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and X2 comprise inosine and do not comprise guanosine nucleosides. In some embodiments, the RLR agonists of the present disclosure have improved biological activity, where the improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0060] In any of the above or related embodiments, the RLR agonist of the present disclosure has the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein X1 and X2 are each 12 nucleotides and contain 1, 2, 3, or 4 inosine nucleosides. In some embodiments, X1 and X2 are each 13 nucleotides and contain 1, 2, 3, 4, or 5 inosine nucleosides. In some embodiments, X1 and X2 are each 14 nucleotides and contain 1, 2, 3, 4, 5, or 6 inosine nucleosides. In some embodiments, X1 and X2 are each 15 nucleotides and contain 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. In some embodiments, X1 and X2 are each 16 nucleotides and contain 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. In some embodiments, X1 and X2 each have 12 nucleotides and contain at least 10%, 20%, 30%, or 40% inosine nucleosides. In some embodiments, the RLR agonists of the present disclosure have improved biological activity, where the improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0061] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 has the sequence motif [AUCN5] x (wherein N5 contains guanosine or inosine, and x is an integer representing the number of sequence motifs, and x is 2 to 4), (viii) X2 has the sequence motif [CN6AU] y (wherein N6 contains guanosine or inosine, and y is an integer representing the number of sequence motifs, y=2 to 4), (ix) L is a linker that operably links the first polynucleotide and the second polynucleotide; optionally, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside base pairs with a cytidine in the hairpin RNA; and Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0062] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (iv) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (v) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (vi) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 has the sequence motif [AUCN5] x (wherein N5 contains guanosine or inosine, and x is an integer representing the number of sequence motifs, and x is 2 to 4), (viii) X2 has the sequence motif [CN6AU] y (wherein N6 contains guanosine or inosine, and y is an integer representing the number of sequence motifs, y=2 to 4), (ix) L is a linker that operably links the first polynucleotide and the second polynucleotide; optionally, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside base pairs with a cytidine in the hairpin RNA; and Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0063] In some embodiments, N5 comprises inosine and N6 comprises inosine. In some embodiments, N5 comprises guanosine and N6 comprises inosine. In some embodiments, N5 comprises inosine and N6 comprises guanosine. In some embodiments, N5 comprises guanosine (G) and N6 comprises guanosine (G). In some embodiments, x=2 and y=2. In some embodiments, x=3 and y=3. In some embodiments, x=4 and y=4. In some embodiments, N1 comprises inosine (I) and N4 comprises cytidine (C). In some embodiments, N2 comprises inosine (I) and N3 comprises cytidine (C). In some embodiments, N3 comprises inosine (I) and N2 comprises cytidine (C). In some embodiments, N4 comprises inosine (I) and N1 comprises cytidine (C). In some embodiments, N1 comprises guanosine (G). In some embodiments, N2 comprises guanosine (G). In some embodiments, N1 comprises cytidine (C). In some embodiments, N2 comprises cytidine (C). In some embodiments, N1 and N2 comprise guanosine (G) and N3 and N4 comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N3 and N4 comprise guanosine (G). In some embodiments, N1 and N2 comprise inosine (I) and N3 and N4 comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N3 and N4 comprise inosine (I). In some embodiments, the RLR agonists of the present disclosure have improved biological activity, wherein the improved biological activity is increased RLR-mediated cytokine production, increased RLR-mediated expression of interferon-stimulated genes, increased RLR-mediated intracellular signaling, increased binding affinity to RLRs, and any combination of the above.

[0064] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises a linker, and the linker is a nucleotide linker or a non-nucleotide linker. In some embodiments, the linker is a non-nucleotide linker. In some embodiments, the linker is a nucleotide linker. In some embodiments, the nucleotide linker comprises a tetraloop, and the nucleotide sequence of the tetraloop is: (a) UNCG (where N=A, C, G, or U), (b) GNRA (where N=A, C, G, or U and R=A or G); (c) ANYA (where N=A, C, G, or U and Y=C or T); (d) CUYG (where Y = C or T), (e) UMAC (where M=A or C), and (f) selected from the group consisting of CUUG.

[0065] In some embodiments, the sequence of the tetraloop is UUCG. In some embodiments, the sequence of the tetraloop is GAUC.

[0066] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises a nucleotide linker, wherein the nucleotide linker comprises the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UGUUU.

[0067] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises a non-nucleotidic linker, wherein the non-nucleotidic linker is (a) an ethylene glycol linker, and (b) an alkyl linker.

[0068] In some embodiments, the non-nucleotidic linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotidic linker is a C9 alkyl linker.

[0069] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises a 5' diphosphate moiety, or a derivative or analog thereof. In some embodiments, the agonist comprises a 5' triphosphate moiety, or a derivative or analog thereof. In some embodiments, the derivative or analog comprises a bioisostere of a phosphate selected from phosphonate, thiophosphonate, phosphorothioate, sulfate, sulfonate, sulfamate, thiazolidinone, carboxylate, malonate, boronic acid, benzoxaborole, boranophosphate, and squaramide.

[0070] In any of the above or related embodiments, the RLR agonist of the present disclosure comprises a modified nucleotide, a modified nucleoside, or a modified nucleic acid base, or a combination thereof. In some embodiments, the agonist comprises a modification to the internucleotide bond or polynucleotide backbone.

[0071] In any of the above or related embodiments, the RLR agonists of the present disclosure have the following properties: (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5, and / or LGP2); (b) increase RLR-mediated cytokine production; (c) increasing RLR-mediated expression of interferon-stimulated genes (ISGs); (d) increasing RLR-dependent intracellular signaling; (e) increasing duplex stability; (f) increasing binding affinity to RLRs; (g) reducing off-target binding; (h) increasing biological half-life; (i) Increase biodistribution and bioavailability; (j) increasing and / or enhancing uptake into cells and / or tissues; (k) reducing immunogenicity; and (l) At least one of the combinations of (a) to (k) is shown.

[0072] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; wherein N1 and N2 each comprise a guanosine, N3 and N4 each comprise a cytidine, X1 and X2 each are 12 nucleotides in length, X1 and X2 each comprise at least one inosine nucleoside, which base pairs with a cytidine in the hairpin RNA, and L comprises a nucleotide linker comprising a tetraloop, the nucleotide sequence of the tetraloop being UUCG; and Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0073] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; wherein N1 and N2 each comprise a guanosine, N3 and N4 each comprise a cytidine, X1 and X2 each are 12 nucleotides in length, X1 and X2 each comprise at least one inosine nucleoside, which base pairs with a cytidine in the hairpin RNA, and L comprises a nucleotide linker comprising a tetraloop, the nucleotide sequence of the tetraloop being UUCG; and Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0074] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; and at least one RLR agonist comprising: N1 comprises an inosine, N2 comprises a guanosine, N3 and N4 each comprise a cytidine, X1 and X2 each are 12 nucleotides in length, X1 and X2 each comprise at least one inosine nucleoside, which inosine nucleoside base pairs with a cytidine in the hairpin RNA, and L comprises a nucleotide linker comprising a tetraloop, wherein the nucleotide sequence of the tetraloop is UUCG; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0075] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; and at least one RLR agonist comprising: N1 comprises an inosine, N2 comprises a guanosine, N3 and N4 each comprise a cytidine, X1 and X2 each are 12 nucleotides in length, X1 and X2 each comprise at least one inosine nucleoside, which inosine nucleoside base pairs with a cytidine in the hairpin RNA, and L comprises a nucleotide linker comprising a tetraloop, wherein the nucleotide sequence of the tetraloop is UUCG; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0076] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; and at least one RLR agonist comprising: N1 and N2 comprise inosine; N3 and N4 comprise cytidine; X1 and X2 are each 12 nucleotides in length; X1 and X2 each comprise at least one inosine nucleoside, which base pairs with a cytidine in the hairpin RNA; and L comprises a nucleotide linker comprising a tetraloop, wherein the nucleotide sequence of the tetraloop is UUCG; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0077] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; and at least one RLR agonist comprising: N1 and N2 comprise inosine; N3 and N4 comprise cytidine; X1 and X2 are each 12 nucleotides in length; X1 and X2 each comprise at least one inosine nucleoside, which base pairs with a cytidine in the hairpin RNA; and L comprises a nucleotide linker comprising a tetraloop, wherein the nucleotide sequence of the tetraloop is UUCG; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0078] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, and having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, X1 and X2 are each 12 nucleotides in length, and the non-nucleotide linker is a C9 alkyl linker; and Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0079] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, and having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, X1 and X2 are each 12 nucleotides in length, and the non-nucleotide linker is a hexaethylene glycol linker; and Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0080] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, wherein the 5'-most nucleotide of the agonist comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0081] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to form a duplex with the second polynucleotide, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide and the second polynucleotide comprising: (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively; and (xxxii) at least one RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97, respectively; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0082] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising at least one nucleotide that includes an inosine that base pairs with a cytidine, the at least one RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0083] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and the first polynucleotide and the second polynucleotide are (i) SEQ ID NOs: 58 and 89, respectively; (ii) SEQ ID NOs: 59 and 89, respectively; and (iii) at least one RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 61 and 91, respectively; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0084] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and the first polynucleotide and the second polynucleotide are (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively; and (xxxii) at least one RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97, respectively; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0085] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, and having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, X1 and X2 are each 12 nucleotides in length, and the non-nucleotide linker is a C9 alkyl linker; and Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0086] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, and having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide; N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, X1 and X2 are each 12 nucleotides in length, and the non-nucleotide linker is a hexaethylene glycol linker; and Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0087] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, wherein the 5'-most nucleotide of the agonist comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0088] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to form a duplex with the second polynucleotide, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, the first polynucleotide and the second polynucleotide comprising: (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively; and (xxxii) at least one RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97, respectively; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0089] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, the at least one RLR agonist comprising a blunt-ended hairpin RNA comprising at least one nucleotide that includes an inosine that base pairs with a cytidine, the at least one RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0090] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and the first polynucleotide and the second polynucleotide are (i) SEQ ID NOs: 58 and 89, respectively; (ii) SEQ ID NOs: 59 and 89, respectively; and (iii) at least one RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 61 and 91, respectively; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0091] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and the first polynucleotide and the second polynucleotide are (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively; and (xxxii) at least one RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97, respectively; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0092] In any of the above or related embodiments, the nucleotide sequence comprising the RLR agonist is not complementary to a genomic DNA sequence or an mRNA sequence, the RLR agonist does not participate in RNA interference, and the RLR agonist does not silence gene expression.

[0093] In any of the above or related aspects, the virus-like particles described herein do not have a lipoprotein-containing envelope.

[0094] In any of the above or related aspects, the virus-like particles described herein are recombinant virus-like particles. In some aspects, the recombinant virus-like particles are (a) Hepatitis B virus recombinant protein, (b) recombinant measles virus proteins; (c) recombinant proteins of Sindbis virus; (d) rotavirus recombinant proteins; (e) recombinant proteins of hand, foot and mouth disease virus; (f) retroviral recombinant proteins; (g) Norwalk virus recombinant protein; (h) recombinant human papillomavirus proteins; (i) BK virus recombinant proteins, (j) bacteriophage recombinant proteins; (k) recombinant proteins of RNA phages; (l) recombinant proteins of Qβ phage; (m) recombinant proteins of GA phage; (n) fr phage recombinant protein, (o) Recombinant proteins of AP205 phage; (p)Ty recombinant protein, and (q) A fragment of any of the recombinant proteins (a) to (p).

[0095] In any of the above or related embodiments, the virus-like particle described herein comprises recombinant proteins of an RNA phage, wherein the RNA phage is selected from the group consisting of: (a) bacteriophage Qβ, (b) bacteriophage R17, (c) bacteriophage fr, (d) bacteriophage GA, (e) bacteriophage SP, (f) bacteriophage MS2, (g) bacteriophage M11, (h) bacteriophage MX1, (i) bacteriophage NL95, (j) bacteriophage f2, (k) bacteriophage PP7, and (l) bacteriophage AP205.

[0096] In any of the above or related embodiments, the virus-like particles described herein comprise recombinant proteins of bacteriophage Qβ. In some embodiments, the recombinant proteins of bacteriophage Qβ comprise a coat protein having the amino acid sequence of SEQ ID NO: 112. In some embodiments, the recombinant proteins of bacteriophage Qβ comprise a coat protein having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 112.

[0097] In some aspects, the present disclosure provides a composition comprising: (a) a virus-like particle of RNA phage Qβ comprising a coat protein having the amino acid sequence of SEQ ID NO: 112; (b) at least one synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), the at least one synthetic RLR agonist comprising a ribonucleic acid (RNA) of 10 to 100 nucleotides in length, wherein the 5'-most nucleotide of the RNA comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0098] In some aspects, the present disclosure provides a composition comprising: (a) Virus-like particles of RNA phage Qβ and (b) at least one synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the at least one synthetic RLR agonist comprises the nucleotide sequence of SEQ ID NO: 23, and wherein the 5'-most nucleotide of the agonist comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; Compositions are provided in which at least one RLR agonist is packaged in a virus-like particle.

[0099] In any of the above or related embodiments, the RLR agonist is non-covalently attached to the viral particle.

[0100] In any of the above or related embodiments, the RLR agonist is attached to a site on the virus-like particle selected from the group consisting of an oligonucleotide-binding site, a DNA-binding site, and an RNA-binding site. In some embodiments, the virus-like particle comprises an arginine-rich repeat.

[0101] In any of the above or related embodiments, the compositions described herein comprise at least one antigen or antigenic determinant bound to a virus-like particle. In some embodiments, the at least one antigen or antigenic determinant is bound to the virus-like particle by at least one covalent bond. In some embodiments, the at least one antigen or antigenic determinant is bound to the virus-like particle by a non-peptide bond. In other embodiments, the antigen or antigenic determinant is fused to the virus-like particle. In some embodiments, the virus-like particle comprises at least one first binding site, and the antigen or antigenic determinant comprises at least one second binding site selected from the group consisting of (a) a binding site that does not naturally occur within the antigen or antigenic determinant, and (b) a binding site that naturally occurs within the antigen or antigenic determinant; Binding of the antigen or antigenic determinant to the virus-like particle occurs via association between a first binding site and a second binding site, optionally via at least one non-peptide bond. In some embodiments, the first binding site comprises an amino group or a lysine residue, and the second binding site comprises a sulfhydryl group or a cysteine ​​residue.

[0102] In some aspects, the present disclosure provides a pharmaceutical composition for stimulating an immune response, treating or slowing the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, the pharmaceutical composition comprising a composition provided by the present disclosure and a pharmaceutically acceptable carrier. In some aspects, the composition is formulated in a polyethyleneimine (PEI) carrier. In some aspects, the PEI carrier is JetPEI®.

[0103] In some aspects, the present disclosure provides methods for increasing RLR-mediated production of one or more cytokines in a cell, the method comprising contacting the cell with a composition provided by the present disclosure, wherein the composition increases RLR-mediated cytokine production in the cell. In some aspects, the composition increases RLR-mediated type I interferon (e.g., IFN-α, IFN-β) production in the cell. In some aspects, the composition increases RLR-mediated IL-1β production in the cell. In some aspects, the composition increases RLR-mediated IP-10 production in the cell. In some aspects, the composition increases RLR-mediated IL-6, IL-12p70, MCP-1, and / or MIP-1β production in the cell.

[0104] In some aspects, the present disclosure provides methods for increasing RLR-mediated expression of one or more interferon-stimulated genes in a cell, the method comprising contacting the cell with a composition provided by the present disclosure, wherein the composition increases RLR-mediated expression of one or more interferon-stimulated genes in the cell.

[0105] In some aspects, the present disclosure provides a method for increasing RLR-dependent intracellular signaling in a cell, the method comprising contacting the cell with a composition provided by the present disclosure, wherein the composition increases RLR-dependent intracellular signaling.

[0106] In some aspects, the present disclosure provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of a composition provided by the present disclosure.

[0107] In some aspects, the present disclosure provides methods of treating or slowing the progression of cancer in a subject, comprising administering to the subject an effective amount of a composition provided by the present disclosure.

[0108] In some aspects, the present disclosure provides methods for reducing or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an effective amount of a composition provided by the present disclosure.

[0109] In some aspects, the present disclosure provides methods for stimulating an immune response, treating or slowing the progression of cancer, or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an effective amount of a composition provided by the present disclosure, wherein the composition increases RLR-mediated production of one or more cytokines in the cell, increases RLR-mediated expression of one or more interferon-stimulated genes in the cell, and / or increases RLR-dependent intracellular signaling in the cell, thereby stimulating the immune response, treating or slowing the progression of cancer, or inhibiting tumor growth.

[0110] In some embodiments, the compositions provided by the present disclosure are administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immunotherapy, cytokine, activator or agonist of costimulatory molecule, inhibitor of inhibitory molecule, vaccine, cellular immunotherapy, or combinations thereof.

[0111] In some embodiments, the compositions provided by the present disclosure are administered prior to or following administration of one or more additional therapeutic agents, or one or more additional therapeutic agents are administered simultaneously with, prior to, or following administration of the agonist or pharmaceutical composition.

[0112] In some embodiments, the one or more additional therapeutic agents are a PD-1 / PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, or a Toll-like receptor 9 (TLR9) agonist.

[0113] In some embodiments, the one or more additional therapeutic agents is an agonist, including a polypeptide (eg, an antibody, or antigen-binding portion thereof) that specifically binds to CD137 (4-1BB).

[0114] In some embodiments, the one or more additional therapeutic agents is an agonist, including a polypeptide (eg, an antibody, or antigen-binding portion thereof) that specifically binds to CD134 (OX40).

[0115] In some embodiments, the one or more additional therapeutic agents are PD-1 / PD-L1 antagonists. In some embodiments, the PD-1 / PD-L1 antagonist is selected from the group consisting of PDR001, KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, the PD-1 / PD-L1 antagonist is selected from the group consisting of FAZ053, TENCENTRIQ® (atezolizumab), BAVENCIO® (avelumab), IMFINZI® (durvalumab), and BMS-936559.

[0116] In some embodiments, the one or more additional therapeutic agents is a TIM-3 antagonist.

[0117] In some embodiments, the one or more additional therapeutic agents is a VISTA antagonist.

[0118] In some embodiments, the one or more additional therapeutic agents is an adenosine A2AR antagonist.

[0119] In some embodiments, the one or more additional therapeutic agents is a B7-H3 antagonist.

[0120] In some embodiments, the one or more additional therapeutic agents is a B7-H4 antagonist.

[0121] In some embodiments, the one or more additional therapeutic agents is a BTLA antagonist.

[0122] In some embodiments, the one or more additional therapeutic agents is a CTLA-4 antagonist.

[0123] In some embodiments, the one or more additional therapeutic agents is an IDO antagonist.

[0124] In some embodiments, the one or more additional therapeutic agents is a KIR antagonist.

[0125] In some embodiments, the one or more additional therapeutic agents is a LAG-3 antagonist.

[0126] In some embodiments, the one or more additional therapeutic agents are Toll-like receptor 3 (TLR3) agonists. In some embodiments, the TLR3 agonist is polyinosinic:polycytidylic acid (poly I:C). In some embodiments, the TLR3 agonist is HILTONOL® (poly ICLC). In some embodiments, the TLR3 agonist is polyadenylic-polyuridylic acid (poly A:U). In some embodiments, the TLR3 agonist is RIBOXXIM® (RGIC® 100). In some embodiments, the TLR3 agonist is RIBOXXON® (RGIC® 50 bioconjugate). In some embodiments, the TLR3 agonist is RIBOXXOL® (RGIC® 50).

[0127] In some embodiments, the one or more additional therapeutic agents are Toll-like receptor 7 (TLR7) agonists. In some embodiments, the TLR7 agonist is GS-9620 (vesatolimod). In some embodiments, the TLR7 agonist is imiquimod (ALDARA™). In some embodiments, the TLR7 agonist is resiquimod (R-848).

[0128] In some embodiments, the one or more additional therapeutic agents are Toll-like receptor 9 (TLR9) agonists. In some embodiments, the TLR9 agonist is a CpG oligodeoxynucleotide (CpG ODN). In some embodiments, the CpG ODN is a class A CpG ODN (CpG-A ODN). In some embodiments, the CpG ODN is a class B CpG ODN (CpG-B ODN). In some embodiments, the CpG ODN is a class C CpG ODN (CpG-C ODN).

