ABSTRACT
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.
(SCIENCE PHOTO LIBRARY/TEK IMAGE/SPL/GETTY IMAGES)
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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.
