Novel immunostimulatory agent based on CpG oligodeoxynucleotide linked to the nontoxic B subunit of cholera toxin.

• In this study, we report the development of a novel, rationally designed immunostimulatory adjuvant based on chemical conjugation of CpG oligodeoxynucleotide (ODN) to the nontoxic B subunit of cholera toxin (CTB). We demonstrate that the immunostimulatory effects of CpG can be dramatically enhanced by conjugation to CTB. Thus, CpG ODN linked to CTB (CTB-CpG) was shown to be a more potent stimulator of proinflammatory cytokine and chemokine responses in murine splenocytes and human PBMCs than those of CpG ODN alone in vitro. The presence of CpG motif, but not modified phosphorothioate ODN backbone, was found to be critical for the enhanced immunostimulatory effects of CTB-CpG. Our mode-of-action studies, including studies on cells from specifically gene knockout mice suggest that similar to CpG, CTB-CpG exerts its immunostimulatory effects through a TLR9/MyD88- and NF-kappaB-dependent pathway. Surprisingly, and as opposed to CpG ODN, CTB-CpG-induced immunity was shown to be independent of endosomal acidification and resistant to inhibitory ODN. Furthermore, preincubation of CTB-CpG with GM1 ganglioside reduced the immunostimulatory effects of CTB-CpG to those of CpG ODN alone. Interestingly, conjugation of CpG ODN to CTB confers an enhanced cross-species activity to CpG ODN. Furthermore, using tetanus toxoid as a vaccine Ag for s.c. immunization, CTB-CpG markedly enhanced the Ag-specific IgG Ab response and altered the specific pattern of Ab isotypes toward a Th1 type response. To our knowledge, CTB is the first nontoxic derivative of microbial toxins discovered that when chemically linked to CpG remarkably augments the CpG-mediated immune responses.

Nyckelord

1-Phosphatidylinositol 3-Kinase

metabolism

Adaptor Proteins

Signal Transducing

metabolism

Adjuvants

Immunologic

chemistry

genetics

pharmacology

Animals

Base Sequence

Chemokines

biosynthesis

Cholera Toxin

chemistry

genetics

pharmacology

Cytokines

biosynthesis

Drug Design

Endosomes

drug effects

metabolism

G(M1) Ganglioside

pharmacology

Humans

Hydrogen-Ion Concentration

Immunoconjugates

chemistry

genetics

pharmacology

Leukocytes

Mononuclear

drug effects

immunology

Mice

Mice

Inbred C3H

Mice

Inbred C57BL

Mice

Knockout

Myeloid Differentiation Factor 88

NF-kappa B

metabolism

Oligodeoxyribonucleotides

chemistry

genetics

pharmacology

Recombinant Proteins

chemistry

genetics

pharmacology

Species Specificity

Toll-Like Receptor 9

metabolism

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