Cysteine-Tailed Class I-Binding Peptides Bind to CpG Adjuvant and Enhance Primary CTL Responses

Abstract

Immunostimulatory CpG motifs in synthetic oligonucleotides can be effective adjuvants for the priming of CTLs. We first observed that a single male-specific peptide (KCSRNRQYL) (HY2) was more efficient than another male-specific peptide (WMHHNMDLI) (HY1) at priming IFN-γ-secreting CTLs in vivo when combined with lipid A and CpG and that it also visibly precipitated CpG. The addition of the six N-terminal residues (KCSRNR) from HY2 to HY1 yielded a peptide, KCSRNR-HY1, that both precipitated CpG and primed increased numbers of HY1-specific CTLs. We refer to this type of peptide as a primotope that includes a class I binding peptide tailed with amino acids that increase priming. Ala residues were substituted for the Arg/Lys residues (ACSANA-HY1), and these substitutions did not reduce in vivo priming potential. However, the substitution of Ala for Cys (KASRNR-HY1) resulted in the complete loss of priming, demonstrating the importance of Cys for in vivo priming when mixed with CpG. This result suggested that increased priming was based in disulfide bonding between Cys residues and internal phosphorothioate groups of synthetic CpG. The addition of Cys-bearing primotopes to radiolabeled CpG with a single thioate group resulted in the appearance of a new band that was inhibited by 1) Cys > Ala substitution and 2) reduction and alkylation of CpG. These results reveal a novel mechanism for complexing class I binding peptides and CpG adjuvant for development of new peptide-adjuvant combinations for vaccines for cancer and infectious diseases.

Conjugation Reactions Involving Maleimides and Phosphorothioate Oligonucleotides

“…Phosphorothioate diesters have been described to react with activated disulfides such as the Ellman reagent, 4 most likely forming mixed thiol-thiophosphate disulfides. 5 Yet, phosphorothioate diesters have also been reported not to react with pyridyldisulfide groups. 6 In fact, there are various reports on the synthesis of disulfide-linked conjugates of phosphorothioated oligonucleotides, 7−9 showing that disulfides between two thiols can be formed in the presence of and with no interference from phosphorothioate groups.…”

Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance

“…1A). It remains unclear which of these two specificities constitutes the major response; D b -Uty + CTL are usually considered quantitatively and qualitatively superior [11,37,38], but other studies have found the converse, with D b -Smcy + CTL predominating [33,39]. The graphs at the top of Fig.…”

Innate and adaptive immune responses in the urinary tract

“…Indeed, modulators of innate immune responses are now frequently being incorporated as ‘adjuvants’ to boost immunity to vaccines [54,55]. TLR ligands such as MPL and CpG‐ODN have been shown to be excellent vaccine adjuvants in animal studies, promoting development of robust antigen specific humoral and cellular responses [56–63]. Thus, immunomodulators used to boost innate immune responses in the urinary tract may be also employed to boost adaptive immune responses.…”