Characterization of Aluminum Hydroxide Gel and Oil-in-Water Emulsion Formulations Containing CpG ODNs

Adjuvant activity can be greatly improved by appropriate formulation of cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs).
Oct 02, 2010


Oligonucleotide sequences known as CpG ODNs are potent TLR9 agonists. The literature shows that formulation of CpG ODNs elicits more potent immune responses than the formulation or the adjuvant molecule alone. Given the importance of formulation on adjuvant biological activity and toxicity, formulation parameters should be well characterized and rationally designed. An understanding of the biophysical phenomena related to adjuvant interactions with formulation components can be obtained through complementary analytical techniques. Microeletrophoresis, UV spectrophotometry, and HPLC are used to characterize the association of CpG ODNs with aluminum hydroxide and oil-in-water emulsion formulations.

Synthetic oligodeoxynucleotides containing cytosine-phosphorothioate-guanine sequences (CpG ODNs) are designed to mimic bacterial genomes that contain a higher proportion of unmethylated CpG motifs compared to vertebrate genomes. CpG ODNs have demonstrated potent vaccine adjuvant activity as TLR9 agonists, inducing higher antibody titers and an array of cellular immune responses.1

Several studies have demonstrated the importance of appropriate formulation of CpG ODNs to maximize adjuvant response, including a recent special issue dedicated to this topic.1 For example, CpG ODN mixed with aluminum hydroxide or MF59 elicited higher hemagglutination inhibition antibody titers compared to CpG or either formulation alone, and a more Th1-biased cytokine response compared to either formulation alone in a mouse influenza model.2 Similarly, adding CpG ODN to MF59 induced higher total antibody levels compared to either component alone and increased IgG2a/IgG1 ratios in mice compared to MF59 alone on immunization with HIV p55 gag antigen.3

It was also shown that CpG ODN formulated with aluminum hydroxide or an oil-in-water emulsion induced significantly higher antibody titers than either component alone in a rabbit model with various antigens or a piglet model with swine streptococcic septicemia killed vaccine.4,5 In addition, total antibody levels and CTL activity induced in mice by a hepatitis B surface antigen formulated with CpG ODN and aluminum hydroxide were significantly higher than CpG or alum alone.6 Finally, encapsulation of CpG ODNs in polymer microparticles has been shown to increase antibody titers to a meningitis antigen in mice compared to non-encapsulated CpG.7

Given that the combination of adjuvant molecules such as CpG ODNs with particulate formulations translates into significantly more potent immune responses or reduced toxicity, it is important to optimize the formulation and presentation of the adjuvant to the immune system. Improving adjuvant formulation design requires an understanding of the basic biophysical principles underlying adjuvant association with the particulate formulation. This topic will be discussed using several specific examples of the formulation of CpG ODNs with particulate formulations commonly employed as vaccine adjuvants, namely aluminum hydroxide gel and oil-in-water emulsion. A particular emphasis has been placed on practical aspects of material properties and characterization methodology.

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