[0129] In some aspects, the present disclosure provides uses of the compositions provided by the present disclosure to stimulate an immune response, treat or slow the progression of cancer, or inhibit tumor growth in a subject in need thereof, optionally in combination with one or more additional therapeutic agents.

[0130] In some embodiments, the present disclosure provides use of a composition provided by the present disclosure in the manufacture of a medicament for stimulating an immune response, treating or slowing the progression of cancer, or inhibiting tumor growth in a subject in need thereof, optionally in combination with one or more additional therapeutic agents. In some embodiments, the composition is administered in combination with one or more additional therapeutic agents, where the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immunotherapy, cytokine, activator of costimulatory molecule, inhibitor of inhibitory molecule, vaccine, cellular immunotherapy, or combinations thereof. In some embodiments, the composition is administered before or after administration of one or more additional therapeutic agents, or the one or more additional therapeutic agents are administered simultaneously with, before, or after administration of the composition.

[0131] In some embodiments, the present disclosure provides kits comprising a composition provided by the present disclosure and instructions for use in stimulating a subject's immune response, treating or slowing the progression of cancer, or inhibiting tumor growth in a subject, optionally in combination with one or more additional therapeutic agents. In some embodiments, the kits comprise instructions for administering the composition in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immunotherapy, cytokine, activator of costimulatory molecule, inhibitor of inhibitory molecule, vaccine, cellular immunotherapy, or combinations thereof. In some embodiments, the composition is administered before or after administration of the one or more additional therapeutic agents, or the one or more additional therapeutic agents are administered simultaneously with, before, or after administration of the composition.

[0132] In any of the above or related embodiments, the one or more additional therapeutic agents is a PD-1 / PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, or a Toll-like receptor 9 (TLR9) agonist.

[0133] In any of the above or related embodiments, the one or more additional therapeutic agents is an agonist comprising a polypeptide (eg, an antibody, or antigen-binding portion thereof) that specifically binds to CD137 (4-1BB).

[0134] In any of the above or related embodiments, the one or more additional therapeutic agents is an agonist comprising a polypeptide (eg, an antibody, or antigen-binding portion thereof) that specifically binds to CD134 (OX40).

[0135] In some aspects, the present disclosure provides a method of making the compositions described herein, comprising: (a) disintegrating virus-like particles; and (b) adding an RLR agonist; (a) reassembling the virus-like particles.

