Stephen Tuck

Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyte activityby a T-helper cell-independent mechanism

Immunostimulatory DNA sequences (ISS) contain unmethylated CpG dinucleotides within a defined motif. Immunization with ISS-based vaccines has been shown to induce high antigen-specific cytotoxic lymphocyte (CTL) activity and a Th1-biased immune response. We have developed a novel ISS-based vaccine composed of ovalbumin (OVA) chemically conjugated to ISS–oligodeoxynucleotide (ODN). Protein–ISS conjugate (PIC) is more potent in priming CTL activity and Th1-biased immunity than other ISS-based vaccines. Cytotoxic lymphocyte activation by ISS–ODN-based vaccines is preserved in both CD4−/− and MHC class II−/− gene-deficient animals. Furthermore, PIC provides protection against a lethal burden of OVA-expressing tumor cells in a CD8+ cell-dependent manner. These results demonstrate that PIC acts through two unique mechanisms: T-helper-independent activation of CTL and facilitation of exogenous antigen presentation on MHC class I. This technology may have clinical applications in cancer therapy and in stimulating host defense in AIDS and chronic immunosuppression.

A minimal human immunostimulatory CpG motif that potently induces IFN‐γ and IFN‐α production

Recent reports have shown that immunostimulatory sequences (ISS) containing CpG motifs have minimal length requirements (≥12 bases) for the exertion of immune‐enhancing function upon mammalian cells. Herein we demonstrate that short ISS (5–7 bases), which exhibit no activity on their own, induce IFN‐γ and IFN‐α secretion from human peripheral blood mononuclear cells when adsorbed to the surface of cationic poly(D,L‐lactide‐co‐glycolide) microparticles (cPLGA). Utilizing this technique, we discovered a minimal ISS sequence for induction of IFN‐γ and IFN‐α from human cells: 5′‐TCGXX‐3′. These short ISS/cPLGA formulations targeted PDC in similar fashion to longer ISS ODN, the activity of which does not require (but is enhanced by) cPLGA. PDC stimulated with short ISS/cPLGA responded with enhanced uptake of ISS and elevated production of cytokines, including IFN‐α. However, ISS‐responsive B cells did not respond to short ISS/cPLGA, underlining the plasmacytoid dendritic cell selectivity of this formulation. These results describe a novel technique for formulating active, but very short, ISS oligodeoxynucleotide that allows for the dissection and characterization of minimal immunostimulatory CpG motifs.