Immunostimulatory oligonucleotides

Abstract

Disclosed herein are immunostimulatory oligonucleotides and compositions and methods of use thereof. More specifically, immunostimulatory oligonucleotides, methods of optimizing the immunostimulatory properties of oligonucleotides, and methods of using the immunostimulatory oligonucleotides to elicit a toll-like receptor 21 (TLR21)-mediated immune response are disclosed.

1 . An immunostimulatory oligonucleotide comprising at least one CpG motif and a guanine nucleotide enriched sequence beginning at or within four nucleotides of the 5′ terminus of the oligonucleotide. 
     
     
         2 . The oligonucleotide of  claim 1 , wherein the guanine nucleotide enriched sequence comprises a first plurality of guanine nucleotides. 
     
     
         3 . The oligonucleotide of  claim 2 , wherein the first plurality of guanine nucleotides comprises three to eight guanine nucleotides. 
     
     
         4 . The oligonucleotide of  claim 3 , wherein the oligonucleotide comprises SEQ ID NO: 16, 17, 18, 19, 20, 21, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 77, 78, 81, 82, 85, 86, 89, 90, 92, 93, 96, 97, 100, 102, 104, 106, 108, 143, or 252. 
     
     
         5 . The oligonucleotide of  claim 1 , wherein the guanine nucleotide enriched sequence comprises TTAGGG, TTAGGGTTAGGG (SEQ ID NO:261), TTTTGGGG, GGGGTTTT, GGGGTTTTGGGG (SEQ ID NO:262), TTAGGG, TTAGGGTTAGGGTTTT (SEQ ID NO:263), TGTGGGTGTGTGTGGG (SEQ ID NO: 268), GGAGG, TGGAGGC, TGGAGGCTGGAGGC (SEQ ID NO:264), or TGGGGT (SEQ ID NO:265). 
     
     
         6 . The oligonucleotide of  claim 1 , wherein the oligonucleotide comprises SEQ ID NO: 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 124, 125, 126, 127, 129, 130, 131, 134, 136, 137, or 138. 
     
     
         7 . The oligonucleotide of  claim 1  further comprising a second plurality of guanine nucleotides between the first plurality of guanine nucleotides and the at least one CpG motif. 
     
     
         8 . The oligonucleotide of  claim 2 , wherein the first plurality of guanine nucleotides, the second plurality of guanine nucleotides, or both comprise a G-quartet sequence. 
     
     
         9 . The oligonucleotide of  claim 7  wherein the first and second pluralities of guanine nucleotides comprise a G-wire sequence. 
     
     
         10 . A vaccine for preventing or treating infectious disease comprising the oligonucleotide of  claim 1 . 
     
     
         11 . A vector comprising the oligonucleotide of  claim 1 . 
     
     
         12 . An immunostimulatory composition comprising the oligonucleotide of  claim 1 . 
     
     
         13 . The immunostimulatory composition of  claim 12  further comprising a pharmaceutically acceptable carrier. 
     
     
         14 . The immunostimulatory composition of  claim 13 , wherein the oligonucleotide and the carrier are linked. 
     
     
         15 . A method of stimulating toll-like receptor 21 (TLR21) comprising:
 administering to a subject in need thereof an immunostimulatory oligonucleotide having at least one CpG motif and a guanine nucleotide enriched sequence beginning at or within four nucleotides of the 5′ terminus of the oligonucleotide, the guanine nucleotide enriched sequence comprising a first plurality of guanine nucleotides.   
     
     
         16 . The method of  claim 15 , wherein the oligonucleotide comprises SEQ ID NO: 110, 111, 112, 113, 114, 115, 116, 117118, 119, 120, 124, 125, 126, 127, 129, 130, 131, 134, 136, 137, or 138. 
     
     
         17 . The method of  claim 16 , wherein the oligonucleotide further comprises a G-wire sequence. 
     
     
         18 . The method of  claim 17 , wherein the oligonucleotide comprises SEQ ID NO:141, 142, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 252, or GCGT-Gwire3. 
     
     
         19 . A method for increasing TLR21-stimulatory activity of an oligonucleotide having at least one CpG motif comprising fusing the 5′ end of the oligonucleotide to a guanine nucleotide enriched sequence. 
     
     
         20 . A method of eliciting an immune response in a subject comprising:
 administering to a subject in need thereof an immunostimulatory composition comprising an oligonucleotide having at least one CpG dinucleotide motif and a guanine nucleotide enriched sequence beginning at or within four nucleotides of the 5′ terminus of the oligonucleotide.