[0136] In some embodiments, the method includes removing nucleic acids from the disassembled virus-like particles. In some embodiments, the method includes purifying the composition after reassembly. In some embodiments, the method includes (d) binding an antigen or antigenic determinant to the virus-like particle. In some embodiments, the antigen or antigenic determinant is bound to the virus-like particle before disassembling the virus-like particle. In other embodiments, the antigen or antigenic determinant is bound to the virus-like particle after reassembling the virus-like particle. The present invention provides, for example, the following items. (Item 1) (a) a virus-like particle; (b) at least one synthetic RIG-I-like receptor (RLR) agonist that specifically binds to an RLR, the RLR agonist comprising a ribonucleic acid (RNA) of 10 to 100 nucleotides in length, wherein the 5'-most nucleotide of the RNA comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof. The composition, wherein the at least one RLR agonist is packaged in the virus-like particle. (Item 2) 2. The composition of claim 1, wherein the RNA is single-stranded. (Item 3) 2. The composition of claim 1, wherein the RNA is double-stranded. (Item 4) 4. The composition according to any one of items 1 to 3, wherein the RNA is 10 to 15, 15 to 20, 20 to 25, 25 to 30, or 30 to 35 nucleotides in length. (Item 5) 2. The composition of claim 1, wherein the RLR agonist comprises a first polynucleotide and a second polynucleotide, and the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex. (Item 6) 6. The composition of claim 5, wherein the duplex comprises a hairpin. (Item 7) 7. The composition according to any one of items 5 to 6, wherein the duplex comprises 10 to 15, 15 to 20, 20 to 25, 25 to 30, or 30 to 35 base pairs. (Item 8) The composition according to any one of Items 5 to 6, wherein the duplex contains fewer than 19 base pairs. (Item 9) 9. The composition according to any one of items 5 to 8, wherein the first polynucleotide is linked to the second polynucleotide by a linker. (Item 10) 10. The composition of any one of items 5 to 9, wherein the RLR agonist comprises a sequence motif that confers at least one biological activity mediated by the RLR compared to an agonist that does not contain the sequence motif. (Item 11) The sequence motif is (i) GT repeat motif, (ii) GA repeat motif; (iii) AUCG repeat motif, (iv) AU repeat motifs; (v) dipyrimidine motif, (vi) ziprine motif, (vii) pyrimidine triplet motif, (viii) Printed triplet motif, (ix) a palindromic sequence motif, and (x) The composition according to item 10, selected from the group consisting of any combination of (i) to (ix). (Item 12) The at least one improved biological activity is (i) Increased RLR-mediated cytokine production; (ii) increased RLR-mediated expression of interferon-stimulated genes; (iii) increased RLR-mediated intracellular signaling; (iv) increased binding affinity to RLRs, and (v) The composition according to any one of items 10 to 11, selected from any combination of (i) to (iv). (Item 13) 13. The composition of items 10 to 12, wherein the sequence motif is a GT repeat motif comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. (Item 14) The GT repeat motif is [GT] n (wherein n=2 to 9). (Item 15) 15. The composition according to any one of items 13 to 14, wherein the GT repeat motif is [GT]7. (Item 16) The composition according to any one of items 13 to 14, wherein the GT repeat motif is [GT]3, and the GT repeat motif is followed by a print triplet motif and a UCG motif, respectively. (Item 17) Item 17. The composition of item 16, wherein the print triplet motif is GGA. (Item 18) 13. The composition of any one of items 10 to 12, wherein the sequence motif is a GA repeat motif comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. (Item 19) The GA repeat motif is [GA] n (wherein n=2 to 9). (Item 20) 20. The composition of claim 19, wherein the GA repeat motif is [GA]7. (Item 21) 13. The composition of any one of items 10 to 12, wherein the sequence motif is an AUCG repeat motif comprising a sequence of less than 19, about 16, about 12-16, about 12, about 8-12, about 6, 16, 12, or 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. (Item 22) The AUCG repeat motif is [AUCG] n (wherein n=2 to 4). (Item 23) 23. The composition of claim 22, wherein the AUCG repeat motif is [AUCG]3. (Item 24) 24. The composition according to any one of items 21 to 23, wherein the AUCG repeat motif is preceded by a CG or dipyrimidine motif. (Item 25) 25. The composition of claim 24, wherein the AUCG repeat motif is preceded by a CG. (Item 26) 25. The composition of claim 24, wherein the dipyrimidine motif is CC. (Item 27) 24. The composition according to any one of items 21 to 23, wherein the AUCG repeat motif is preceded by a dipurine motif. (Item 28) 28. The composition of claim 27, wherein the dipurine motif is GA. (Item 29) 28. The composition according to item 27, wherein the dipurine motif is II. (Item 30) 30. The composition according to any one of items 21 to 29, wherein the U constituting the AUCG repeat motif is substituted with a modified nucleoside. (Item 31) 31. The composition of claim 30, wherein the modified nucleoside is ribothymidine (T). (Item 32) 30. The composition according to any one of items 21 to 29, wherein the G constituting the AUCG repeat motif is substituted with a modified nucleoside. (Item 33) 33. The composition of claim 32, wherein the modified nucleoside is inosine (I). (Item 34) 24. The composition of any one of items 21 to 23, wherein the AUCG repeat motif is preceded by IG. (Item 35) 24. The composition according to any one of items 21 to 23, wherein the G constituting the AUCG repeat is replaced with inosine (I), and the AUCG repeat is preceded by inosine (I). (Item 36) 36. The composition of claim 35, wherein the 5'-most nucleotide of the first polynucleotide is inosine (I). (Item 37) 23. The composition according to any one of items 21 to 22, wherein the AUCG repeat motif is [AUCG]2. (Item 38) 38. The composition of claim 37, wherein the AUCG repeat motif is preceded by a dipurine motif. (Item 39) 39. The composition of claim 38, wherein the dipurine motif is GG. (Item 40) 38. The composition of claim 37, wherein the AUCG repeat motif is preceded by a print triplet. (Item 41) Item 41. The composition of item 40, wherein the print replica is GGG. (Item 42) The composition according to item 37, wherein the AUCG repeat motif is preceded by CCCCCG. The composition according to item 37, wherein the AUCG repeat motif is preceded by TCGUCG. 13. The composition of any one of items 10 to 12, wherein the sequence motif is a palindromic sequence comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, linked in any order to generate a palindrome. (Item 45) 45. The composition according to any one of items 9 to 44, wherein the linker is flanked by AU. (Item 46) The linker is flanked by AU repeat motifs, and the AU repeat motifs are [AU] n (wherein n=2 to 3). (Item 47) 47. The composition of claim 46, wherein the AU repeat motif is [AU]2. (Item 48) (a) a virus-like particle; (b) at least one RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide, at least one of N1, N2, N3, and N4 is inosine, and / or at least one of X1 and / or X2 comprises at least one inosine nucleoside, said inosine nucleoside base-pairing with a cytidine in said hairpin RNA; The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 49) 49. The composition of item 48, wherein N1 comprises inosine and N4 comprises cytidine. (Item 50) 49. The composition of item 48, wherein N1 comprises cytidine and N4 comprises inosine. (Item 51) 49. The composition of claim 48, wherein N2 comprises inosine and N3 comprises cytidine. (Item 52) 49. The composition of item 48, wherein N2 comprises cytidine and N3 comprises inosine. (Item 53) 49. The composition of item 48, wherein N1 comprises guanosine. (Item 54) 49. The composition of claim 48, wherein N2 comprises guanosine. (Item 55) 49. The composition of claim 48, wherein N1 comprises cytidine. (Item 56) 49. The composition of claim 48, wherein N2 comprises cytidine. (Item 57) 49. The composition of item 48, wherein N1 and N2 comprise guanosine and N3 and N4 comprise cytidine. (Item 58) 49. The composition of item 48, wherein N1 and N2 comprise cytidine and N3 and N4 comprise guanosine. (Item 59) 49. The composition of item 48, wherein N1 and N2 comprise inosine and N3 and N4 comprise cytidine. (Item 60) 49. The composition of item 48, wherein N1 and N2 comprise cytidine and N3 and N4 comprise inosine. (Item 61) 49. The composition of item 48, wherein N1 comprises inosine, N4 comprises cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 62) 49. The composition of item 48, wherein N2 comprises inosine, N3 comprises cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 63) 49. The composition of item 48, wherein N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 64) 49. The composition of item 48, wherein N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. (Item 65) 49. The composition of item 48, wherein N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise at least one inosine. (Item 66) The composition according to Item 48, wherein N1 and N2 contain guanosine, N3 and N4 contain cytidine, and X1 and X2 each contain inosine, and the composition does not contain a guanosine nucleoside. (Item 67) The composition according to Item 48, wherein N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise inosine, and the composition does not comprise a guanosine nucleoside. (Item 68) 49. The composition of item 48, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 69) 49. The composition of item 48, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. (Item 70) 49. The composition of item 48, wherein N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and / or X2 each comprise at least one inosine. (Item 71) 49. The composition of claim 48, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 comprise inosine and do not comprise guanosine nucleosides. (Item 72) 49. The composition of claim 48, wherein N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and X2 comprise inosine, and the composition does not include guanosine nucleosides. (Item 73) 49. The composition of item 48, wherein X1 and X2 are each 12 nucleotides and contain 1, 2, 3 or 4 inosine nucleosides. (Item 74) 49. The composition of item 48, wherein X1 and X2 are each 13 nucleotides and contain 1, 2, 3, 4 or 5 inosine nucleosides. (Item 75) 49. The composition of item 48, wherein X1 and X2 are each 14 nucleotides and contain 1, 2, 3, 4, 5, or 6 inosine nucleosides. (Item 76) 49. The composition of item 48, wherein X1 and X2 are each 15 nucleotides and contain 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. (Item 77) 49. The composition of item 48, wherein X1 and X2 are each 16 nucleotides and each contain 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. (Item 78) 49. The composition of item 48, wherein X1 and X2 are each 12 nucleotides and contain at least 10%, 20%, 30%, or 40% inosine nucleosides. (Item 79) (a) a virus-like particle; (b) at least one synthetic RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 has the sequence motif [AUCN5] x (wherein N5 contains guanosine or inosine, and x is an integer representing the number of sequence motifs, and x is 2 to 4), (viii) X2 has the sequence motif [CN6AU] y (wherein N6 contains guanosine or inosine, and y is an integer representing the number of sequence motifs, y=2 to 4), (ix) L is a linker that operably links the first polynucleotide and the second polynucleotide; optionally, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside base pairs with a cytidine in the hairpin RNA; The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 80) 80. The composition of item 79, wherein N5 comprises inosine and N6 comprises inosine. (Item 81) 81. The composition of item 80, wherein N5 comprises guanosine and N6 comprises inosine. (Item 82) 82. The composition of item 81, wherein N5 comprises inosine and N6 comprises guanosine. (Item 83) 83. The composition of claim 82, wherein N5 comprises guanosine (G) and N6 comprises guanosine (G). (Item 84) 84. The composition according to any one of items 79 to 83, wherein (i) x=2 and y=2, or (ii) x=3 and y=3. (Item 85) 84. The composition according to any one of items 79 to 83, wherein x=4 and y=4. (Item 86) 84. The composition of any one of items 79 to 83, wherein N1 comprises inosine (I) and N4 comprises cytidine (C). (Item 87) 84. The composition of any one of items 79 to 83, wherein N2 comprises inosine (I) and N3 comprises cytidine (C). (Item 88) 84. The composition of any one of items 79 to 83, wherein N3 comprises inosine (I) and N2 comprises cytidine (C). (Item 89) 84. The composition of any one of items 79 to 83, wherein N4 comprises inosine (I) and N1 comprises cytidine (C). (Item 90) 84. The composition of any one of items 79 to 83, wherein N1 comprises guanosine (G). (Item 91) 84. The composition of any one of items 79 to 83, wherein N2 comprises guanosine (G). (Item 92) 84. The composition of any one of items 79 to 83, wherein N1 comprises cytidine (C). (Item 93) 84. The composition of any one of items 79 to 83, wherein N2 comprises cytidine (C). (Item 94) 84. The composition of any one of items 79 to 83, wherein N1 and N2 comprise guanosine (G) and N3 and N4 comprise cytidine (C). (Item 95) 84. The composition of any one of items 79 to 83, wherein N1 and N2 comprise cytidine (C) and N3 and N4 comprise guanosine (G). (Item 96) 84. The composition of any one of items 79 to 83, wherein N1 and N2 comprise inosine (I) and N3 and N4 comprise cytidine (C). (Item 97) 84. The composition according to any one of items 79 to 83, wherein N1 and N2 comprise cytidine (C) and N3 and N4 comprise inosine (I). (Item 98) (a) a virus-like particle; (b) at least one synthetic RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, and having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker covalently linking the first polynucleotide and the second polynucleotide; When inosine is present, it base pairs with cytidine, The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 99) 99. The composition of item 98, wherein N1 comprises inosine and N4 comprises cytidine. (Item 100) 99. The composition of item 98, wherein N1 comprises cytidine and N4 comprises inosine. (Item 101) 99. The composition of item 98, wherein N2 comprises inosine and N3 comprises cytidine. (Item 102) 99. The composition of item 98, wherein N2 comprises cytidine and N3 comprises inosine. (Item 103) 99. The composition of item 98, wherein N1 comprises guanosine. (Item 104) 99. The composition of item 98, wherein N2 comprises guanosine. (Item 105) 99. The composition of item 98, wherein N1 comprises cytidine. (Item 106) 99. The composition of item 98, wherein N2 comprises cytidine. (Item 107) 99. The composition of item 98, wherein N1 and N2 comprise guanosine and N3 and N4 comprise cytidine. (Item 108) 99. The composition of item 98, wherein N1 and N2 comprise cytidine and N3 and N4 comprise guanosine. (Item 109) 99. The composition of item 98, wherein N1 and N2 comprise inosine and N3 and N4 comprise cytidine. (Item 110) 99. The composition of item 98, wherein N1 and N2 comprise cytidine and N3 and N4 comprise inosine. (Item 111) 99. The composition of item 98, wherein N1 comprises inosine, N4 comprises cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 112) 99. The composition of item 98, wherein N2 comprises inosine, N3 comprises cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 113) 99. The composition of item 98, wherein N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 114) 99. The composition of item 98, wherein N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. (Item 115) 99. The composition of item 98, wherein N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise at least one inosine. (Item 116) The composition according to Item 98, wherein N1 and N2 comprise guanosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise inosine, and the composition does not comprise a guanosine nucleoside. (Item 117) The composition according to Item 98, wherein N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise inosine, and the composition does not comprise a guanosine nucleoside. (Item 118) 99. The composition of item 98, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine. (Item 119) 99. The composition of item 98, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. (Item 120) 99. The composition of item 98, wherein N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and / or X2 each comprise at least one inosine. (Item 121) 99. The composition of item 98, wherein N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 comprise inosine and do not comprise guanosine nucleosides. (Item 122) 99. The composition of item 98, wherein N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and X2 comprise inosine, and the composition does not include guanosine nucleosides. (Item 123) 99. The composition of item 98, wherein X1 and X2 are each 12 nucleotides and contain 1, 2, 3, or 4 inosine nucleosides. (Item 124) 99. The composition of item 98, wherein X1 and X2 are each 13 nucleotides and contain 1, 2, 3, 4, or 5 inosine nucleosides. (Item 125) 99. The composition of item 98, wherein X1 and X2 are each 14 nucleotides and contain 1, 2, 3, 4, 5, or 6 inosine nucleosides. (Item 126) 99. The composition of item 98, wherein X1 and X2 are each 15 nucleotides and contain 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. (Item 127) 99. The composition of item 98, wherein X1 and X2 are each 16 nucleotides and each contain 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. (Item 128) 99. The composition of item 98, wherein X1 and X2 are each 12 nucleotides and contain at least 10%, 20%, 30%, or 40% inosine nucleosides. (Item 129) 98. The composition according to any one of items 9 to 97, wherein the linker is a nucleotide linker or a non-nucleotide linker. (Item 130) 130. The composition of claim 129, wherein the linker is a non-nucleotidic linker. (Item 131) Item 130. The composition of item 129, wherein the linker is a nucleotide linker. (Item 132) the nucleotide linker comprises a tetraloop, and the nucleotide sequence of the tetraloop is (a) UNCG (where N=A, C, G, or U), (b) GNRA (where N=A, C, G, or U and R=A or G); (c) ANYA (where N=A, C, G, or U and Y=C or T); (d) CUYG (where Y = C or T), (e) UMAC (where M=A or C), and (f) The composition according to item 131, selected from the group consisting of CUUG. (Item 133) Item 132. The composition of item 131, wherein the nucleotide linker comprises the nucleotide sequence UUUGAU or UGUUU. (Item 134) Item 133. The composition of item 132, wherein the sequence of the tetraloop is UUCG. (Item 135) Item 133. The composition of item 132, wherein the sequence of the tetraloop is GAUC. (Item 136) Item 134. The composition of item 133, wherein the nucleotide linker comprises the nucleotide sequence UUUGAU. (Item 137) Item 134. The composition of item 133, wherein the nucleotide linker comprises the nucleotide sequence UGUUU. (Item 138) the non-nucleotide linker (a) an ethylene glycol linker, and (b) the composition according to any one of items 98 to 128 and 130, selected from the group consisting of alkyl linkers. (Item 139) 139. The composition of claim 138, wherein the non-nucleotidic linker is a hexaethylene glycol linker. (Item 140) Item 139. The composition of item 138, wherein the non-nucleotidic linker is a C9 alkyl linker. (Item 141) 141. The composition of any one of items 1 to 140, wherein the agonist comprises a 5' diphosphate moiety, or a derivative or analogue thereof. (Item 142) 141. The composition of any one of items 1 to 140, wherein the agonist comprises a 5' triphosphate moiety, or a derivative or analogue thereof. (Item 143) 143. The composition according to item 141 or 142, wherein the derivative or analog thereof comprises a bioequivalent of a phosphate selected from phosphonates, thiophosphonates, phosphorothioates, sulfates, sulfonates, sulfamates, thiazolidinones, carboxylates, malonates, boronic acids, benzoxaboroles, boranophosphates, squaramides. (Item 144) 144. The composition of any one of items 1 to 143, wherein the agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleobase, or a combination thereof. (Item 145) 145. The composition of any one of items 1 to 144, wherein the agonist comprises a modification to an internucleotide bond or polynucleotide backbone. (Item 146) The agonist has the following characteristics: (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5, and / or LGP2); (b) increase RLR-mediated cytokine production; (c) increasing RLR-mediated expression of interferon-stimulated genes (ISGs); (d) increasing RLR-dependent intracellular signaling; (e) increasing duplex stability; (f) increasing binding affinity to RLRs; (g) reducing off-target binding; (h) increasing biological half-life; (i) Increase biodistribution and bioavailability; (j) increasing and / or enhancing uptake into cells and / or tissues; (k) reducing immunogenicity; and (l) The composition according to any one of items 1 to 145, exhibiting at least one or more of any combination of (a) to (k). (Item 147) (a) a virus-like particle; (b) at least one synthetic RLR agonist that specifically binds to an RLR, wherein the 5'-most nucleotide of the agonist comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-36; The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 148) (a) a virus-like particle; (b) at least one synthetic RLR agonist that specifically binds to an RLR, the synthetic RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to form a duplex with the second polynucleotide, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, the agonist comprising the sequence motif that confers at least one improved biological activity mediated by the RLR compared to an agonist that does not comprise the sequence motif, and the first polynucleotide and the second polynucleotide (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively; and (xxxii) the at least one synthetic RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97, respectively; The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 149) (a) a virus-like particle; (b) at least one synthetic RLR agonist that specifically binds to an RLR, the at least one synthetic RLR agonist comprising a blunt-ended hairpin RNA comprising at least one nucleotide that includes an inosine that base pairs with a cytidine, the at least one synthetic RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25; The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 150) (a) a virus-like particle; (b) at least one synthetic RLR agonist that specifically binds to an RLR, the synthetic RLR agonist comprising a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, the first polynucleotide and the second polynucleotide being: (i) SEQ ID NOs: 58 and 89, respectively; (ii) SEQ ID NOs: 59 and 89, respectively; and (iii) each comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 61 and 91; The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 151) (a) a virus-like particle; (b) at least one synthetic RLR agonist that specifically binds to an RLR, the synthetic RLR agonist comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide are (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively; and (xxxii) each comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97; and The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 152) 2. The composition of any one of the preceding items, wherein the nucleotide sequence comprising the agonist is not complementary to a genomic DNA sequence or an mRNA sequence, wherein the RLR agonist is not involved in RNA interference, and wherein the RLR agonist does not silence gene expression. (Item 153) Item 10. The composition of any one of the preceding items, wherein the virus-like particle does not have a lipoprotein-containing envelope. (Item 154) 153. The composition of any one of items 1 to 152, wherein the virus-like particle is a recombinant virus-like particle. (Item 155) the recombinant virus-like particle (a) Hepatitis B virus recombinant protein, (b) recombinant measles virus proteins; (c) recombinant proteins of Sindbis virus; (d) rotavirus recombinant proteins; (e) recombinant proteins of hand, foot and mouth disease virus; (f) retroviral recombinant proteins; (g) Norwalk virus recombinant protein; (h) recombinant human papillomavirus proteins; (i) BK virus recombinant proteins, (j) bacteriophage recombinant proteins; (k) recombinant proteins of RNA phages; (l) recombinant proteins of Qβ phage; (m) recombinant proteins of GA phage; (n) fr phage recombinant protein, (o) Recombinant proteins of AP205 phage; (p)Ty recombinant protein, and (q) The composition according to Item 154, wherein the recombinant protein is selected from the group consisting of a fragment of any of (a) to (p). (Item 156) 153. The composition of any one of items 1 to 152, wherein the virus-like particle comprises recombinant proteins of an RNA phage, and the RNA phage is selected from the group consisting of (a) bacteriophage Qβ, (b) bacteriophage R17, (c) bacteriophage fr, (d) bacteriophage GA, (e) bacteriophage SP, (f) bacteriophage MS2, (g) bacteriophage M11, (h) bacteriophage MX1, (i) bacteriophage NL95, (j) bacteriophage f2, (k) bacteriophage PP7, and (l) bacteriophage AP205. (Item 157) 157. The composition of claim 156, wherein the virus-like particle comprises recombinant proteins of bacteriophage Qβ. (Item 158) 153. The composition of any one of items 1 to 152, wherein the virus-like particle comprises recombinant proteins of RNA phage Qβ, or a fragment thereof. (Item 159) (a) Virus-like particles of RNA phage Qβ and (b) at least one synthetic RLR agonist that specifically binds to an RLR, the at least one synthetic RLR agonist comprising a ribonucleic acid (RNA) of 10 to 100 nucleotides in length, wherein the 5'-most nucleotide of the RNA comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 160) (a) Virus-like particles of RNA phage Qβ and (b) at least one synthetic RLR agonist that specifically binds to an RLR, the agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to form a duplex with the second polynucleotide, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first oligonucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprising: (i) GT repeat motif, (ii) GA repeat motif; (iii) AUCG repeat motif, (iv) AU repeat motifs; (v) dipyrimidine motif, (vi) ziprine motif, (vii) pyrimidine triplet motif, (viii) Printed triplet motif, (ix) a palindromic sequence motif, and (x) the at least one synthetic RLR agonist comprising a sequence motif selected from any combination of (i) to (ix); The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 161) (a) Virus-like particles of RNA phage Qβ and (b) at least one synthetic RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3' [in the formula, (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 contains the sequence motif [AUCN5]x (wherein N5 contains guanosine or inosine, and x is an integer representing the number of sequence motifs, and x = 2 to 4); (viii) X2 contains a sequence motif [CN6AU]y (wherein N6 contains guanosine or inosine, and y is an integer representing the number of sequence motifs, y=2 to 4); (ix) L is a linker that operably links the first polynucleotide and the second polynucleotide; optionally, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside base pairs with a cytidine in the hairpin RNA; The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 162) (a) Virus-like particles of RNA phage Qβ and (b) at least one synthetic RLR agonist that specifically binds to an RLR, said at least one synthetic RLR agonist comprising the nucleotide sequence of SEQ ID NO: 23, wherein the 5'-most nucleotide of said agonist comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; The composition, wherein said at least one RLR agonist is packaged in said virus-like particle. (Item 163) 163. The composition of any one of items 159 to 162, wherein the virus-like particles comprise RNA phage Qβ coat proteins each having the amino acid sequence of SEQ ID NO: 112. (Item 164) 163. The composition of any one of items 159 to 162, wherein the virus-like particle comprises an RNA phage Qβ coat protein having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 112, respectively. (Item 165) The composition of any one of the preceding items, wherein the RLR agonist is non-covalently bound to the virus-like particle. (Item 166) 165. The composition of any one of items 1 to 164, wherein the RLR agonist is bound to a site on the virus-like particle selected from the group consisting of an oligonucleotide binding site, a DNA binding site, and an RNA binding site. (Item 167) 167. The composition of claim 166, wherein the virus-like particle site comprises an arginine-rich repeat. (Item 168) Item 10. The composition of any one of the preceding items, further comprising at least one antigen or antigenic determinant bound to the virus-like particle. (Item 169) 169. The composition of claim 168, wherein the at least one antigen or antigenic determinant is bound to the virus-like particle by at least one covalent bond. (Item 170) 169. The composition of claim 168, wherein the at least one antigen or antigenic determinant is attached to the virus-like particle by a non-peptide bond. (Item 171) 169. The composition of claim 168, wherein the antigen or antigenic determinant is fused to the virus-like particle. (Item 172) the virus-like particle comprises at least one first binding site, and the antigen or antigenic determinant comprises at least one second binding site selected from the group consisting of: (a) a binding site that does not naturally occur within the antigen or antigenic determinant, and (b) a binding site that naturally occurs within the antigen or antigenic determinant; 169. The composition of claim 168, wherein the binding of the antigen or antigenic determinant to the virus-like particle is via association between the first binding site and the second binding site, and optionally the binding is via at least one non-peptide bond. (Item 173) 173. The composition of claim 172, wherein the first binding site comprises an amino group or a lysine residue and the second binding site comprises a sulfhydryl group or a cysteine ​​residue. (Item 174) 174. A pharmaceutical composition for stimulating an immune response, treating or slowing the progression of cancer, or reducing or inhibiting tumor growth in a subject in need thereof, comprising the composition according to any one of items 1 to 173 and a pharmaceutically acceptable carrier. (Item 175) Item 176. The pharmaceutical composition of Item 174, formulated in a polyethyleneimine (PEI) carrier. Item 177. The pharmaceutical composition of Item 175, wherein the PEI carrier is JetPEI®. 174. A method for increasing RLR-mediated production of one or more cytokines in a cell, comprising contacting said cell with the composition of any one of items 1 to 173, wherein said agonist increases RLR-mediated cytokine production in the cell. (Item 178) 174. A method for increasing RLR-mediated expression of one or more interferon-stimulated genes in a cell, comprising contacting the cell with the composition of any one of items 1 to 173, wherein the agonist increases RLR-mediated expression of one or more interferon-stimulated genes in the cell. (Item 179) 174. A method for increasing RLR-dependent intracellular signaling in a cell, comprising contacting said cell with the composition of any one of items 1 to 173, wherein said agonist increases RLR-dependent intracellular signaling. (Item 180) 176. A method for stimulating an immune response in a subject, comprising administering to the subject an effective amount of the composition according to any one of items 1 to 173, or the pharmaceutical composition according to any one of items 174 to 176. (Item 181) 176. A method for treating or delaying the progression of cancer in a subject, comprising administering to the subject an effective amount of the composition according to any one of items 1 to 173, or the pharmaceutical composition according to any one of items 174 to 176. (Item 182) 176. A method for reducing or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an effective amount of the composition according to any one of items 1 to 173 or the pharmaceutical composition according to any one of items 174 to 176. (Item 183) A method for stimulating an immune response, treating or delaying the progression of cancer, or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an effective amount of the composition according to any one of items 1 to 173, or the pharmaceutical composition according to any one of items 174 to 176, wherein the agonist increases RLR-mediated production of one or more cytokines in a cell, increases RLR-mediated expression of one or more interferon-stimulated genes in a cell, and / or increases RLR-dependent intracellular signaling in a cell, thereby stimulating the immune response, treating or delaying the progression of cancer, or inhibiting tumor growth. (Item 184) 184. The method of any one of items 180 to 183, wherein the composition is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immunotherapy, cytokine, activator or agonist of costimulatory molecule, inhibitor of inhibitory molecule, vaccine, cellular immunotherapy, or combination thereof. (Item 185) 185. The method of claim 184, wherein the composition is administered before or after the administration of the one or more additional therapeutic agents, or the one or more additional therapeutic agents are administered simultaneously with, before, or after the administration of the agonist or pharmaceutical composition. (Item 186) 186. The method of item 184 or 185, wherein the one or more additional therapeutic agents is a PD-1 / PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, or a Toll-like receptor 9 (TLR9) agonist. (Item 187) 186. The method of item 184 or 185, wherein the one or more additional therapeutic agents is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD137 (4-1BB). (Item 188) 186. The method of paragraph 184 or 185, wherein the one or more additional therapeutic agents is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD134 (OX40). (Item 189) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a PD-1 / PD-L1 antagonist. (Item 190) 189. The method of claim 189, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of PDR001, KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. (Item 191) 189. The method of item 189, wherein the PD-1 / PD-L1 antagonist is selected from the group consisting of FAZ053, TENCENTRIQ® (atezolizumab), BAVENCIO® (avelumab), IMFINZI® (durvalumab), and BMS-936559. (Item 192) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a TIM-3 antagonist. (Item 193) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a VISTA antagonist. (Item 194) 187. The method of claim 186, wherein the one or more additional therapeutic agents is an adenosine A2AR antagonist. (Item 195) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a B7-H3 antagonist. (Item 196) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a B7-H4 antagonist. (Item 197) Item 187. The method of item 186, wherein the one or more additional therapeutic agents is a BTLA antagonist. (Item 198) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a CTLA-4 antagonist. (Item 199) 187. The method of claim 186, wherein the one or more additional therapeutic agents is an IDO antagonist. (Item 200) 187. The method of item 186, wherein the one or more additional therapeutic agents is a KIR antagonist. (Item 201) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a LAG-3 antagonist. (Item 202) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a Toll-like receptor 3 (TLR3) agonist. (Item 203) 203. The method of claim 202, wherein the TLR3 agonist is polyinosinic:polycytidylic acid (poly I:C). (Item 204) 203. The method of claim 202, wherein the TLR3 agonist is HILTONOL® (poly ICLC). (Item 205) 203. The method of claim 202, wherein the TLR3 agonist is polyadenylic acid-polyuridylic acid (poly A:U). (Item 206) Item 203. The method of item 202, wherein the TLR3 agonist is RIBOXXIM® (RGIC® 100). (Item 207) 203. The method of claim 202, wherein the TLR3 agonist is RIBOXXON® (RGIC® 50 bioconjugate). (Item 208) Item 203. The method of item 202, wherein the TLR3 agonist is RIBOXXOL® (RGIC® 50). (Item 209) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a Toll-like receptor 7 (TLR7) agonist. (Item 210) 209. The method of claim 209, wherein the TLR7 agonist is GS-9620 (vesatolimod). (Item 211) 209. The method of claim 209, wherein the TLR7 agonist is imiquimod (ALDARA™). (Item 212) 209. The method of claim 209, wherein the TLR7 agonist is resiquimod (R-848). (Item 213) 187. The method of claim 186, wherein the one or more additional therapeutic agents is a Toll-like receptor 9 (TLR9) agonist. (Item 214) 214. The method of item 213, wherein the TLR9 agonist is a CpG oligodeoxynucleotide (CpG ODN). (Item 215) 215. The method of item 214, wherein the CpG ODN is a class A CpG ODN (CpG-A ODN). (Item 216) 215. The method of item 214, wherein the CpG ODN is a class B CpG ODN (CpG-B ODN). (Item 217) 215. The method of item 214, wherein the CpG ODN is a class C CpG ODN (CpG-C ODN). (Item 218) 177. Use of the composition according to any one of items 1 to 173 or the pharmaceutical composition according to any one of items 174 to 176 for stimulating an immune response, treating or delaying the progression of cancer, or inhibiting tumor growth in a subject in need thereof, optionally in combination with one or more further therapeutic agents. (Item 219) 176. Use of the composition according to any one of items 1 to 173 or the pharmaceutical composition according to any one of items 174 to 176 in the manufacture of a medicament for stimulating an immune response, treating or delaying the progression of cancer, or inhibiting tumor growth in a subject in need thereof, optionally in combination with one or more further therapeutic agents. (Item 220) A kit comprising the composition according to any one of items 1 to 173 or the pharmaceutical composition according to any one of items 174 to 176 and instructions for use in stimulating an immune response in a subject, or treating or slowing the progression of cancer, or inhibiting tumor growth in a subject, optionally together with instructions for use in combination with one or more further therapeutic agents. (Item 221) 221. The use of item 218 or the kit of item 220, wherein the agonist or pharmaceutical composition is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents are selected from the group consisting of chemotherapy, targeted anti-cancer therapy, oncolytic agent, cell death inducer, opsonizing agent (e.g., opsonizing antibody), cytotoxic agent, immunotherapy, cytokine, activator of costimulatory molecule, inhibitor of inhibitory molecule, vaccine, cellular immunotherapy, or combination thereof. (Item 222) 222. The use or kit according to item 221, wherein the agonist or pharmaceutical composition is administered before or after the administration of the one or more additional therapeutic agents, or the one or more additional therapeutic agents are administered simultaneously with, before or after the administration of the agonist or pharmaceutical composition. (Item 223) 223. The use of any one of items 218, 219, 221, or 222, or the kit of items 220 to 222, wherein the one or more additional therapeutic agents is a PD-1 / PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, a Toll-like receptor 9 (TLR9) agonist. (Item 224) 223. The use of any one of items 218, 219, 221, or 222, or the kit of items 220 to 222, wherein the one or more additional therapeutic agents is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD137(4-1BB). (Item 225) 223. The use of any one of items 218, 219, 221, or 222, or the kit of items 220-222, wherein the one or more additional therapeutic agents is an agonist comprising a polypeptide (e.g., an antibody, or an antigen-binding portion thereof) that specifically binds to CD134 (OX40). (Item 226) A method for producing the composition according to any one of items 1 to 173, (a) degrading the virus-like particles; (b) adding the RLR agonist; (a) reassembling the virus-like particles. (Item 227) 227. The method of claim 226, further comprising removing nucleic acid from the degraded virus-like particles. (Item 228) 228. The method of claim 226 or 227, further comprising purifying the composition after reassembly. (Item 229) (d) attaching an antigen or antigenic determinant to said virus-like particle. (Item 230) 230. The method of claim 229, wherein the antigen or antigenic determinant is bound to the virus-like particle prior to disassembling the virus-like particle. (Item 231) 230. The method of claim 229, wherein the antigen or antigenic determinant is attached to the virus-like particle after the virus-like particle is reassembled. [Brief explanation of the drawings]

[0137] [Figure 1]1 shows a bar graph depicting the quantification of secretion of the cytokine IFN-α2a from human PBMCs treated with 0.4 nM, 2 nM, and 10 nM of RLR agonists with various modifications. [Figure 2] 1 shows a bar graph depicting quantification of IFN-α secretion from human PBMCs treated with RIG 50c (X24907) and inosine-substituted RIG 27c (X24935) at concentrations of 0.2 nM, 2 nM, 20 nM, and 200 nM. [Figure 3] 1 shows a bar graph depicting quantification of IFN-α secretion from human PBMCs treated with Qβ-RIG27 (RIG 27c packaged in VLPs containing RNA phage Qβ coat protein) at concentrations of 2 nM, 20 nM, 200 nM, and 600 nM. DETAILED DESCRIPTION OF THE INVENTION

[0138] overview RIG-I-like receptors (RLRs) are a family of cytosolic pattern recognition receptors essential for detecting viral RNA and initiating innate immune responses. The RLR family includes three members: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (Laboratory of Genetics and Physiology 2). These receptors are expressed in both immune and non-immune cell types and regulate signaling pathways that promote IRF3- and IRF7-dependent expression of type I and III interferons (IFNs) and NF-κB-dependent expression of proinflammatory cytokines.

[0139] All three RLR family receptors contain a DExD / H-box RNA helicase domain with ATPase activity, which, together with the adjacent C-terminal domain, is required for RNA binding. Furthermore, the C-terminal domains of RIG-I and LGP2 have been shown to act as repressor domains, maintaining these receptors in an inactive conformation until activating RNA binds.

[0140] The present disclosure provides RLR agonists, including synthetic RNA molecules that fold to form double-stranded dsRNA and contain one or more sequence motifs that confer one or more improved biological activities. The present disclosure also provides compositions that include at least one RLR agonist packaged in a VLP, which have improved immunostimulatory efficacy, such as the induction of cytokine expression, compared to the VLP alone. These RIG-VLPs provide improved immunostimulatory compositions that can be used, for example, in preventive or therapeutic regimens for tumors.

[0141] RIG-I-like receptors and their ligands The present disclosure provides synthetic RNA ligands that specifically bind to and stimulate RIG-I-like receptors (RLRs) (RLR agonists). In some aspects, the present disclosure provides RLR agonists that are useful for treating cancer. In some aspects, the present disclosure provides RLR agonists that are useful for treating infectious diseases. In some embodiments, the RLR agonists induce cytokine production. In some embodiments, the RLR agonists increase the number of CD8+ T cells in the tumor microenvironment. In some aspects, the RLR agonists induce protective anti-tumor immunity.

