Formulation of vaccines containing CpG oligonucleotides and alum

Abstract

CpG oligodeoxynucleotides are potent immunostimulants. For parenterally delivered alum-based vaccines, the immunostimulatory effect of CpG depends on the association of the CpG and antigen to the alum. We describe effects of buffer components on the binding of CPG 7909 to aluminum hydroxide (Alhydrogel), assays for measuring binding of CPG 7909 to alum and CPG 7909 induced dissociation of antigen from the alum. Free CPG 7909 is a potent inducer of IP-10 in mice. However the lack of IP-10 production from formulations containing bound CPG 7909 suggested that CPG 7909 does not rapidly dissociate from the alum after injection. It also suggests that IP-10 assays are not a good basis for potency assays for alum-based vaccines containing CPG 7909.

Fig. 1

Stability of an AMA1-C1/Alhydrogel vaccine following CPG 7909 addition. Reducing silver stained SDS-PAGE gels are shown. Prior to CpG addition, a sample of vaccine was centrifuged and the supernatant loaded onto the gel (B and C, lanes 2). Clinical grade CpG containing phosphate was added (final concentration 1 mg/ml) to 2-yr old (A and B) or freshly prepared (C) 160 μg/ml AMA1-C1/Alhydrogel vaccine. A: Samples were analyzed at t=0 (lane 1) and after incubation at room temperature for 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, and 4 h (lanes 2-8). B and C: Samples were taken at t=0 (lanes 3) and after storage for 6 h on ice at 0°C (lanes 4) and room temperature (lanes 5). For comparison, 1.8 μg (the amount of protein that would be present had all of it dissociated from Alhydrogel) of reference AMA1-C1 is in A lane 9, and 0.1 μg of reference AMA1-C1 is in B lane 1. Molecular weight markers are indicated in kDa.

Fig. 2

Analysis of the stability of freshly formulated and 3-yr old (aged) Pvs25H/Alhydrogel vaccines containing CPG 7909 by SDS-PAGE with silver staining on three reducing gels. Before CpG was added, a sample of each vaccine was centrifuged and the supernatant loaded onto the gel (lanes 1). Preclinical grade CpG was added (final concentration 1 mg/ml) to freshly formulated or aged vaccines containing either 10 μg/ml, 40 μg/ml or 160 μg/ml of protein. Samples were taken at t=0 (lanes 2), after a 6-hr incubation on ice at 0°C (lanes 3), and after a 6-hr incubation at room temperature.