Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen

Abstract

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.

Prophylactic vaccine delivery systems against epidemic infectious diseases

“…After combining FUC-HTCC nanoparticles with the approved anthrax vaccine AVA, the novel anthrax nanovaccine significantly increased the IgG antibody titers and provided complete protection compared with CpG plus AVA (75%) or AVA alone (50%) in mice exposed to anthrax lethal toxin challenge [162] . However, some other polymer particles (e.g., sucrose polymer, chitosan or its derivative, and others) need an addition of an adjuvant (e.g., CpG, mast cell activator compound 48/80 (C48/80), Poly I:C) to elicit an enhanced immune response [163] , [164] , [231] . Although mice immunized with these liposomes or polymer-based vaccines can achieve a high protection rate after challenge, the use of adjuvants reduces their competitiveness compared with viral vectors.…”

Nanoparticle Vaccines Adopting Virus-like Features for Enhanced Immune Potentiation

“…In a maleimide-thiol reaction, the nucleophilic thiolate anion attacks the π-bond of maleimide, forming the enolate intermediate and yielding the desired conjugate. This technique has been widely used to assemble nanoparticle vaccines [184][185][186]. In a study on refining a liposomal formulation of HIV vaccine, thiol chemistry was exploited to control the physiological conformation and density of the target antigen to modulate immune responses [186].…”

Display of DNA on Nanoparticles for Targeting Antigen Presenting Cells

“…We estimated a conjugation ratio of 16 ± 5 CpG/E2 nanoparticle (Supporting Information), within the range achieved for a synthetic nanoparticle system, when adjusting for the difference in particle diameter. 24 Depending on the conformation of the PEG ( i.e. brush or mushroom), the polymer may not be fully extended and the maleimide may not be easily accessible to react with the thiol-terminated CpG.…”

“…We estimated a conjugation ratio of 16 ± 5 CpG/E2 nanoparticle (Supporting Information), within the range achieved for a synthetic nanoparticle system, when adjusting for the difference in particle diameter. 24 Depending on the conformation of the PEG (i.e., brush or mushroom), the polymer may not be fully extended and the maleimide may not be easily accessible to react with the thiol-terminated CpG. 25 Dynamic light scattering (DLS) revealed no significant changes in average particle sizes (Figure 1), demonstrating that conjugation of PEG and ssDNA to E2 did not cause large increases in particle size or aggregation.…”