Nanoparticulate delivery systems for vaccine adjuvants, designed to enhance targeting of secondary lymphoid organs and activation of APCs, have shown substantial promise for enhanced immunopotentiation. We investigated the adjuvant activity of synthetic oligonucleotides containing CpG-rich motifs (CpG-ODN) linked to the sucrose polymer Ficoll, forming soluble 50 nm particles (DV230-Ficoll), each containing over 100 molecules of the TLR9 ligand, DV230. DV230-Ficoll was evaluated as an adjuvant for a candidate vaccine for anthrax using a recombinant form of protective antigen (rPA) from Bacillus anthracis. A single immunization with rPA plus DV230-Ficoll induced 10-fold higher titers of toxin-neutralizing antibodies in cynomolgus monkeys at 2 weeks compared with animals immunized with equivalent amounts of monomeric DV230. Monkeys immunized either once or twice with rPA plus DV230-Ficoll were completely protected from challenge with 200 LD50 aerosolized anthrax spores. In mice, DV230-Ficoll was more potent than DV230 for the induction of innate immune responses at the injection site and draining lymph nodes. DV230-Ficoll was preferentially co-localized with rPA in key antigen-presenting cell populations and induced greater maturation marker expression (CD69 and CD86) on these cells and stronger germinal center B and T cell responses, relative to DV230. DV230-Ficoll was also preferentially retained at the injection site and draining lymph nodes and produced fewer systemic inflammatory responses. These findings support the development of DV230-Ficoll as an adjuvant platform, particularly for vaccines such as for anthrax, for which rapid induction of protective immunity and memory with a single injection is very important.
We have synthesized and characterized
a novel phosphorothioate
CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate
adjuvant, termed DV230-Ficoll. This adjuvant was constructed from
an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and
the CpG-ODN DV230. Herein, we describe the evaluation of the purity
and reactivity of linkers of different lengths for CpG-ODN-Ficoll
conjugation, optimization of linker coupling, and conjugation of thiol-functionalized
CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical
characterization of independently produced lots of DV230-Ficoll reveal
a bioconjugate with a particle size of approximately 50 nm and covalent
attachment of more than 100 molecules of CpG per Ficoll. Solutions
of purified DV230-Ficoll were stable for at least 12 months at frozen
and refrigerated temperatures and stability was further enhanced in
lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll
conjugate demonstrated improved in vitro potency for induction of
IFN-α from human peripheral blood mononuclear cells and induced
higher titer neutralizing antibody responses against coadministered
anthrax recombinant protective antigen in mice. The processes described
here establish a reproducible and robust process for the synthesis
of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant
suitable for manufacture and use in vaccines.
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