[0142] RIG-I-like receptors (RLRs) comprise a family of DExD / H-box RNA helicases that function as cytosolic pattern recognition receptors (PRRs) that detect the presence of pathogens through the recognition of pathogen-associated molecular patterns (PAMPs). In particular, the intracellular presence of non-self (e.g., viral) RNA is detected by infected cells through RNA binding to RLRs, leading to the initiation and regulation of antiviral immunity. Like many viral RNAs, endogenous mRNAs and RNA polymerase III transcripts are 5' triphosphorylated. However, eukaryotic mRNAs have a 5' cap structure linked to an N7-methylated guanosine, which prevents RIG-I activation. These structural differences between viral and self RNAs, along with their differential subcellular localization, may enable RIG-I to effectively function as a defense mechanism against viral infection by selectively detecting viral RNA. Molecular recognition and binding of non-self RNA ligands to RLRs propagates specific intracellular signaling events, resulting in the activation of transcription factors that induce type 1 interferon (IFN) production and antiviral gene expression. RLR-mediated induction of IFN and proinflammatory cytokine production and antiviral gene expression triggers immune responses to suppress and control viral infection (Yoneyama et al., (2015) Curr Opin Immunol 32:48-53).

[0143] Three RLR family members have been identified: RIG-I (retinoic acid-inducible gene I) (the major and best-characterized member of the RLR family), MDA5 (melanoma differentiation-associated factor 5), and LGP2 (laboratory of genetics and physiology 2, a homolog of mouse D11 lgp2). RIG-I is a key component of the innate immune system and plays a crucial role in defense against infection with RNA viruses. Unlike the Toll-like receptors TLR3, TLR7, TLR8, and TLR9, which detect nucleic acids in the endosomes of a subset of immune cells, RIG-I is a cytosolic innate immune receptor expressed in all cell types (Kato et al.). (Hornung et al., (2006) Nature 441(7089):101-105; Loo et al., (2008) J Virol 82(1):335-345). Two early studies independently established that RIG-I specifically detects and activates viral RNA (Hornung et al., (2006) Science 314(5801):994-997; Pichlmair et al., (2006) Science 314(5801):997-1001).

[0144] High-resolution structures of RIG-I / ligand complexes provide molecular details of RIG-I binding to RNA ligands, particularly the activating ligand double-stranded 5' triphosphorylated RNA (ppp-dsRNA) (Civril et al., (2011) EMBO Reports 12(11):1127-1134; Jiang et al., (2011) Nature 479(7373):423-427; Kowalinski et al., (2011) Cell 147(2):423-435; Lu et al., (2010) Structure 18(8): 1032-1043; Luo et al. (2011) Cell 147(2)409-422; Wang et al., (2010) Nature Structural & Molecular Biology 17(7):781-787; Hornung et al., (2006) Science 314(5801):994-997; Pichlmair et al., (2006) Science 314(5801):997-1001; Schlee et al., (2009) Immunity 31(1):25-34). Crystal structures of RIG-I / RNA complexes show protein binding to the backbone but not the bases, suggesting that the RNA sequence does not affect RIG-I binding or that the RNA sequence may exhibit uncharacterized effects or activities. To date, no evidence for sequence-dependent differential interaction or affinity with and activation of RIG-I-like receptors has been described in the art (Schlee and Hartmann (2010) Molecular Therapy 18(7):1254-1262).

[0145] Accordingly, the present disclosure provides synthetic RIG-I-like receptor (RLR) agonists, including non-naturally occurring, synthetic, and / or engineered RNA ligands of RLRs. In some embodiments, the RLR agonists comprise ribonucleic acid (RNA) of 10-100 nucleotides in length. In some aspects, the RNA is 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, or 95-100 nucleotides in length.

[0146] In some embodiments, the RLR agonist may be a single-stranded; a single-stranded oligonucleotide containing self-complementary sequences capable of forming a duplex, stem-loop, or hairpin structure; a double-stranded oligonucleotide; or a partially double-stranded oligonucleotide.

[0147] In some embodiments, the double-stranded oligonucleotide is fully double-stranded, in which case the oligonucleotide is composed of two single-stranded oligonucleotides that have the same length and 100% complementary sequences to each other.

[0148] In some embodiments, double-stranded oligonucleotide is partially double-stranded.In this case, the two strands that form oligonucleotide have different lengths, or have sequences that are not 100% complementary to each other, or both.In other words, at least one complete double-stranded part of oligonucleotide is linked at one or both ends with single-stranded structure.

[0149] In some embodiments, the duplex, hairpin, or stem-loop structure comprises 10-15, 15-20, 20-25, 25-30, 30-35, 30-35, 35-40, 40-45, 45-50, 50-55 base pairs.

[0150] In some forms, the oligonucleotides form duplexes containing fewer than 19 base pairs. In some embodiments, the complementary bases of the duplex are linked by a nucleotide or non-nucleotide linker.

[0151] In some embodiments, the oligonucleotide is single-stranded, single-stranded containing a self-complementary sequence, or double-stranded, and the length of the oligonucleotide is the length of a single strand.

[0152] In some embodiments, the oligonucleotide is partially double-stranded, and the length of the oligonucleotide is the length of the longer strand. Thus, oligonucleotides of the invention include partially double-stranded oligonucleotides in which at least one strand is 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, or 85-90 nucleotides in length.

[0153] In some embodiments, the oligonucleotide is a double-stranded or partially double-stranded oligonucleotide, wherein at least one strand contains at least one 5' di- or triphosphate group. When both strands contain a 5' di- or triphosphate group, the number of phosphate groups can be the same or different in the two strands. In some embodiments, the oligonucleotide is a partially double-stranded oligonucleotide, wherein the at least one ribonucleotide at the 5' end that comprises at least one 5' di- or triphosphate can be on either the longer strand or the shorter strand, and wherein at least the longer strand is 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, or 85-90 nucleotides in length.

[0154] In some embodiments, the degree of complementarity is preferably at least 50%, 60%, or 70%, more preferably at least 75%, 80%, 85%, or 90%, even more preferably at least 95%, 96%, 97%, 98%, or 99%, and most preferably 100%. As used in the art, the term "degree of complementarity" between two oligonucleotides / polynucleotides refers to the percentage of complementary bases in the overlapping region of the two oligonucleotides. Two bases are complementary to each other if they can form base pairs through hydrogen bonds. Base pairs include both Watson-Crick base pairs and wobble base pairs. Watson-Crick base pairs include AT, CG, and AU, while wobble base pairs include GU, IU, IA, and IC. The degree of complementarity can be determined manually or automatically by various engines, such as BLAST, using any method known in the art. For example, ATCG has 100% complementarity to CGAT and CGATGG, and 75% complementarity to CGTT and CGTTGG.

[0155] In some aspects, the disclosure provides an RIG-I-like receptor (RLR) agonist that specifically binds to an RLR, the RLR agonist comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, the first polynucleotide being sufficiently complementary to form a duplex with the second polynucleotide, the duplex comprising fewer than 19 base pairs, the 5'-most nucleotide of the first polynucleotide comprising a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and the agonist comprising a sequence motif that confers at least one improved biological activity mediated by the RLR compared to an agonist that does not comprise the sequence motif.

[0156] In some embodiments, the RLR agonist of the present disclosure is: (i) GT repeat motif, (ii) GA repeat motif; (iii) AUCG repeat motif, (iv) AU repeat motifs; (v) dipyrimidine motif, (vi) ziprine motif, (vii) pyrimidine triplet motif, (viii) Printed triplet motif, (ix) a palindromic sequence motif, and (x) Contains a sequence motif selected from the group consisting of any combination of (i) to (ix).

[0157] In some embodiments, the RLR agonists of the present disclosure comprise at least one improved biological activity, wherein the improved biological activity is one of the following: (i) Increased RLR-mediated cytokine production; (ii) increased RLR-mediated expression of interferon-stimulated genes; (iii) increased RLR-mediated intracellular signaling; (iv) increased binding affinity to RLRs, and (v) A combination of any one of (i) to (iv) is selected.

[0158] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is a GT repeat motif comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the GT repeat motif is [GT] n (where n=2 to 9). In some embodiments, the GT repeat motif is [GT]. In some embodiments, the GT repeat motif is [GT], where the GT repeat motif is followed by a print triplet and a UCG triplet. In some embodiments, the print triplet is GGA.

[0159] In some embodiments, the sequence motif is a GA repeat motif comprising a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the GA repeat motif is [GA] n (where n=2 to 9). In some embodiments, the GA repeat motif is [GA]7.

[0160] In some embodiments, the RLR agonists of the present disclosure comprise a sequence motif, wherein the sequence motif is an AUCG repeat motif comprising a sequence of less than 19, about 16, about 12-16, about 12, about 8-12, about 6, about 16, 12, or 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof.

[0161] In some embodiments, the AUCG repeat motif is [AUCG] n(wherein n=2 to 4). In some embodiments, the AUCG repeat motif is [AUCG]3.

[0162] In some embodiments, the AUCG repeat motif is preceded by a CG or dipyrimidine motif. In some embodiments, the AUCG repeat motif is preceded by a CG. In some embodiments, the dipyrimidine motif is CC. In some embodiments, the AUCG repeat motif is preceded by a dipurine motif. In some embodiments, the dipurine motif is GA. In some embodiments, the dipurine motif is GG.

[0163] In some embodiments, the RLR agonists of the present disclosure comprise an AUGC repeat motif, in which one or more uridine nucleosides (U) are replaced with modified nucleosides. In some embodiments, the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC repeat motif is [AUCG]3, in which one or more uridine nucleosides (U) comprising the AUCG repeat motif are replaced with modified nucleosides, the modified nucleoside is ribothymidine (T). In some embodiments, the AUCG repeat motif is [AUCG]3, in which one or more uridine nucleosides (U) comprising the AUCG repeat motif are replaced with modified nucleosides, the modified nucleoside is ribothymidine (T), and the AUGC repeat motif is preceded by GG.

[0164] In some embodiments, the RLR agonist of the present disclosure comprises an AUGC repeat motif, in which one or more guanosine nucleosides (G) are replaced with modified nucleosides. In some embodiments, the modified nucleoside is inosine (I). In some embodiments, the AUGC repeat motif is [AUCG]3, in which one or more guanosine nucleosides (G) constituting the AUCG repeat motif are replaced with modified nucleosides, in which the modified nucleoside is ribothymidine (T), and in which GG precedes the AUGC repeat motif.

[0165] In some embodiments, the RLR agonists of the present disclosure comprise an AUCG repeat motif, where the motif is preceded by IG. In some embodiments, the AUCG repeat motif is [AUCG]3, where the motif is preceded by IG.

[0166] In some embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat, wherein one or more guanosine nucleosides (G) are substituted with inosine (I), and the AUCG repeat is preceded by an inosine (I). In some embodiments, the guanosine nucleosides (G) comprising the AUCG repeat are substituted with inosine (I), the AUCG repeat is preceded by an inosine (I), and the 5'-most nucleotide of the first polynucleotide comprises an inosine (I).

[0167] In some embodiments, the 5'-most nucleotide of the first polynucleotide comprises an inosine (I).

[0168] In some embodiments, the RLR agonist of the present disclosure comprises an AUCG repeat sequence motif, wherein the AUCG repeat motif is [AUCG]2. In some embodiments, the AUCG repeat motif is preceded by a dipurine motif. In some embodiments, the dipurine motif is GG. In some embodiments, the AUCG repeat motif is preceded by a print triplet. In some embodiments, the print triplet is GGG. In some embodiments, the AUCG repeat motif is preceded by CCCCCG. In some embodiments, the AUCG repeat motif is preceded by TCGUCG.

[0169] In some embodiments, the RLR agonists of the present disclosure comprise a palindromic sequence, which comprises a sequence of less than 19, about 15-18, about 15, about 10-15, about 10, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, linked in any order to create a palindrome.

[0170] In some embodiments, the linker is flanked by AU repeat motifs, and the AU repeat motifs are [AU] n (where n=2 to 3). In some embodiments, the AU repeat motif is [AU]2.

[0171] In some aspects, the disclosure provides an RLR agonist that specifically binds to an RLR, comprising a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein: (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that covalently links the first polynucleotide and the second polynucleotide.

[0172] In another aspect, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', [wherein: (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2 and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide.

[0173] In some embodiments, inosine, when present in the RLR agonist, base pairs with cytidine.

[0174] In some embodiments, the linker (L) is a nucleotide linker or a non-nucleotide linker.

[0175] In some embodiments, the present disclosure provides an RLR agonist that specifically binds to an RLR, the RLR agonist comprising a blunt-ended hairpin RNA containing a nucleotide or non-nucleotide linker. RNA hairpins are one of the most common RNA secondary structural elements, and the hybridized portion, or "stem," of the hairpin is often capped by an RNA tetraloop. RNA tetraloops are composed of four characteristic loop nucleotides that form a compact and stable structure. While they can be formed by many different nucleotide sequences, the UNCG (N = A, C, G, or U), GNRA (R = A or G), and CUUG tetraloops are the most common. Tetraloops typically help initiate the RNA folding process and promote the assembly of ribonucleoprotein particles by providing three-dimensional contacts within and between RNAs and sites for protein binding. Further description of tetraloops can be found in Cheong, H., Kim, N., and Cheong, C. (2015). RNA Structure: Tetraloops. In: eLS, John Wiley & Sons, Ltd. (Ed.).

[0176] Thus, in some embodiments, the RLR agonists of the present disclosure comprise a nucleotide linker comprising a tetraloop. In some embodiments, the nucleotide sequence of the tetraloop is: (a) UNCG (where N=A, C, G, or U), (b) GNRA (where N=A, C, G, or U and R=A or G); (c) ANYA (where N=A, C, G, or U and Y=C or T); (d) CUYG (where Y = C or T), (e) UMAC (where M=A or C), and (f) selected from the group consisting of CUUG.

[0177] In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the sequence of the tetraloop is UUCG. In some embodiments, the sequence of the tetraloop is GAUC. In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UGUUU.

[0178] In other embodiments, the RLR agonists of the present disclosure comprise a non-nucleotide linker. As described herein, nucleic acid loops (e.g., tetraloops) are common elements found in nucleic acid secondary structures. Nucleotide loops occur within folded domains that occur in intrastrand duplexes. Synthetic nucleic acids designed to contain hairpin loops containing non-nucleotide linking groups (e.g., non-nucleotide linkers) can replace several nucleotides that bridge folded duplex structures. Non-nucleotide groups have also been used as linkers within unfolded structures. Such linking groups can be useful replacements for natural nucleotide linkers (e.g., tetraloops). Such linking groups can shorten several steps in the synthesis of nucleic acids with desired secondary structures, for example, by replacing several individual nucleotides that would normally constitute a loop with a single, relatively long non-nucleotide linking group. Such non-natural loops or linkers (e.g., non-nucleotide linkers) can confer resistance to degradation by nucleases that normally act on natural loop structures in biological situations (e.g., within a subject's cells upon administration or in the circulation). Non-nucleotide linking groups also have the potential to provide more stable folded structures than those achieved with nucleotide loops and / or linkers. Further description of non-nucleotide linkers can be found in Rumney and Kool (1995) J Am Chem Soc 117:5635-5646, which is incorporated herein by reference in its entirety.

[0179] Thus, in some embodiments, the RLR agonists of the present disclosure: (a) an ethylene glycol linker, and (b) comprises a non-nucleotidic linker selected from the group consisting of alkyl linkers.

[0180] In some embodiments, the non-nucleotidic linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotidic linker is a C9 alkyl linker.

[0181] In some embodiments, the RLR agonist comprises a 5' diphosphate moiety, or a derivative or analog thereof. In some embodiments, the agonist comprises a 5' triphosphate moiety, or a derivative or analog thereof. In some embodiments, the derivative or analog comprises a bioisostere of a phosphate selected from phosphonate, thiophosphonate, phosphorothioate, sulfate, sulfonate, sulfamate, thiazolidinone, carboxylate, malonate, boronic acid, benzoxaborole, boranophosphate, and squaramide.

[0182] In some embodiments, the agonist comprises a modified nucleotide, modified nucleoside, or modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification to the internucleotide linkage or polynucleotide backbone.

[0183] In some embodiments, the RLR agonists of the present disclosure have the following properties: (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5, and / or LGP2); (b) increase RLR-mediated cytokine production; (c) increasing RLR-mediated expression of interferon-stimulated genes (ISGs); (d) increasing RLR-dependent intracellular signaling; (e) increasing duplex stability; (f) increasing binding affinity to RLRs; (g) reducing off-target binding; (h) increasing biological half-life; (i) Increase biodistribution and bioavailability; (j) increasing and / or enhancing uptake into cells and / or tissues; (k) reducing immunogenicity; and (l) At least one of the combinations of (a) to (k) is shown.

[0184] In some aspects, the present disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2, and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 and X2 are each an oligonucleotide containing a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (viii) X1 is complementary to X2; (ix) X1 and X2 are each 12 to 16 nucleotides in length and are the same length; (x) L is a linker that operably links the first polynucleotide and the second polynucleotide; at least one of N1, N2, N3, and N4 is inosine, and / or at least one of X1 and / or X2 contains at least one inosine nucleoside, which base pairs with a cytidine in the hairpin RNA.

[0185] In some embodiments, N1 comprises inosine and N4 comprises cytidine. In some embodiments, N1 comprises cytidine and N4 comprises inosine. In some embodiments, N2 comprises inosine and N3 comprises cytidine. In some embodiments, N2 comprises cytidine and N3 comprises inosine. In some embodiments, N1 comprises guanosine. In some embodiments, N2 comprises guanosine. In some embodiments, N1 comprises cytidine. In some embodiments, N2 comprises cytidine. In some embodiments, N1 and N2 comprise guanosine and N3 and N4 comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise guanosine. In some embodiments, N1 and N2 comprise inosine and N3 and N4 comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise inosine. In some embodiments, N1 comprises an inosine, N4 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N2 comprises an inosine, N3 comprises a cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a cytidine, N3 and N4 comprise a guanosine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise a guanosine, N3 and N4 comprise a cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise guanosine, and X1 and X2 each comprise inosine and do not comprise a guanosine nucleoside. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and / or X2 each comprise at least one inosine.In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and / or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine, N3 and N4 comprise cytidine, and X1 and X2 comprise inosine and do not include guanosine nucleosides. In some embodiments, N1 and N2 comprise cytidine, N3 and N4 comprise inosine, and X1 and X2 comprise inosine and do not include guanosine nucleosides. In some embodiments, X1 and X2 each comprise 12 nucleotides and include 1, 2, 3, or 4 inosine nucleosides. In some embodiments, X1 and X2 each comprise 13 nucleotides and include 1, 2, 3, 4, or 5 inosine nucleosides. In some embodiments, X1 and X2 are each 14 nucleotides and contain 1, 2, 3, 4, 5, or 6 inosine nucleosides. In some embodiments, X1 and X2 are each 15 nucleotides and contain 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. In some embodiments, X1 and X2 are each 16 nucleotides and contain 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. In some embodiments, X1 and X2 are each 12 nucleotides and contain at least 10%, 20%, 30%, or 40% inosine nucleosides.

[0186] In some aspects, the present disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), comprising a blunt-ended hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (i) (N1-N2-X1) comprises a first polynucleotide comprising linked nucleotides N1, N2, and X1; (ii) (X2-N3-N4) comprises a second polynucleotide comprising linked nucleotides X2, N3 and N4; (iii) N1, N2, N3, and N4 each comprise a single nucleotide comprising a nucleoside selected from the group consisting of adenosine, guanosine, cytidine, 5-methyluridine, uridine, and inosine; (iv) N1 base pairs with N4; (v) N2 base pairs with N3; (vi) N1 comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof; (vii) X1 has the sequence motif [AUCN5] x (wherein N5 contains guanosine or inosine, and x is an integer representing the number of sequence motifs, and x=3 or 4), (viii) X2 has the sequence motif [CN6AU] y (wherein N6 contains guanosine or inosine, and y is an integer whose value indicates the number of sequence motifs, and y=3 or 4), (ix) L is a linker that operably links the first polynucleotide and the second polynucleotide; Optionally, at least one of N1, N2, N3, and N4 is inosine, and the inosine nucleoside base pairs with a cytidine in the hairpin RNA. In some embodiments, N5 comprises inosine and N6 comprises inosine. In some embodiments, N5 comprises guanosine and N6 comprises inosine. In some embodiments, N5 comprises inosine and N6 comprises guanosine. In some embodiments, N5 comprises guanosine (G) and N6 comprises guanosine (G). In some embodiments, x=3 and y=3. In some embodiments, x=4 and y=4. In some embodiments, N1 comprises inosine (I) and N4 comprises cytidine (C). In some embodiments, N2 comprises inosine (I) and N3 comprises cytidine (C). In some embodiments, N3 comprises inosine (I) and N2 comprises cytidine (C). In some embodiments, N4 comprises inosine (I) and N1 comprises cytidine (C). In some embodiments, N1 comprises guanosine (G). In some embodiments, N2 comprises guanosine (G). In some embodiments, N1 comprises cytidine (C). In some embodiments, N2 comprises cytidine (C). In some embodiments, N1 and N2 comprise guanosine (G) and N3 and N4 comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N3 and N4 comprise guanosine (G). In some embodiments, N1 and N2 comprise inosine (I) and N3 and N4 comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N3 and N4 comprise inosine (I).

[0187] In some embodiments, the linker (L) is a nucleotide linker or a non-nucleotide linker. In some embodiments, the linker (L) is a nucleotide linker comprising a tetraloop, and the nucleotide sequence of the tetraloop is: (a) UNCG (where N=A, C, G, or U), (b) GNRA (where N=A, C, G, or U and R=A or G); (c) ANYA (where N=A, C, G, or U and Y=C or T); (d) CUYG (where Y = C or T), (e) UMAC (where M=A or C), and (f) selected from the group consisting of CUUG.

[0188] In some embodiments, the linker (L) is a nucleotide linker comprising the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UGUUU.

[0189] In some embodiments, the linker (L) is a nucleotide linker comprising a tetraloop, and the sequence of the tetraloop is UUGG. In some embodiments, the sequence of the tetraloop is GAUC.

[0190] In some embodiments, the linker (L) is (a) an ethylene glycol linker, and (b) a non-nucleotidic linker selected from the group consisting of alkyl linkers.

[0191] In some embodiments, the non-nucleotidic linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotidic linker is a C9 alkyl linker.

[0192] In some embodiments, the RLR agonist comprises a 5' diphosphate moiety, or a derivative or analog thereof. In some embodiments, the agonist comprises a 5' triphosphate moiety, or a derivative or analog thereof. In some embodiments, the derivative or analog comprises a bioisostere of a phosphate selected from phosphonate, thiophosphonate, phosphorothioate, sulfate, sulfonate, sulfamate, thiazolidinone, carboxylate, malonate, boronic acid, benzoxaborole, boranophosphate, and squaramide.

[0193] In some embodiments, the RLR agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification to the internucleotide bond or the polynucleotide backbone.

[0194] In some embodiments, the RLR agonist has the following properties: (a) specifically binds to one or more RLRs (e.g., RIG-1, MDA5, and / or LGP2); (b) increase RLR-mediated cytokine production; (c) increasing RLR-mediated expression of interferon-stimulated genes (ISGs); (d) increasing RLR-dependent intracellular signaling; (e) increasing duplex stability; (f) increasing binding affinity to RLRs; (g) reducing off-target binding; (h) increasing biological half-life; (i) Increase biodistribution and bioavailability; (j) increasing and / or enhancing uptake into cells and / or tissues; (k) reducing immunogenicity; and (l) At least one of the combinations of (a) to (k) is shown.

[0195] In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to form a duplex with the second polynucleotide, the duplex comprising fewer than 19 base pairs, and wherein the 5'-most nucleotide of the first polynucleotide is a 5' diphosphate or triphosphate moiety, or derivatives thereof. or analogs thereof, wherein the agonist comprises a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, and further wherein the agonist comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36.

[0196] In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to an RIG-I-like receptor (RLR), comprising a blunt-ended hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to form a duplex with the second polynucleotide, wherein the duplex comprises fewer than 19 base pairs, and wherein the 5'-most nucleotide of the first polynucleotide comprises a 5' diphosphate or triphosphate moiety, or a derivative or analog thereof, and wherein the agonist comprises a sequence motif that confers at least one improved biological activity mediated by an RLR compared to an agonist that does not comprise the sequence motif, and wherein the first polynucleotide and the second polynucleotide are (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively; and (xxxii) Provided are synthetic RLR agonists comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97, respectively.

[0197] In some aspects, the present disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), the synthetic RLR agonist comprising a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, the synthetic RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 25.

[0198] In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), comprising a blunt-ended hairpin RNA comprising at least one or more nucleotides including an inosine that base pairs with a cytidine, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide are (i) SEQ ID NOs: 58 and 89, respectively; (ii) SEQ ID NOs: 59 and 89, respectively; and (iii) providing a synthetic RLR agonist comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 61 and 91, respectively.

[0199] In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), comprising a blunt-ended hairpin RNA comprising a non-nucleotide linker, the hairpin RNA having the formula: 5'-(N1-N2-X1)-L-(X2-N3-N4)-3', wherein (N1-N2-X1) comprises a first polynucleotide and (X2-N3-N4) comprises a second polynucleotide, and the first polynucleotide and the second polynucleotide are (i) SEQ ID NOs: 37 and 68, respectively; (ii) SEQ ID NOs: 38 and 69, respectively; (iii) SEQ ID NOs: 39 and 70, respectively; (iv) SEQ ID NOs: 40 and 71, respectively; (v) SEQ ID NOs: 41 and 72, respectively; (vi) SEQ ID NOs: 42 and 73, respectively; (vii) SEQ ID NOs: 43 and 74, respectively; (viii) SEQ ID NOs: 44 and 75, respectively; (ix) SEQ ID NOs: 45 and 76, respectively; (x) SEQ ID NOs: 46 and 77, respectively; (xi) SEQ ID NOs: 47 and 78, respectively; (xii) SEQ ID NOs: 48 and 79, respectively; (xiii) SEQ ID NOs: 49 and 80, respectively; (xiv) SEQ ID NOs: 50 and 81, respectively; (xv) SEQ ID NOs: 51 and 82, respectively; (xvi) SEQ ID NOs: 52 and 83, respectively; (xvii) SEQ ID NOs: 53 and 84, respectively; (xviii) SEQ ID NOs: 54 and 85, respectively; (xix) SEQ ID NOs: 55 and 86, respectively; (xx) SEQ ID NOs: 56 and 87, respectively; (xxi) SEQ ID NOs: 57 and 88, respectively; (xxii) SEQ ID NOs: 58 and 89, respectively; (xxiii) SEQ ID NOs: 59 and 89, respectively; (xxiv) SEQ ID NOs: 60 and 90, respectively; (xxv) SEQ ID NOs: 61 and 91, respectively; (xxvi) SEQ ID NOs: 62 and 92, respectively; (xxvii) SEQ ID NOs: 63 and 91, respectively; (xxviii) SEQ ID NOs: 64 and 93, respectively; (xxix) SEQ ID NOs: 65 and 94, respectively; (xxx) SEQ ID NOs: 66 and 95, respectively; (xxxi) SEQ ID NOs: 67 and 96, respectively; and (xxxii) Provided are synthetic RLR agonists comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63 and 97, respectively.

[0200] In some aspects, the present disclosure provides RLR agonists, wherein the nucleotide sequence comprising the agonist is not complementary to a genomic DNA sequence or an mRNA sequence, wherein the RLR agonist does not participate in RNA interference, and wherein the RLR agonist does not silence gene expression.

[0201] RLR agonists containing modified nucleobases, nucleosides, or nucleotides In some embodiments, the RLR agonists of the present disclosure comprise one or more modified nucleic acid bases, nucleosides, or nucleotides. In some embodiments, modified RLR agonists may have useful properties, including increased stability in cells into which the RLR agonist is introduced, increased intracellular retention, increased target binding, and / or increased induction of innate immune responses, compared to a reference unmodified RLR agonist. Thus, the use of modified RLR agonists can not only reduce immunogenicity but also increase the efficiency of target binding and the intracellular retention of nucleic acids. In one embodiment, the agonists provided by the present disclosure comprise one or more oligonucleotides containing at least one region modified to enhance target binding affinity. The affinity of an oligonucleotide for its target polypeptide (e.g., an RLR receptor) can be determined, for example, by measuring fluorescence polarization (FP) upon binding of a fluorescently labeled oligonucleotide to its target (Moerke (2009) Curr Protoc Chem Biol 1(1):1-15).

[0202] In another embodiment, the RLR agonist provided by the present disclosure comprises at least one oligonucleotide, which comprises at least one region that comprises at least one modified nucleobase, nucleoside or nucleotide that enhances the stability of duplex.The stability of duplex can be determined by measuring the Tm of duplex, which is the temperature at which the two oligonucleotide strands that constitute duplex dissociate, and dissociation is detected by spectrophotometry.The higher the Tm, the higher the stability of duplex.

[0203] In one embodiment, the region of the oligonucleotide modified to increase duplex stability contains at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro modified nucleotide. In another embodiment, the oligonucleotide constituting the RLR agonist is also modified to increase nuclease resistance. Cells contain various exonucleases and endonucleases that can degrade nucleic acids. Many nucleotide and nucleoside modifications have been shown to increase the resistance of the incorporated oligonucleotide to nuclease digestion compared to unmodified oligonucleotides. Nuclease resistance is routinely measured by incubating the oligonucleotide with a cell extract or isolated nuclease solution and measuring the degree of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides modified to increase nuclease resistance remain intact for longer periods than unmodified oligonucleotides. Various oligonucleotide modifications have been shown to increase or confer nuclease resistance. In one embodiment, oligonucleotides containing at least one phosphorothioate modification are used. In certain cases, oligonucleotide modifications that increase target binding affinity can also independently increase nuclease resistance (De Mesmaeker et al., 1995, Acc. Chem. 28:366-374).

[0204] Specific examples of oligonucleotides relevant to the present disclosure include those containing modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages. In some embodiments, oligonucleotides with phosphorothioate backbones (including those synthesized stereospecifically) and oligonucleotides with heteroatom backbones are used, particularly CH2-NH-O-CH2, CH2-N(CH3)-O-CH2 (known as the methylene(methylimino) or MMI backbone), CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2, and ON(CH3)-CH2-CH2 backbones (the natural phosphodiester backbone is represented as OPO-CH2). The amide backbone disclosed by De Mesmaeker et al. (1995, Acc. Chem. Res. 28:366-374) is also used in some embodiments. Oligonucleotides may contain one or more substituted sugar moieties. In some embodiments, the oligonucleotide has one of the following at the 2-position: OH, SH, SCH, F, OCN, OCHOCH, OCHO(CH), CH, O(CH)NH, or O(CH)CH (where n is 1 to about 10); C1-C10 lower alkyl, alkoxyalkoxy (also known in the art as O-alkyl-O-alkyl), substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF; OCF; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH; SOCH; ONO; NO; N; NH; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; reporter group; intercalator; group that improves the pharmacokinetic properties of the oligonucleotide; or group that improves the pharmacodynamic properties of the oligonucleotide and other substituents with similar properties.In one embodiment, the modification includes 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486). In some embodiments, the modification includes 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2CH2CH3), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3'-terminal nucleotide and the 5' position on the 5'-terminal nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyls, in place of pentofuranosyl groups.

[0205] Oligonucleotides may also or alternatively include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are found infrequently or only transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also called 5-methyl-2' deoxycytosine, often referred to in the art as 5-me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases such as 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine. Kornberg, A., DNA Replication, W.H. Freeman & Co., San Francisco, 1980, pp. 75-77; Gebeyehu, G., et al., 1987, Nucl. Acids Res. 15:4513). "Universal" bases, such as inosine, well known in the art, may also be included. The 5-me-C substitution has been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, Y.S., in Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is currently used as a base substitution in some embodiments.

[0206] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that increase the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholesteryl moieties (Letsinger et al., Proc. Natl. Acad. Sci. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., Proc. Natl. Acad ... al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipids, polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651). Oligonucleotides containing lipophilic moieties and methods for preparing such oligonucleotides are well known in the art, for example, U.S. Patent Nos. 5,138,045, 5,218,105 and 5,459,255.

[0207] The oligonucleotides of the invention may be provided as prodrugs, generally containing one or more moieties that are cleaved in the body to yield the active oligonucleotide. One example of the prodrug approach is described by Imbach et al. in International Publication No. WO 94 / 26764.

[0208] It is not necessary for all positions within a particular oligonucleotide to be uniformly modified, and in fact more than one of the above modifications may be incorporated within a single oligonucleotide or even a single nucleoside within an oligonucleotide.

[0209] Oligonucleotides according to the invention are preferably from about 8 to about 50 nucleotides in length, which in the context of the present invention is understood to include the non-natural oligomers described herein above having 8 to 50 monomers.

[0210] The oligonucleotides used in accordance with the present invention can be conveniently and routinely prepared by well-known methods of solid-phase synthesis. Equipment for such synthesis is commercially available from several vendors, including Applied Biosystems. Any other means for such synthesis can be used. The actual synthesis of oligonucleotides is well within the knowledge and skill of those skilled in the art. It is also well known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives. It is also well known to synthesize other modified oligonucleotides, such as fluorescently labeled oligonucleotides, biotinylated oligonucleotides, or cholesterol-modified oligonucleotides, using similar techniques and commercially available modified amidites, such as biotin, fluorescein, acridine, or psoralen-modified amidites and / or CPG (commercially available from Glen Research, Sterling, Va.).

[0211] In some embodiments, the RLR agonist comprises one or more (e.g., 1, 2, 3, or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the RLR agonist comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified RLR agonist can be reduced in degradation in cells into which the RLR agonist is introduced, compared to the corresponding unmodified RLR agonist.

[0212] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (φ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyluridine (m 3 U), 5-methoxyuridine (mo 5 U), uridine 5-hydroxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e., having a deoxythymine nucleobase), 1-methyl-pseudouridine (m 1 ψ), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thio-pseudo-uridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0213] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thiozebularine, 2-thiozebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4 Cm), N4,2'-O-dimethyl-cytidine (m 4 Cm), 5-formyl-2'-O-methyl-cytidine (f 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m 4 2Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0214] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenines include α-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halopurine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azidoadenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6-methyladenosine (m 6A), 2-methylthio-N6-methyl-adenosine (ms 2 m 6 A), N6-isopentenyl-adenosine (i 6 A), 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A), N6-(cis-hydroxyisopentenyl)adenosine (io 6 A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A), N6-glycinylcarbamoyl-adenosine (g 6 A), N6-threonylcarbamoyl-adenosine (t 6 A), N6-methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6 A), N6,N6-dimethyl-adenosine (m 6 2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn 6 A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms 2 hn 6 A), N6-acetyl-adenosine (ac 6 A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m 6 2Am), 1,2'-O-dimethyl-adenosine (m 1 Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0215] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include α-thio-guanosine, inosine (I), 1-methyl-inosine (m), 1 I), wiosin (imG), methylwiosin (mimG), 4-dimethyl-wiosin (imG-14), isowyosin (imG2), wibutosin (yW), peroxywibutosin (o2yW), hydroxywibutosin (OhyW), unmodified hydroxywibutosin (OhyW*), 7-deaza-guanosine, quosin (Q), epoxyquosin (oQ), galactosyl-quosin (galQ), mannosyl-quosin (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deazaguanosine (preQ1), and archaeosin (G + ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7 G), N2,N2,7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Im), 2'-O-ribosylguanosine(phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0216] In some embodiments, the RLR agonists of the present disclosure comprise a combination of one or more of the foregoing modified nucleobases (eg, a combination of two, three, or four of the foregoing modified nucleobases).

[0217] In certain embodiments, the RLR agonists of the present disclosure are uniformly modified (i.e., fully modified and modified throughout the entire sequence) for a particular modification. For example, the RLR agonist may be a 5-methyl-cytidine (m 5 C), which means that all cytosine residues in the mRNA sequence are uniformly modified with 5-methyl-cytidine (mC). 5 C). Similarly, the RLR agonists of the present disclosure can be uniformly modified at any type of nucleoside residue present in the sequence by substitution with modified residues such as those described above.

[0218] Examples of nucleoside modifications and combinations thereof that may be present in the RLR agonists of the present disclosure include, but are not limited to, those described in PCT Patent Application Publications WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.

[0219] The RLR agonists of the present disclosure can include combinations of modifications to the sugar, nucleobase, and / or internucleoside linkages, which can include any one or more of the modifications described herein.

[0220] Examples of modified nucleosides and combinations of modified nucleosides are shown in Tables 1 and 2 below. These combinations of modified nucleotides can be used to form the RLR agonists of the present disclosure. In certain embodiments, modified nucleosides can be partially or completely substituted for natural nucleotides in the RLR agonists of the present disclosure. As a non-limiting example, the natural nucleotide uridine can be substituted with a modified nucleoside described herein. In another non-limiting example, the natural nucleoside uridine can be partially substituted with at least one of the modified nucleosides disclosed herein (e.g., about 0.1%, 1%, 5%, 1%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, or 99.9% of the natural uridine).

[0221] [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0222] In accordance with the present disclosure, polynucleotides of the present disclosure can be synthesized to contain any combination or single modification of Table 1 or Table 2.

[0223] When a single modification is listed, the listed nucleoside or nucleotide represents 100% of that modified A, U, G, or C nucleotide or nucleoside. When percentages are listed, they represent the percentage of that particular A, U, G, or C nucleobase triphosphate relative to the total amount of A, U, G, or C triphosphates present. For example, the combination 25% 5-aminoallyl-CTP + 75% CTP / 25% 5-methoxy-UTP + 75% UTP refers to a polynucleotide in which 25% of the cytosine triphosphates are 5-aminoallyl-CTP and 75% of the cytosines are CTP, while 25% of the uracils are 5-methoxy-UTP and 75% of the uracils are UTP. When no modified UTP is listed, natural ATP, UTP, GTP, and / or CTP are used at 100% of these nucleotide positions found in the polynucleotide. In this example, all of the GTP and ATP nucleotides remain unmodified.

[0224] Method for producing RLR agonists The RLR agonists of the present disclosure can be produced by means available in the art, including, but not limited to, in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT) methods, solid-phase methods, liquid-phase methods, complex synthesis methods, small-area synthesis methods, and ligation methods can be used. In one embodiment, the RLR agonist is produced using IVT enzymatic synthesis. Methods for producing polynucleotides by IVT are well known in the art and are described in International Application PCT / US2013 / 30062, the entire contents of which are incorporated herein by reference. Therefore, the present disclosure also includes polynucleotides, such as DNA constructs and vectors, that can be used to in vitro transcribe the RLR agonists described herein.

[0225] Unnatural modified nucleobases can be introduced into polynucleotides, such as RNA, during or after synthesis. In certain embodiments, modifications can be made to internucleoside linkages, purine or pyrimidine bases, or sugars. In certain embodiments, modifications can be introduced at the termini of a polynucleotide chain or anywhere else in a polynucleotide chain, using chemical synthesis or polymerase enzymes. Examples of modified nucleic acids and their synthesis are disclosed in PCT application PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0226] Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or regions thereof with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0227] The synthesis of oligonucleotides, polynucleotides, and their conjugation and ligation are further described in Taskova et al., (2017) Chembiochem 18(17):1671-1682; Gooding et al., (2016) Eur J Pharm Biopharm 107:321-40; Menzi et al., (2015) Future Med Chem 7(13):1733-49; Winkler J., (2013) Ther Deliv. (7):791-809; Singh et al., (2010) Chem Soc Rev 39(6):2054-70; and Lu et al., (2010) Bioconjug Chem 21(2):187-202.

[0228] Virus-like particles (VLPs) In some embodiments, the present disclosure provides a composition comprising at least one RLR agonist described and a virus-like particle (VLP). In some embodiments, the RLR agonist is bound to the VLP. In some aspects, the RLR agonist is packaged within the VLP.

[0229] In the context of this application, a virus-like particle refers to a structure that resembles a virus particle but is not pathogenic. Virus-like particles generally lack the viral genome and are therefore non-infectious. Furthermore, virus-like particles can be produced in large quantities by heterologous expression and can be easily purified.

[0230] Exemplary virus-like particles suitable for use in the compositions described herein are set forth in PCT Publication No. WO2003 / 024481 and International Publication No. WO2004 / 084940, the entire contents of each of which are incorporated herein by this reference.

[0231] In some embodiments, the virus-like particle is a recombinant virus-like particle. One skilled in the art can generate VLPs using recombinant DNA technology and the viral coding sequences described herein, which are readily available to the public. For example, coding sequences for viral envelope or core proteins can be engineered for expression in commercially available baculovirus expression vectors, with appropriate modifications to the sequences to enable operably linking the coding sequence to regulatory sequences under the regulatory control of the viral promoter. Viral envelope or core protein coding sequences can also be engineered for expression in, for example, bacterial expression vectors.

[0232] Examples of VLPs include, but are not limited to, hepatitis B virus (Ulrich, et al., Virus Res. 50:141-182 (1998)), measles virus (Warnes, et al., Gene 160:173-178 (1995)), Sindbis virus, rotavirus (U.S. Pat. Nos. 5,071,651 and 5,374,426), foot-and-mouth disease virus (Twomey, et al., Vaccine 13:1603-1610, (1995)), Norwalk virus (Jiang, X., et al., Science 250:1580-1583 (1990); Matsui, S. M., et al., J. Clin. Invest. 87:1456-1461 (1991)), and the like. (1991)), capsid protein of retroviral GAG protein (PCT Patent Application No. WO96 / 30523), retrotransposon Ty protein p1, surface protein of hepatitis B virus (WO92 / 11291), human papillomavirus (WO98 / 15631), human polyomavirus (Sasnauskas K., et al., Biol. Chem. 380(3):381-386 (1999); Sasnauskas K., et al., Generation of recombinant virus-like particles of different polyomaviruses in yeast 3 rdInternational Workshop “Virus-like particles as vaccines.” Berlin, September 26-29, 2001), RNA phages, Ty, fr phages, GA phages, AP205 phages, and especially Qβ phages.

[0233] As will be readily apparent to one skilled in the art, the VLPs of the present disclosure are not limited to any particular form. The particles can be synthesized chemically or by biological processes, which can be natural or non-natural. By way of example, this type of embodiment includes virus-like particles or recombinant forms thereof. In some embodiments, the VLP comprises a recombinant polypeptide of rotavirus, a recombinant polypeptide of Norwalk virus, a recombinant polypeptide of alphavirus, a recombinant protein forming bacterial pili or pilus-like structures, a recombinant polypeptide of foot-and-mouth disease virus, a recombinant polypeptide of measles virus, a recombinant polypeptide of Sindbis virus, a recombinant polypeptide of retrovirus, a recombinant polypeptide of hepatitis B virus (e.g., HBcAg), a recombinant polypeptide of tobacco mosaic virus, a recombinant polypeptide of Flock House virus, a recombinant polypeptide of human papillomavirus, a recombinant polypeptide of polyomavirus, particularly a recombinant polypeptide of human polyomavirus, and particularly a recombinant polypeptide of BK virus, a recombinant polypeptide of bacteriophage, a recombinant polypeptide of RNA phage, a recombinant polypeptide of Ty, a recombinant polypeptide of fr phage, a recombinant polypeptide of GA phage, a recombinant polypeptide of AP205 phage, and particularly a recombinant polypeptide of Qβ phage. The virus-like particle may further comprise, or alternatively consist of, one or more fragments of such polypeptides and variants of such polypeptides. Variants of polypeptides can, for example, share at least 80%, 85%, 90%, 95%, 97%, or 99% identity at the amino acid level with their wild-type polypeptides.

[0234] In some embodiments, the virus-like particle comprises recombinant proteins, or fragments thereof, of an RNA phage, hi some embodiments, the RNA phage is selected from the group consisting of: a) bacteriophage Qβ, b) bacteriophage R17, c) bacteriophage fr, d) bacteriophage GA, e) bacteriophage SP, f) bacteriophage MS2, g) bacteriophage M11, h) bacteriophage MX1, i) bacteriophage NL95, k) bacteriophage f2, and l) bacteriophage PP7.

[0235] In some embodiments, the virus-like particle comprises recombinant proteins of RNA bacteriophage Qβ or RNA bacteriophage fr, or fragments thereof. In some embodiments, the virus-like particle comprises recombinant proteins of RNA bacteriophage Qβ, or fragments thereof.

[0236] In some embodiments, the recombinant protein comprises a coat protein of an RNA phage.

[0237] Thus, capsid- or VLP-forming RNA phage coat proteins, or fragments of bacteriophage coat proteins adapted for self-assembly into capsids or VLPs, are further embodiments of the present disclosure. The coat proteins of bacteriophage Qβ can be recombinantly expressed, for example, in E. coli. Upon such expression, these proteins naturally form capsids. Furthermore, these capsids form structures with unique repetitive organization.

[0238] Examples of bacteriophage coat proteins that can be used to prepare the compositions of the present disclosure include bacteriophage Qβ (SEQ ID NO: 112: PIR database, accession number VCBPQb, which refers to Qβ CP, and SEQ ID NO: 113: accession number AAA16663, which refers to Qβ A1 protein), bacteriophage R17 (SEQ ID NO: 114: PIR accession number VCBPR7), bacteriophage fr (SEQ ID NO: 115: PIR accession number VCBPFR), bacteriophage GA (SEQ ID NO: 116: GenBank accession number NP-040754), bacteriophage SP (SEQ ID NO: 117: GenBank accession number CAA30374, which refers to SPCP, and SEQ ID NO: 118: SP Examples of coat proteins include those of RNA bacteriophages such as bacteriophage Qβ (accession numbers referring to the A1 protein), bacteriophage MS2 (SEQ ID NO: 119; PIR accession number VCBPM2), bacteriophage M11 (SEQ ID NO: 120; GenBank accession number AAC06250), bacteriophage MX1 (SEQ ID NO: 121; GenBank accession number AAC14699), bacteriophage NL95 (SEQ ID NO: 122; GenBank accession number AAC14704), bacteriophage f2 (SEQ ID NO: 123; GenBank accession number P03611), and bacteriophage PP7 (SEQ ID NO: 124). Additionally, the A1 protein of bacteriophage Qβ, or C-terminal truncations thereof lacking up to 100, 150, or 180 amino acids from the C-terminus, can be incorporated into the capsid assembly of Qβ coat protein. Generally, the percentage of Qβ A1 protein relative to Qβ CP in the capsid assembly is limited in order to ensure encapsidation.

[0239] Qβ coat protein has also been found to self-assemble into capsids when expressed in Escherichia coli (Kozlovska T M. et al., GENE 137:133-137 (1993)). The resulting capsids or virus-like particles exhibited a quasi-symmetric, icosahedral, phage-like capsid structure with a diameter of 25 nm and a T=3. Furthermore, the crystal structure of phage Qβ has been elucidated. The capsid contains 180 copies of the coat protein, which are covalently linked as pentamers and hexamers by disulfide bridges (Golmohammadi, R. et al., Structure 4:543-5554 (1996)), resulting in high capsid stability of Qβ coat protein. However, capsids or VLPs formed from recombinant Qβ coat protein may contain subunits that are not linked to other subunits within the capsid via disulfide links or that are incompletely linked. Thus, when recombinant Qβ capsid is subjected to non-reducing SDS-PAGE, bands corresponding to monomeric Qβ coat protein as well as bands corresponding to hexamers or pentamers of Qβ coat protein are visible. Incompletely disulfide-linked subunits may appear as dimer, trimer, or tetramer bands in non-reducing SDS-PAGE. Qβ capsid protein also exhibits exceptional resistance to organic solvents and denaturing agents. DMSO and acetonitrile concentrations as high as 30% and guanidinium concentrations as high as 1 M have been observed to have no effect on capsid stability. The high stability of Qβ coat protein capsids is an advantageous attribute, particularly for their use in the immunization and vaccination of mammals and humans according to the present invention.

[0240] When expressed in E. coli, the N-terminal methionine of Qβ coat protein is usually removed, as observed by N-terminal Edman sequencing as described in Stoll, E. et al. J. Biol. Chem. 252:990-993 (1977). VLPs composed of Qβ coat protein in which the N-terminal methionine has not been removed, or VLPs containing a mixture of Qβ coat proteins in which the N-terminal methionine has been truncated or is present, are also within the scope of the present disclosure.

[0241] Coat proteins of additional RNA phages have also been shown to self-assemble when expressed in bacterial hosts (Kastelein, R A. et al., Gene 23: 245-254 (1983), Kozlovskaya, T. M. et al., Dokl. Akad. Nauk SSSR 287: 452-455 (1986), Adhin, M. R. et al., Virology 170: 238-242 (1989), Ni, C. Z. et al., Protein Sci. 5: 2485-2493 (1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995)). In addition to the coat protein, the Qβ phage capsid contains the so-called readthrough protein A1 and the maturation protein A2. A1 is generated by suppression at the UGA stop codon and is 329 amino acids long. In some embodiments, the capsid of recombinant coat protein of phage Qβ used in the present disclosure does not contain A2 lysis protein but contains host-derived RNA. The coat protein of RNA phages is an RNA-binding protein that interacts with the stem-loop of the ribosome-binding site of the replicase gene, which acts as a translational repressor during the viral life cycle. The sequence and structural elements of this interaction are well known (Witherell, G. W. & Uhlenbeck, O. C. Biochemistry 28:71-76 (1989); Lim F. et al., J. Biol. Chem. 271: 31839-31845 (1996)). Stem-loops and RNA are generally known to be involved in virus assembly (Golmohammadi, R. et al., Structure 4:543-5554 (1996)).

[0242] In some embodiments, the virus-like particle comprises a recombinant protein or fragment thereof of an RNA phage, wherein the recombinant protein comprises a mutant coat protein of an RNA phage, preferably a mutant coat protein of an RNA phage as described above. In some embodiments, the mutant coat protein of an RNA phage has been modified by removal of at least one lysine residue by substitution or by addition of at least one lysine residue by substitution, or the mutant coat protein of an RNA phage has been modified by deletion of at least one lysine residue or by addition of at least one lysine residue by insertion.

[0243] In some embodiments, the virus-like particle comprises recombinant proteins or fragments thereof of RNA bacteriophage Qβ, wherein the recombinant proteins comprise a coat protein having the amino acid sequence of SEQ ID NO: 112, or a mixture of coat proteins having the amino acid sequences of SEQ ID NO: 112 and SEQ ID NO: 113, or a variant of SEQ ID NO: 113, preferably with the N-terminal methionine truncated.

[0244] In some embodiments, the virus-like particle comprises a recombinant Qβ protein or fragment thereof, wherein the recombinant protein comprises a mutant Qβ coat protein. In some embodiments, these mutant coat proteins are modified by removing at least one lysine residue by substitution or by adding at least one lysine residue by substitution. Alternatively, these mutant coat proteins are modified by deleting at least one lysine residue or by adding at least one lysine residue by insertion.

[0245] The Qβ coat protein has four exposed lysine residues on the surface of the capsid. Qβ mutants in which the exposed lysine residues are replaced by arginines can also be used in the present invention. Thus, the following Qβ coat protein mutants and mutant Qβ VLPs can be used in the practice of the present invention: "Qβ240" (Lys13-Arg, SEQ ID NO: 125), "Qβ-243" (Asn10-Lys, SEQ ID NO: 126), "Qβ-250" (Lys2-Arg, Lys13-Arg, SEQ ID NO: 127), "Qβ-251" (SEQ ID NO: 128), and "Qβ-259" (Lys2-Arg, Lys16-Arg, SEQ ID NO: 129). Thus, in some embodiments, the virus-like particle comprises a recombinant protein of a mutant Qβ coat protein, comprising a protein having an amino acid sequence selected from the group consisting of a) the amino acid sequence of SEQ ID NO: 125, b) the amino acid sequence of SEQ ID NO: 126, c) the amino acid sequence of SEQ ID NO: 127, d) the amino acid sequence of SEQ ID NO: 128, and e) the amino acid sequence of SEQ ID NO: 129. The structure, expression, and purification of the above-described Qβ coat protein, mutant Qβ coat protein VLP, and capsid are each disclosed in U.S. Publication No. US 2003-0175290, which is incorporated herein by reference in its entirety. For details, reference is made herein to Example 18 of the above application.

[0246] In some embodiments, the virus-like particle comprises a recombinant protein of Qβ or a fragment thereof, wherein the recombinant protein comprises a mixture of any one of the above Qβ mutants and the corresponding A1 protein.

[0247] In some embodiments, the virus-like particle comprises recombinant proteins of RNA phage AP205, or fragments thereof.

[0248] The AP205 genome consists of maturation proteins, coat proteins, replicase, and two open reading frames not present in related phages, with the lysis gene and open reading frame responsible for translating the maturation gene (Klovins, J., et al., J. Gen. Virol. 83:1523-33 (2002)). The AP205 coat protein can be expressed from the plasmid pAP283-58 (SEQ ID NO:79), which is a derivative of pQb10 (Kozlovska, TM et al., Gene 137:133-37 (1993)) and contains the AP205 ribosome binding site. Alternatively, the AP205 coat protein can be cloned into pQb185 downstream of the ribosome binding site present in the vector. Either approach results in protein expression and capsid formation as described in U.S. Patent No. 7,138,252, the entire contents of which are incorporated by reference. The vectors pQb10 and pQb185 are derived from the pGEM vector, and expression of genes cloned into these vectors is controlled by the trp promoter (Kozlovska, TM et al., Gene 137:133-37 (1993)). Plasmid pAP283-58 (SEQ ID NO: 130) contains a putative AP205 ribosome binding site within the following sequence: tctagaATTTTCTGCGCACCCATCCCGGGTGGCGCCCAAAGTGAGGAAAATCACatg (SEQ ID NO: 131), located downstream of the XbaI site and immediately upstream of the ATG start codon of the AP205 coat protein. Vector pQb185 contains a Shine-Dalgarno sequence downstream of the XbaI site and upstream of the start codon (tctagaTTAACCCAACGCGTAGGAGTCAGGCCatg, the Shine-Dalgarno sequence is underlined; SEQ ID NO: 132).

[0249] In some embodiments, the virus-like particle comprises recombinant coat proteins, or fragments thereof, of RNA phage AP205.

[0250] In some embodiments, the AP205 coat protein forms capsids. Such proteins are recombinantly expressed or prepared from natural sources. AP205 coat protein produced in bacteria spontaneously forms capsids, as demonstrated by electron microscopy (EM) and immunodiffusion. The structural features of capsids formed by the AP205 coat protein (SEQ ID NO: 133) and those of the AP205 RNA phage coat protein are nearly indistinguishable under electron microscopy. AP205 VLPs are highly immunogenic and can be linked to antigens and / or antigenic determinants to generate vaccine constructs that display antigens and / or antigenic determinants in a repetitive orientation. High titers against such displayed antigens indicate that the bound antigens and / or antigenic determinants are accessible for interaction with antibody molecules and are immunogenic.

[0251] In some embodiments, the virus-like particle comprises a recombinant mutant coat protein, or a fragment thereof, of RNA phage AP205.

[0252] In some embodiments, self-assembling mutant forms of AP205 VLPs are used in the practice of the present disclosure, including the AP205 coat protein with a proline to threonine substitution at amino acid 5 (SEQ ID NO: 134). These VLPs, AP205 VLPs derived from natural sources, or AP205 viral particles are capable of binding antigens to generate ordered and repetitive arrays of antigens according to the present invention.

[0253] The AP205 P5-T mutant coat protein can be expressed from the plasmid pAP281-32 (SEQ ID NO: 135), which contains the mutant AP205 coat protein gene in place of the Qβ coat protein gene, derived directly from pQb185. The AP205 coat protein expression vector is transfected into E. coli to express the AP205 coat protein.

[0254] In some embodiments, the present disclosure provides compositions comprising proteins having amino acid sequences that are at least 80%, 85%, 90%, 95%, 97%, or 99% identical to wild-type proteins that form an ordered array and have a unique repeat structure.

[0255] In some embodiments, the disclosure provides nucleic acid molecules encoding proteins used to prepare compositions of the invention.

[0256] In some embodiments, the compositions described herein include a protein comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to any of the amino acid sequences set forth in SEQ ID NOs: 112-129.

[0257] Proteins suitable for use in the present disclosure also include C-terminal truncation mutants of proteins that form capsids or capsid-like structures, or VLPs. Specific examples of such truncation mutants include proteins having the amino acid sequence set forth in any of SEQ ID NOS: 112-129, with 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids removed from the C-terminus. Generally, these C-terminal truncation mutants retain the ability to form capsids or capsid-like structures.

[0258] Additional proteins suitable for use in the present disclosure also include N-terminal truncation mutants of proteins that form capsids or capsid-like structures. Specific examples of such truncation mutants include proteins having the amino acid sequence set forth in any of SEQ ID NOS: 112-129, with 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids removed from the N-terminus. Generally, these N-terminal truncation mutants retain the ability to form capsids or capsid-like structures.

[0259] Additional proteins suitable for use in the present disclosure also include N- and C-terminal truncation mutants that form capsids or capsid-like structures. Suitable truncation mutants include proteins having the amino acid sequence set forth in any of SEQ ID NOS: 112-129, with 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids removed from the N-terminus and 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids removed from the C-terminus. Generally, these N- and C-terminal truncation mutants retain the ability to form capsids or capsid-like structures.

[0260] Fragments of VLPs that retain the ability to induce an immune response may comprise or consist of polypeptides of about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 amino acids in length, although this will obviously depend on the length of the sequences of the subunits that make up the VLP. Examples of such fragments include fragments of the proteins discussed herein that are suitable for the preparation of compositions that enhance an immune response.

[0261] In some embodiments, the VLP does not comprise a lipoprotein envelope or a lipoprotein-containing envelope, hi some embodiments, the VLP does not comprise an envelope at all.

[0262] The absence of a lipoprotein envelope or a lipoprotein-containing envelope, particularly the complete absence of an envelope, results in virus-like particles with a more defined structure and composition. Therefore, such more defined virus-like particles can minimize side effects. Furthermore, the absence of a lipoprotein-containing envelope, or particularly the complete absence of an envelope, can prevent or minimize the incorporation of potentially toxic molecules and pyrogens into the virus-like particles.

[0263] In some embodiments, the particles used in the compositions of the present disclosure are composed of hepatitis B capsid (core) protein (HBcAg) or a fragment of HBcAg modified to eliminate or reduce the number of free cysteine ​​residues. Zhou et al. (J. Virol. 66:5393-5398 (1992)) have shown that HBcAg modified to remove naturally occurring cysteine ​​residues retains the ability to bind and form multimeric structures. Thus, core particles suitable for use in the compositions of the present disclosure include those containing modified HBcAg or fragments thereof in which one or more naturally occurring cysteine ​​residues have been deleted or replaced with another amino acid residue (e.g., serine residue).

[0264] HBcAg is a protein produced by processing of hepatitis B core antigen precursor protein. Many isotypes of HBcAg have been identified, and their amino acid sequences are readily available to those skilled in the art. For example, the HBcAg protein having the amino acid sequence set forth in SEQ ID NO: 136 is 185 amino acids long and is produced by processing of the 212 amino acid hepatitis B core antigen precursor protein. This processing removes 29 amino acids from the N-terminus of the hepatitis B core antigen precursor protein. Similarly, the 185 amino acid long HBcAg protein is produced by processing of the 214 amino acid hepatitis B core antigen precursor protein.

[0265] In some embodiments, compositions of the present disclosure are prepared using a processed form of HBcAg (ie, HBcAg from which the N-terminal leader sequence of the Hepatitis B core antigen precursor protein has been removed).

[0266] Furthermore, when HBcAg is produced under conditions in which processing does not occur, the HBcAg is generally expressed in a "processed" form. For example, bacterial systems such as E. coli do not generally remove leader sequences (also called "signal peptides") from proteins normally expressed in eukaryotic cells. Thus, when the HBcAg of the present disclosure is produced using an E. coli expression system that expresses proteins in the cytoplasm, these proteins are generally expressed such that the N-terminal leader sequence of the hepatitis B core antigen precursor protein is absent.

[0267] Preparation of Hepatitis B virus-like particles that can be used in the present disclosure are disclosed, for example, in WO 00 / 32227, particularly Examples 17-19 and 21-24 thereof, and in WO 01 / 85208, particularly Examples 17-19, 21-24, 31, and 41 thereof, and further in pending U.S. Publication No. US 2003-0175290. For the latter application, see particularly Examples 23, 24, 31, and 51. All three of these documents are expressly incorporated herein by reference.

[0268] The present disclosure also includes HBcAg variants that have been modified to delete or substitute one or more additional cysteine ​​residues. Thus, vaccine compositions of the present invention include compositions comprising an HBcAg in which cysteine ​​residues not present in the amino acid sequence set forth in SEQ ID NO: 136 have been deleted.

[0269] It is well known in the art that free cysteine ​​residues can be involved in many chemical side reactions. These side reactions include disulfide exchange, for example, reactions with chemicals or metabolites injected or formed during combined therapy with other substances, or direct oxidation and reactions with nucleotides due to exposure to UV light. Therefore, toxic adducts may be generated, especially considering the fact that HBcAg has a strong tendency to bind to nucleic acids. Therefore, toxic adducts may be distributed among multiple species, each of which may exist at low concentrations, but together may reach toxic levels.

[0270] In view of the above, one advantage of using in a composition an HBcAg that has been modified to remove naturally occurring cysteine ​​residues is that the number of sites at which toxic species can bind when an antigen or antigenic determinant binds is reduced or completely eliminated.

[0271] Many naturally occurring HBcAg variants suitable for use in the practice of the present disclosure have been identified. For example, Yuan et al. (J. Virol. 73:10122-10128 (1999)) describe variants in which the isoleucine residue at position 97 of SEQ ID NO: 137 is replaced with either a leucine or a phenylalanine residue.The amino acid sequences of many HBcAg variants, as well as several Hepatitis B core antigen precursor variants, are available from GenBank reports AAF121240 (SEQ ID NO: 138), AF121239 (SEQ ID NO: 139), X85297 (SEQ ID NO: 140), X02496 (SEQ ID NO: 141), X85305 (SEQ ID NO: 142), X85303 (SEQ ID NO: 143), AF151735 (SEQ ID NO: 144), X85259 (SEQ ID NO: 145), AF151735 (SEQ ID NO: 146), X85297 (SEQ ID NO: 147), X02496 (SEQ ID NO: 148), X85305 (SEQ ID NO: 149), X85303 (SEQ ID NO: 150), AF151735 (SEQ ID NO: 151), X85259 (SEQ ID NO: 152), X02496 (SEQ ID NO: 153), X85305 (SEQ ID NO: 154), X85303 (SEQ ID NO: 155), AF151735 (SEQ ID NO: 156), X85259 (SEQ ID NO: 157), X02496 (SEQ ID NO: 158), X85305 (SEQ ID NO: 159), X85303 (SEQ ID NO: 160), AF151735 (SEQ ID NO: 161), X85259 (SEQ ID NO: 162), X02496 (SEQ ID NO: 163), X85305 (SEQ ID NO: 164), X853 ), X85286 (SEQ ID NO: 146), X85260 (SEQ ID NO: 147), X85317 (SEQ ID NO: 148), X85298 (SEQ ID NO: 149), AF043593 (SEQ ID NO: 150), M20706 (SEQ ID NO: 151), X85295 (SEQ ID NO: 152), X80925 (SEQ ID NO: 153), X85284 (SEQ ID NO: 154), X85275 (SEQ ID NO: 155), X72702 (SEQ ID NO: 156), X85291 (SEQ ID NO: 157), X65258 (SEQ ID NO: 158), X85302 (SEQ ID NO: 159), No. 159), M32138 (SEQ ID NO: 160), X85293 (SEQ ID NO: 161), X85315 (SEQ ID NO: 162), U95551 (SEQ ID NO: 163), X85256 (SEQ ID NO: 164), X85316 (SEQ ID NO: 165), X85296 (SEQ ID NO: 166), AB033559 (SEQ ID NO: 167), X59795 (SEQ ID NO: 168), X85299 (SEQ ID NO: 169), X85307 (SEQ ID NO: 170), X65257 (SEQ ID NO: 171), X85311 (SEQ ID NO: 172), X853 01 (SEQ ID NO: 173), X85314 (SEQ ID NO: 174), X85287 (SEQ ID NO: 175), X85272 (SEQ ID NO: 176), X85319 (SEQ ID NO: 177), AB010289 (SEQ ID NO: 178), X85285 (SEQ ID NO: 179), AB010289 (SEQ ID NO: 180), AF121242 (SEQ ID NO: 181), M90520 (SEQ ID NO: 182), P03153 (SEQ ID NO: 183), AF110999 (SEQ ID NO: 184), and M95589 (SEQ ID NO: 185).These HBcAg variants differ in amino acid sequence at a number of positions, including amino acid residues corresponding to amino acid residues located at positions 12, 13, 21, 22, 24, 29, 32, 33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80, 81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126, 130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and 183 of SEQ ID NO:77. Additional HBcAg variants suitable for use in the compositions of the present invention, and which can be further modified in accordance with the disclosure herein, are described in WO00 / 198333, WO00 / 177158 and WO00 / 214478.

[0272] HBcAgs suitable for use in the present disclosure may be derived from any organism capable of encapsulating or binding or otherwise attaching an RLR agonist, particularly one capable of packaging an RLR agonist to induce an immune response.

[0273] In some embodiments, compositions comprise HBcAg variants that are capable of associating to form dimeric or multimeric structures, hi some embodiments, compositions comprise HBcAg polypeptides that comprise amino acid sequences that are at least 80%, 85%, 90%, 95%, 97%, or 99% identical to any of the wild-type amino acid sequences, and, where appropriate, forms of these proteins that have been processed to remove the N-terminal leader sequence.

[0274] Whether the amino acid sequence of a polypeptide has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to any wild-type amino acid sequence, or portion thereof, can be determined in a conventional manner using known computer programs such as the Bestfit program. When using Bestfit or other sequence alignment programs to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, parameters are set so that the percent identity is calculated over the entire length of the reference amino acid sequence and allows gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence.

[0275] HBcAg variants and precursors having the amino acid sequences set forth in SEQ ID NOS: 138-181 and 182-185 are relatively similar to one another. Thus, when referring to an amino acid residue of an HBcAg variant located at a position corresponding to a particular position in SEQ ID NOS: 186, this refers to the amino acid residue present at that position in the amino acid sequence set forth in SEQ ID NOS: 186. The homology between these HBcAg variants is sufficiently high for the most part among hepatitis B viruses that infect mammals that a person skilled in the art would have little difficulty identifying the "corresponding" amino acid residues by examining the amino acid sequences set forth in SEQ ID NOS: 186 and 136, as well as the amino acid sequences of specific HBcAg variants. Furthermore, the HBcAg amino acid sequence set forth in SEQ ID NOS: 182, which represents the amino acid sequence of an HBcAg derived from a virus that infects woodchucks, has sufficient homology to the HBcAg having the amino acid sequence set forth in SEQ ID NOS: 186, such that it is immediately apparent that an insert consisting of three amino acid residues exists in SEQ ID NOS: 182 between amino acid residues 155 and 156 of SEQ ID NOS: 186.

[0276] As noted above, removal of free cysteine ​​residues reduces the number of sites at which toxic components can bind to HBcAg and also eliminates potential sites for cross-linking between lysine and cysteine ​​residues on the same or adjacent HBcAg molecules. Thus, in some embodiments, one or more cysteine ​​residues in the Hepatitis B virus capsid protein are deleted or substituted with another amino acid residue.

[0277] In some embodiments, the compositions described herein comprise an HBcAg in which the C-terminal region (e.g., amino acid residues 145-185 or 150-185 of SEQ ID NO: 186) has been removed. Accordingly, additional modified HBcAgs suitable for use in the practice of the present disclosure include C-terminal truncation mutants. Suitable truncation mutants include HBcAgs in which 1, 5, 10, 15, 20, 25, 30, 34, or 35 amino acids have been removed from the C-terminus.

[0278] Accordingly, HBcAgs suitable for use in the practice of the present disclosure also include N-terminal truncation mutants, such as modified HBcAgs having 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids removed from the N-terminus.

[0279] Additional HBcAgs suitable for use in the practice of the present disclosure include N- and C-terminal truncation mutants. Suitable truncation mutants include HBcAgs with 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 amino acids removed from the N-terminus and 1, 5, 10, 15, 20, 25, 30, and 34 amino acids removed from the C-terminus.

[0280] In some embodiments, compositions comprising HBcAg polypeptides comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to the truncation mutants described above.

[0281] In some embodiments, lysine residues are introduced into an HBcAg polypeptide to mediate binding of an antigen or antigenic determinant to a VLP of the HBcAg. In some embodiments, the compositions described herein are prepared using an HBcAg comprising amino acids 1-144, 1-149, or 1-185 of SEQ ID NO: 186, modified such that the amino acids corresponding to positions 79 and 80 are replaced with a peptide having the amino acid sequence Gly-Gly-Lys-Gly-Gly (SEQ ID NO: 187). These compositions are particularly useful in embodiments in which an antigenic determinant is attached to a VLP of the HBcAg. In some embodiments, the cysteine ​​residues at positions 48 and 107 of SEQ ID NO: 186 are mutated to serine. In some embodiments, the compositions described herein comprise the corresponding polypeptide having the amino acid sequence set forth in any of SEQ ID NOs: 138-183, also having the amino acid modifications set forth above. Further included within the scope of this disclosure are additional HBcAg variants capable of binding to form capsids or VLPs and having the amino acid modifications set forth above. Thus, the present disclosure further includes compositions comprising HBcAg polypeptides comprising amino acid sequences that are at least 80%, 85%, 90%, 95%, 97% or 99% identical to any of the wild-type amino acid sequences, and, where appropriate, forms of these proteins that have been processed to remove the N-terminal leader sequence and modified to have the modifications set forth above.

[0282] In some embodiments, the compositions described herein contain a mixture of different HBcAgs. Thus, these compositions may be comprised of HBcAgs that differ in amino acid sequence. For example, compositions can be prepared that contain a "wild-type" HBcAg and a modified HBcAg in which one or more amino acid residues have been modified (e.g., deleted, inserted, or substituted).

[0283] The crystal structures of several RNA bacteriophages have been determined (Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using such information, surface-exposed residues can be identified, and thus RNA phage coat proteins can be modified to insert one or more reactive amino acid residues by insertion or substitution. As a result, modified forms of bacteriophage coat proteins can also be used in the present disclosure. Thus, variants of proteins that form capsids or capsid-like structures (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophage MS2, bacteriophage AP205) can also be used to prepare the compositions described herein.

[0284] Although the sequences of the above-described variant proteins differ from those of their wild-type proteins, these variant proteins generally retain the ability to form capsids or capsid-like structures. Accordingly, the present invention further includes compositions comprising variants of proteins that form capsids or capsid-like structures, as well as methods for preparing such compositions, individual protein subunits used to prepare such compositions, and nucleic acid molecules encoding these protein subunits. Thus, included within the scope of the present disclosure are variant forms of wild-type proteins that retain the ability to form capsids or capsid-like structures and associate to form capsids or capsid-like structures.

[0285] Antigens and antigenic determinants In some embodiments, the compositions described herein comprise an antigen or antigenic determinant bound to a virus-like particle. The present disclosure provides different compositions depending on the antigen or antigenic determinant selected based on the desired therapeutic effect. Exemplary antigens or antigenic determinants suitable for use in the present invention are disclosed in U.S. Patent Nos. 7,229,624, 6,964,769, and 7,264,810, the disclosures of which are incorporated herein by reference in their entireties.

[0286] The antigen may be any antigen of known or unknown origin. The antigen may be isolated from bacteria, viruses or other pathogens, or may be a recombinant antigen obtained by expression of a suitable nucleic acid encoding the antigen. Antigens may also be isolated from prions, tumors, self-molecules, non-peptide hapten molecules, allergens and hormones. In some embodiments, the antigen is a recombinant antigen. The choice of antigen will, of course, depend on the desired immunological response and the host.

[0287] In some embodiments, an immune response is elicited against the VLP itself, hi some embodiments, the virus-like particle is conjugated, fused, or otherwise attached to an antigen / immunogen against which an enhanced immune response is desired.

[0288] In some embodiments, at least one antigen or antigenic determinant is fused to a virus-like particle. As outlined above, VLPs are usually composed of at least one subunit that assembles to form a VLP. Thus, in some embodiments, an antigen or antigenic determinant is fused to at least one subunit of a protein that can be incorporated into a virus-like particle or VLP, forming a chimeric VLP-subunit-antigen fusion.

[0289] Fusion of the antigen or antigenic determinant can be achieved by insertion into the VLP subunit sequence or by fusion to either the N- or C-terminus of the VLP subunit or a protein that can be incorporated into the VLP. Hereinafter, reference to a fusion protein of a VLP subunit and a peptide includes fusion to both termini of the subunit sequence or internal insertion of the peptide within the subunit sequence.

[0290] Fusions can also be achieved by inserting an antigen or antigenic determinant sequence into variants of VLP subunits in which a portion of the subunit sequence has been deleted, further referred to as truncation mutants. Truncation mutants can have deletions at the N-terminus or C-terminus of the VLP subunit sequence, or internal deletions of a portion of the sequence. For example, a particular VLP HBcAg having a deletion of amino acid residues 79-81 is a truncation mutant with an internal deletion. In some embodiments, an antigen or antigenic determinant is fused to either the N-terminus or C-terminus of a truncation mutant VLP subunit. Similarly, fusion of an epitope into the sequence of a VLP subunit can also be achieved by substitution, e.g., in a particular VLP HBcAg, amino acids 79-81 are replaced with a foreign epitope. Thus, fusions, as referred to hereinafter, can be achieved by inserting an antigen or antigenic determinant sequence into the sequence of a VLP subunit, by substituting an antigen or antigenic determinant into a portion of the VLP subunit sequence, or by a combination of deletion, substitution, or insertion.

[0291] Chimeric antigen-VLP subunits or chimeric antigenic determinant-VLP subunits can generally self-assemble to form VLPs. VLPs displaying epitopes fused to VLP subunits are also referred to herein as chimeric VLPs. As indicated, virus-like particles comprise or are composed of at least one VLP subunit. In some embodiments, virus-like particles comprise or are composed of a mixture of chimeric VLP subunits and non-chimeric VLP subunits (i.e., VLP subunits to which an antigen is not fused), resulting in so-called mosaic particles. This can be advantageous for ensuring VLP formation and assembly. In these embodiments, the proportion of chimeric VLP subunits can be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95%, or more.

[0292] Flanking amino acid residues can be added to either end of the peptide or epitope sequence fused to either end of the VLP subunit sequence, or to internally insert such peptide sequences into the VLP subunit sequence. Glycine and serine residues are particularly preferred amino acids for use in flanking sequences added to fused peptides. Glycine residues can provide additional flexibility and reduce the potential destabilizing effect of fusing foreign sequences to the VLP subunit sequence.

[0293] In some embodiments, at least one antigen or antigenic determinant is fused to the Qβ coat protein. Fusion protein constructs in which an epitope is fused to the C-terminus of a truncated form of the Qβ A1 protein or inserted within the A1 protein have been described (Kozlovska, TM, et al., Intervirology, 39:9-15 (1996)). The A1 protein is generated by suppression at the UGA stop codon and has a length of 329 amino acids, or 328 amino acids if the N-terminal methionine cleavage is taken into account. Cleavage of the N-terminal methionine before the alanine (the second amino acid encoded by the Qβ CP gene) is typically performed in E. coli, as is the case with the N-terminus of the Qβ coat protein. The portion of the A1 gene 3'-terminal to the UGA amber codon encodes a CP extension having a length of 195 amino acids. Insertion of at least one antigen or antigenic determinant between positions 72 and 73 of the CP extension represents a further embodiment of the present invention (Kozlovska, TM, et al., Intervirology 39:9-15 (1996)). Fusion of an antigen or antigenic determinant at the C-terminus of a C-terminally truncated Qβ A1 protein represents a further embodiment of the present invention. For example, Kozlovska et al. (Intervirology, 39:9-15 (1996)) describe a Qβ A1 fusion protein in which an epitope is fused to the C-terminus of the CP extension of Qβ truncated at position 19.

[0294] Kozlovska et al.(Intervirology,39: 9-15 As described by [End Page 110] (1996), assembly of particles displaying fusion epitopes typically requires the presence of both the A1 protein-antigen fusion and the wild-type CP to form mosaic particles. However, virus-like particles, and herein specifically, embodiments comprising VLPs of the coat protein of RNA phage Qβ composed solely of VLP subunits to which at least one antigen or antigenic determinant has been fused, are also included within the scope of the present disclosure.

[0295] The generation of mosaic particles can be achieved in a number of ways. Kozlovska et al., Intervirology, 39:9-15 (1996) described three methods, all of which can be used to practice the present disclosure. In the first approach, efficient display of the fusion epitope on the VLP is mediated by expression of a plasmid encoding a Qβ A1 fusion protein with a UGA stop codon between the CP and CP extension in an E. coli strain harboring a plasmid encoding a cloned UGA suppressor tRNA, which results in translation of the UGA codon to Trp (pISM3001 plasmid (Smiley BK, et al., Gene 134:33-40 (1993))). In another approach, the CP gene stop codon is modified to UAA and cotransformed with a second plasmid expressing the A1 protein-antigen fusion. The second plasmid encodes a different antibiotic resistance and has a replication origin compatible with that of the first plasmid (Kozlovska, T.M., et al., Intervirology 39:9-15 (1996)). In a third approach, as shown in FIG. 1 of Kozlovska et al., Intervirology 39:9-15 (1996), the CP and A1 protein antigen fusion are encoded in a bicistronic form and operably linked to a promoter such as the Trp promoter.

[0296] In some embodiments, recombinant DNA technology can be used to fuse heterologous proteins to VLP proteins (Kratz, PA, et al., Proc. Natl. Acad. Sci. USA 96:1915 (1999)). For example, the present disclosure encompasses VLPs recombinantly fused or chemically coupled (including both covalent and non-covalent linkages) to antigens (or portions thereof, preferably at least 10, 20, or 50 amino acids) to generate fusion proteins or conjugates. Fusion need not necessarily be direct but can occur via linker sequences. More generally, when epitopes fused, linked, or otherwise attached to virus-like particles are used as antigens in accordance with the present invention, spacer or linker sequences are added to one or both ends of the epitope. Such linker sequences preferably include sequences recognized by proteases of the proteasome, endosome, or other vesicular compartment of the cell.

[0297] One method of attachment is via a peptide bond, and the conjugate can be a continuous polypeptide, i.e., a fusion protein. In some embodiments, different peptides or polypeptides are linked in-frame to each other to form a continuous polypeptide. Thus, a first portion of the fusion protein contains an antigen or immunogen, and a second portion of the fusion protein, N-terminal or C-terminal to the first portion, contains a VLP. Alternatively, internal insertion into a VLP can be used in accordance with the present invention, with any linking sequence at both ends of the antigen.

[0298] A flexible linker sequence (e.g., a polyglycine / polyserine-containing sequence such as [Gly4 Ser]2 (Huston et al., Meth. Enzymol. 203:46-88 (1991)) can be inserted between the antigen and ligand of the fusion protein. Fusion proteins can also be constructed to contain an "epitope tag" that allows the fusion protein to be bound by an antibody (e.g., a monoclonal antibody), for example, for labeling or purification purposes. One example of an epitope tag is the Glu-Glu-Phe tripeptide, which is recognized by the monoclonal antibody YL1 / 2.

[0299] The present disclosure also relates to chimeric DNA comprising a VLP-encoding sequence and an antigen / immunogen-encoding sequence. The DNA can be expressed, for example, in insect cells transformed with baculovirus, in yeast, or in bacteria. There is no limitation regarding the expression system, and a wide selection of expression systems is available for routine use. Preferably, a system capable of expressing large amounts of protein is used. Bacterial expression systems are generally used due to their efficiency. Examples of bacterial expression systems suitable for use within the scope of the present invention include those described in Clarke et al., J. Gen. Virol. 71:1109-1117 (1990), Borisova et al., J. Virol. 67:3696-3701 (1993), and Studier et al., Methods Enzymol. 185:60-89 (1990). One example of a suitable yeast expression system is that described in Emr, Methods Enzymol. 185:231-3 (1990). The baculovirus system, which has traditionally been used to prepare capsid proteins, is also suitable. Constitutive or inducible expression systems can also be used. The form in which the protein is obtained can be controlled by the choice and possible modifications of the available expression system.

[0300] In some embodiments, at least one antigen or antigenic determinant is attached to the virus-like particle by at least one covalent bond. In some embodiments, at least one antigen or antigenic determinant is attached to the virus-like particle by at least one covalent bond, which is a non-peptide bond, resulting in antigen or antigenic determinant arrays and antigen or antigenic determinant-VLP conjugates, respectively. The antigen or antigenic determinant arrays and conjugates typically preferably have a repetitive and regular structure, respectively, due to the directed attachment of at least one antigen or antigenic determinant to the VLP. In some embodiments, 120 or more, 180 or more, 270 or more, and 360 or more antigens are attached to the VLP. The formation of repetitive and regular antigen or antigenic determinant-VLP arrays and conjugates is ensured by the directed, directional, and defined attachment and linkage of at least one antigen or antigenic determinant to the VLP, respectively, as will become apparent below. Furthermore, the typical inherent highly repetitive and organized structure of VLPs advantageously contributes to the presentation of antigens or antigenic determinants in a highly ordered and repetitive manner, giving highly organized and repetitive antigen or antigenic determinant-VLP arrays and conjugates, respectively.

[0301] VLPs or capsids of Qβ coat protein display a defined number of lysine residues on their surface, with a defined topology in which three lysine residues face the interior of the capsid and interact with RNA, and the other four lysine residues are exposed to the exterior of the capsid. These defined properties favor antigen binding to the exterior of the particle rather than binding to the interior of the particle where the lysine residues interact with RNA. VLPs of other RNA phage coat proteins also have a defined number of lysine residues on their surface and a defined topology of these lysine residues.

[0302] In some embodiments, the first binding site is a lysine residue and / or the second binding site comprises a sulfhydryl group or a cysteine ​​residue, hi some embodiments, the first binding site is a lysine residue and the second binding site is a cysteine ​​residue.

[0303] In some embodiments, the antigen or antigenic determinant is attached via a cysteine ​​residue to a lysine residue of the VLP of the coat protein of an RNA phage, in particular the VLP of the coat protein of Qβ.

[0304] The use of VLPs as carriers allows the formation of stable antigen arrays and conjugates with different antigen densities, respectively. In particular, the use of VLPs of RNA phages, and in this specification, particularly VLPs of RNA phage Qβ coat protein, can achieve extremely high epitope densities. In particular, for example, by binding human Aβ1-6 peptide to VLPs of Qβ coat protein, a density of more than 1.5 epitopes per subunit has been achieved (WO2004 / 016282). The preparation of compositions of VLPs of RNA phage coat protein with high epitope densities can be achieved using the teachings of the present application. In some embodiments, when antigens or antigenic determinants are attached to a VLP Qβ coat protein, an average number of antigens or antigenic determinants per subunit of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or more are used.

[0305] A second binding site, as defined herein, can be naturally or non-naturally present on an antigen or antigenic determinant. If a suitable natural second binding site does not exist on the antigen or antigenic determinant, a non-natural second binding site must be added to the antigen.

[0306] As mentioned above, four lysine residues are exposed on the surface of the VLP of Qβ coat protein. These residues are typically derivatized by reacting with a cross-linking molecule. If not all of the exposed lysine residues are capable of binding to antigens, the lysine residues that reacted with the cross-linking agent will still have the cross-linking molecule attached to their quaternary amino groups after the derivatization step. This can result in the loss of one or more positive charges, which can be detrimental to the solubility and stability of the VLP. Substituting some of the lysine residues with arginine, as in the Qβ coat protein mutants disclosed below, prevents excessive loss of positive charges because the arginine residues do not react with the cross-linking agent. Furthermore, substituting lysine residues with arginine can provide a more defined antigen array because fewer sites are available for antigen reaction.

[0307] In some embodiments, exposed lysine residues are substituted with arginine in the following Qβ coat protein mutants and variant Qβ VLPs disclosed herein: Qβ-240 (Lys13-Arg, SEQ ID NO: 125), Qβ-250 (Lys2-Arg, Lys13-Arg, SEQ ID NO: 127) and Qβ-259 (Lys2-Arg, Lys16-Arg, SEQ ID NO: 129).

[0308] In some embodiments, the Qβ mutant coat protein contains an additional lysine residue suitable for obtaining even higher-density antigen arrays. This mutant Qβ coat protein, Qβ-243 (Asn 10-Lys, SEQ ID NO: 126), was cloned, expressed, and capsids or VLPs were isolated and purified, demonstrating that the introduction of an additional lysine residue is compatible with the self-assembly of subunits into capsids or VLPs. Thus, antigen or antigenic determinant arrays and conjugates, respectively, can be prepared using the VLPs of Qβ coat protein mutants. A particularly preferred method for conjugating antigens to VLPs, particularly VLPs of RNA phage coat proteins, is to link a lysine residue present on the surface of the VLP of the RNA phage coat protein with a cysteine ​​residue added to the antigen. For a cysteine ​​residue to be effective as a second conjugation site, a sulfhydryl group must be available for conjugation. Therefore, the cysteine ​​residue must be in its reduced state. That is, a free cysteine ​​or a cysteine ​​residue with a free sulfhydryl group must be present. If the cysteine ​​residue serving as the second binding site is oxidized, e.g., if it forms a disulfide bridge, reduction of this disulfide bridge with, for example, DTT, TCEP, or β-mercaptoethanol is required. The concentration of the reducing agent and the molar excess of the reducing agent relative to the antigen must be adjusted for each antigen. Starting with a low concentration of 10 μM or less, a titration range of reducing agent was tested, if necessary, ranging from 10 to 20 mM or more to evaluate antigen binding to the carrier. As described in WO 02 / 056905, low concentrations of reducing agent are compatible with the coupling reaction. However, as those skilled in the art will understand, higher concentrations inhibit the coupling reaction, and in such cases, the reducing agent must be removed or its concentration reduced, for example, by dialysis, gel filtration, or reverse-phase HPLC. The pH of the dialysis or equilibration buffer is advantageously below 7, preferably 6. Compatibility of low pH buffers with antigen activity or stability must be tested.

[0309] The epitope density on the VLP of RNA phage coat protein can be adjusted by selecting the cross-linker and other reaction conditions. For example, the cross-linkers Sulfo-GMBS and SMPH usually allow for high epitope density. Because derivatization is positively affected by high concentrations of reactants, manipulation of reaction conditions can be used to control the number of antigens bound to the VLP of RNA phage coat protein, particularly the VLP of Qβ coat protein.

[0310] Prior to designing a non-native second binding site, it is necessary to select a location where the second binding site will be fused, inserted, or generally engineered. The selection of the location of the second binding site can be based, for example, on the crystal structure of the antigen. Such a crystal structure of the antigen can provide information on the availability of the C- or N-termini of the molecule (e.g., as measured by their solvent accessibility) or the solvent exposure of residues suitable for use as second binding sites, such as cysteine ​​residues. Exposed disulfide bridges, as in the case of Fab fragments, can also serve as a source of second binding sites, as they can generally be converted to a single cysteine ​​residue by mild reduction with, for example, 2-mercaptoethylamine, TCEP, β-mercaptoethanol, or DTT. Mild reduction conditions are selected that do not affect the immunogenicity of the antigen. Generally, when immunization with an autoantigen aims to inhibit the interaction of this autoantigen with its natural ligand, the second binding site is added to allow for the generation of antibodies against the interaction site with the natural ligand. Thus, the location of the second binding site is selected to prevent steric hindrance by the second binding site or any amino acid linker that includes it. In further embodiments, an antibody response against a site different from the interaction site between the autoantigen and its natural ligand is desired. In such embodiments, the second binding site can be selected to prevent the generation of antibodies against the interaction site between the autoantigen and its natural ligand.

[0311] Other criteria for selecting the location of the second binding site include the oligomerization state of the antigen, the oligomerization site, the presence of cofactors, and the existence of experimental evidence disclosing sites in the antigen structure and sequence where modification of the antigen is compatible with the function of the autoantigen or with the generation of antibodies that recognize the autoantigen.

[0312] In some embodiments, the antigen or antigenic determinant comprises a single secondary binding site or single reactive binding site capable of binding to a primary binding site on the core particle and VLP or VLP subunit, respectively. This further ensures defined, uniform binding and association of at least one, and generally more than one, preferably more than 10, 20, 40, 80, 120, 150, 180, 210, 240, 270, 300, 360, 400, 450 antigens with the core particle and VLP, respectively. Thus, providing a single secondary binding site or single reactive binding site on an antigen ensures a single, uniform type of binding and association, providing a highly ordered, repeating sequence, respectively. For example, if the binding is via interactions of lysine (as the first binding site) and cysteine ​​(as the second binding site), respectively, according to one embodiment of the present invention, only one cysteine ​​residue per antigen can bind to the first binding site of the VLP and core particle, respectively, regardless of whether this cysteine ​​residue is naturally or non-naturally occurring on the antigen.

[0313] In some embodiments, engineering a second binding site onto an antigen requires the incorporation of an amino acid linker containing an amino acid suitable as a second binding site according to the present disclosure. Thus, in some embodiments, the amino acid linker is attached to the antigen or antigenic determinant via at least one covalent bond. In some embodiments, the amino acid linker comprises the second binding site. In some embodiments, the amino acid linker comprises a sulfhydryl group or a cysteine ​​residue. In some embodiments, the amino acid of the linker is cysteine.

[0314] In some embodiments, the virus-like particle comprises at least one first binding site, and the antigen or antigenic determinant comprises at least one second binding site. In some embodiments, the first binding site comprises an amino group or a lysine residue. In some embodiments, the second binding site is selected from the group consisting of (a) a binding site not naturally occurring in the antigen or antigenic determinant, and (b) a binding site naturally occurring in the antigen or antigenic determinant. In some embodiments, the second binding site comprises a sulfhydryl group or a cysteine ​​residue. In some embodiments, the antigen or antigenic determinant binds to the virus-like particle via binding between the first binding site and the second binding site, the binding being via at least one peptide bond, and the antigen or antigenic determinant and the virus-like particle interact through the bond to form an ordered and repetitive array. In some embodiments, the first binding site is a lysine residue, and the second binding site is a cysteine ​​residue. In some embodiments, the first binding site is an amino group, and the second binding site is a sulfhydryl group.

[0315] The present disclosure is applicable to a wide range of antigens. In some embodiments, the antigen is a protein, polypeptide, or peptide. In some embodiments, the antigen is DNA. The antigen may also be a lipid, carbohydrate, or organic molecule, particularly a small organic molecule such as nicotine.

[0316] Methods for producing VLPs and packaging RLR agonists into VLPs Methods for expression of coat proteins and mutant coat proteins, respectively, that result in self-assembly into VLPs are described in U.S. Patent No. 7,138,252, which is incorporated by reference in its entirety. Suitable E. coli strains include, but are not limited to, E. coli K802, JM 109, and RR1. Suitable vectors and strains, and combinations thereof, can be identified by SDS-PAGE and encapsidation and assembly, optionally by first purifying the capsids by gel filtration, followed by immunodiffusion assays (Ouchterlony test) or electron microscopy (Kozlovska, TM et al., Gene These mutant coat proteins can be identified by examining their expression in the IL-16 β -antigen (IL-16 β -antigen) and mutant coat proteins, respectively.

[0317] An advantage of using VLPs derived from RNA phages is their high expression yield in bacteria, which allows for the production of large amounts of material at affordable cost. Methods for producing the virus-like particles described herein, including methods scalable to commercial scale, are described in U.S. Patent Nos. 9,518,095 and 9,657,065, respectively, which are incorporated herein by reference in their entireties.

[0318] The present disclosure also provides a method for producing a composition comprising a VLP and an RLR agonist packaged within the VLP, the method comprising incubating the VLP with an RLR agonist, adding an RNase, and purifying the composition. In some embodiments, the method further comprises binding an antigen or antigenic determinant to the virus-like particle. In some embodiments, the antigen or antigenic determinant is bound to the virus-like particle before incubating the virus-like particle with the RLR agonist. In some embodiments, the antigen or antigenic determinant is bound to the virus-like particle after purifying the composition. In some embodiments, the method comprises incubating the VLP with an RNase, adding an RLR agonist, and purifying the composition. In some embodiments, the method further comprises binding an antigen or antigenic determinant to the virus-like particle. In some embodiments, the antigen or antigenic determinant is bound to the virus-like particle before incubating the virus-like particle with the RNase. In some embodiments, the antigen or antigenic determinant is bound to the virus-like particle after purifying the composition. In some embodiments, the VLPs are expressed in a bacterial expression system. In another embodiment, the RNase is RNase A.

[0319] The present disclosure further provides methods for producing a composition comprising an RLR agonist packaged within a VLP, the method comprising disassembling the VLP, adding the RLR agonist, and reassembling the VLP. In some embodiments, the disassembled VLPs are generated during VLP production. In some embodiments, the disassembled VLPs comprise isolated coat protein dimers (e.g., Qβ dimers). In some embodiments, the isolated dimers assemble around the RLR agonist to form a VLP, packaging the agonist within the VLP. The method can further comprise removing nucleic acid from the disassembled VLPs and / or purifying the composition after reassembly. In some embodiments, the method further comprises binding an antigen or antigenic determinant to the virus-like particle. In some embodiments, the antigen or antigenic determinant is bound to the virus-like particle before disassembling the virus-like particle. In some embodiments, the antigen or antigenic determinant is bound to the virus-like particle after reassembly, preferably after purifying the composition.

[0320] The present disclosure provides methods for attaching antigens or antigenic determinants to VLPs. As indicated, in some embodiments, at least one antigen or antigenic determinant is attached to a VLP by chemical cross-linking, typically by using a heterobifunctional cross-linker. Several heterobifunctional cross-linkers are known in the art. In some embodiments, the heterobifunctional cross-linker contains a first binding site, i.e., a functional group capable of reacting with the side chain amino group of a lysine residue on a VLP or at least one VLP subunit, and a second binding site, i.e., a cysteine ​​residue fused to the antigen or antigenic determinant, optionally made available for reaction by reduction. The first step of this procedure, commonly referred to as derivatization, is the reaction of the VLP with the cross-linker. The product of this reaction is an activated VLP, also referred to as an activated carrier. In a second step, unreacted cross-linker is removed using conventional methods such as gel filtration or dialysis. In a third step, the antigen or antigenic determinant is reacted with the activated VLP, commonly referred to as the coupling step. Unreacted antigen or antigenic determinant can optionally be removed in a fourth step, for example, by dialysis. Several heterobifunctional crosslinkers are known in the art. These include the crosslinkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, and SVSB, SIA, and other crosslinkers with one functional group reactive toward amino groups and one functional group reactive toward cysteine ​​residues, such as those sold by Pierce Chemical Company (Rockford, Ill., USA). All of the above crosslinkers form thioether bonds. Another suitable class of crosslinkers is characterized by the introduction of a disulfide bond between the antigen or antigenic determinant and the VLP upon coupling. In one embodiment, crosslinkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce).The degree of derivatization of VLPs with cross-linking agents can be influenced by various experimental conditions such as the concentration of each reaction partner, the excess of one reagent over the other, pH, temperature, ionic strength, etc. The degree of coupling, i.e., the amount of antigen or antigenic determinant per VLP subunit, can be adjusted by varying the above-mentioned experimental conditions to suit the vaccine requirements.

[0321] In some embodiments, methods of attaching an antigen or antigenic determinant to a VLP involve linking a lysine residue on the surface of the VLP to a cysteine ​​residue on the antigen or antigenic determinant, which may require fusion of an amino acid linker containing a cysteine ​​residue to the antigen or antigenic determinant as or part of a second attachment site for coupling to the VLP.

[0322] In some embodiments, flexible amino acid linkers are used. Examples of amino acid linkers include (a) CGG, (b) an N-terminal γ1-linker, (c) an N-terminal γ3-linker, (d) an Ig hinge region, (e) an N-terminal glycine linker, (f) (G)kC(G)n, where n=0-12 and k=0-5, (g) an N-terminal glycine-serine linker, (h) (G)kC(G)m(S)l(GGGGS)n, where n=0-3, k=0-5, m=0-10, and l=0-2 (SEQ ID NO: 188), (i) GGC (k) GGC-NH2, (l) C-terminal γ1-linker, (m) C-terminal γ3 linker, (n) C-terminal glycine linker, (o) (G)nC(G)k, where n=0-12 and k=0-5, (p) C-terminal glycine-serine linker, (q) (G)m(S)l(GGGGS)n(G)oC(G)k, where n=0-3, k=0-5, m=0-10, 1=0-2, and o=0-8 (SEQ ID NO: 189).

[0323] Further examples of amino acid linkers include the hinge region of an immunoglobulin, a glycine-serine linker (GGGGS)n (SEQ ID NO: 190), and a glycine linker (G)n, all of which contain a cysteine ​​residue as the second attachment site and optionally an additional glycine residue. Common examples of amino acid linkers include N-terminal γ1:CGDKTHTSPP (SEQ ID NO: 191), C-terminal γ1:DKTHTSPPCG (SEQ ID NO: 192), N-terminal γ3:CGGPKPSTPPGSSGGAP (SEQ ID NO: 193), C-terminal γ3:PKPSTPPGSSGGAPGGCG (SEQ ID NO: 194), N-terminal glycine linker:GCGGGG (SEQ ID NO: 195), C-terminal glycine linker:GGGGCG (SEQ ID NO: 196), C-terminal glycine-lysine linker:GKKKGC (SEQ ID NO: 197), and N-terminal glycine-lysine linker:CGKKGG (SEQ ID NO: 198).

[0324] In some embodiments, other amino acid linkers when a hydrophobic antigen or antigenic determinant is attached to the VLP include CGKKGG (SEQ ID NO: 199), or CGDEGG (SEQ ID NO: 200) for N-terminal linkers, or GGKKGC (SEQ ID NO: 201) and GGEDGC (SEQ ID NO: 202) for C-terminal linkers. In the case of C-terminal linkers, the terminal cysteine ​​is optionally C-terminally amidated.

[0325] In some embodiments, GGCG (SEQ ID NO: 203), GGC, or GGC-NH2 ("NH2" denotes amidation) linkers are used as amino acid linkers at the C-terminus of the peptide, or CGG at its N-terminus. Generally, a glycine residue is inserted between the bulky amino acid and the cysteine ​​used as the second attachment site to prevent potential steric hindrance of the bulkier amino acid in the coupling reaction. In some embodiments, the amino acid linker GGC-NH2 is fused to the C-terminus of the antigen or antigenic determinant.

[0326] Cysteine ​​residues present in the antigen or antigenic determinant must be in their reduced state to react with the heterobifunctional cross-linker on the activated VLP. That is, a free cysteine ​​or a cysteine ​​residue with a free sulfhydryl group must be present. If the cysteine ​​residue that serves as the binding site is in an oxidized form, e.g., if it forms a disulfide bridge, reduction of the disulfide bridge, e.g., with DTT, TCEP, or β-mercaptoethanol, is required. As described in WO 02 / 05690, low concentrations of reducing agents are compatible with coupling, but as will be understood by those skilled in the art, higher concentrations will inhibit the coupling reaction, in which case the reducing agent must be removed or its concentration reduced prior to coupling, e.g., by dialysis, gel filtration, or reverse-phase HPLC.

[0327] Attaching an antigen or antigenic determinant to a...

Claims

[Claim 1] The invention described in the specification.

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