That depot formation is important for alum adjuvant activity was first proposed by Glennyet al.(1,2) in 1925. sequence of this response; furthermore, they induced comparable antigen-specific T-cell activationin vivo. Notably, removal of the injection site and associated alum depot, as early as 2 h after administration, had no appreciable effect on antigen-specific T- and B-cell responses. This study clearly rules out a role for depot formation in alum adjuvant activity.Hutchison, S., Benson, R. A., Gibson, V. B., Pollock, A. H., Garside, P., Brewer, J. M. Antigen depot is not required for alum adjuvanticity. Keywords:vaccine,in vivo Antigen depot is frequently cited as the principal mechanism of action of vaccine adjuvants, in particular clinically applicable adjuvants, such as alum. That depot formation is usually important for alum adjuvant activity was first proposed by Glennyet al.(1,2) in 1925. Since then, our understanding of microbial adjuvant activity has progressed greatly, particularly in the past 15 yr, with the description of Toll-like receptor (TLR) recognition of microbial patterns, directly activating dendritic cell (DC) antigen presentation and T-cell activation (3). More recently, the induction of inflammation by sensors of endogenous danger signals has been proposed as a mechanism where nonmicrobial adjuvants may have similar effects on DCs to enhance subsequent T-cell responses (4). However, the role of these mechanisms in alum adjuvant activity has remained controversial (5,6). In the absence of a definitive mechanism of action, alum has remained HLY78 in constant clinical use for the past 80 yr, and throughout this period, the depot theory of alum adjuvant activity has persisted. However, no evidence exists in the literature to demonstrate the importance, or otherwise, of the antigen depot in the enhancement of antigen presentation and subsequent primary T-cell and B-cell responses by alum adjuvants (7,8). As there is an urgent need for the development of new adjuvants with improved immunogenicity and safety profiles, a clearer understanding of the role that this depot plays in alum adjuvant activity will clearly contribute to the rational design of these important vaccine components. == MATERIALS AND METHODS == == Mice == Homozygous DO11.10x4getmice were prepared from4get(9) and DO11.10 BALB/c TcR transgenic (tg) mice (10). Cell suspensions from secondary lymphoid organs of DO11.10x4getwere labeled with the fluorescent dye Cell Tracker Orange 9-(4-(and 5)-chloromethyl-2-carboxyphenyl)-7-chloro-6-oxo-1,2,2,4-tetramethyl-1,2-dihydropyrido[2,3-6]xanthene (CMRA); Invitrogen, Paisley, UK; ref.11], then 3 106T cells were transferred i.v. to 6- to 8-wk-old female BALB/c mice (Harlan, Bicester, UK). Procedures were performed according to the UK Home Office regulations. == Antigens and adjuvants == BALB/c mice were immunized with chromatographically purified chicken ovalbumin (OVA; Worthington Biochemical, Lakewood, NJ, USA), while C57BL/6 mice received EGFP (12). Preparation of EGFP and associated experimental protocols have been clearly described previously (13). Adjuvants were a 1% alum suspension (Brenntag Biosector, Frederikssund, Denmark), or 100 g/ml CpG (CpG-ODN 1826; Coley Pharmacuetical Group, Ottawa, ON, Canada) or a combination of both. Mice received 100 l s.c., 50 l in the footpad or 10 l in the ear pinna. Following ear pinna administration, the injection site (0.5 cm2) was removed under general anesthetic. == Flow cytometry == Cell suspensions were prepared from draining lymph nodes, as described above, and analyzed using the appropriate combinations of the following antibodies: CD4, KJ1.26, B220, CD11c, CD69, CD62L, or Y-Ae (BD Biosciences, Oxford, UK) in 100 l of FACS buffer (PBS, 2% fetal calf serum, and 0.05% NaN3) containing Fc Block (2.4G2 hybridoma supernatant). Antigen-presenting cell (APC) populations were identified as B220-expressing B cells, CD11c-positive conventional DCs, and B220/CD11c-expressing plasmacytoid DCs (pDCs), as described previously (14,15). Data were acquired on Mouse monoclonal to Dynamin-2 a FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). == Enzyme-linked immunosorbent assay (ELISA) and multiplex bead cytokine analysis == Antigen-specific IgG1 and IgG2a titers were decided in serum samples as described previously (16). Cytokine levels were decided in supernatants fromin HLY78 vitrorestimulated, draining lymph node cell cultures by multiplex bead cytokine analysis (Invitrogen), according to the manufacturer’s instructions. == Statistical analysis == Results are expressed as means sd. Intergroup significance was decided as appropriate, by Student’s 2-tailed unpairedttest or 1-way ANOVA using Prism (GraphPad Software, HLY78 La Jolla, CA, USA). A value ofP 0.05 was considered significant. == RESULTS == == Characterization of the adjuvant activity of alum and CpG == We analyzed the kinetics of T-cell activation, division, and differentiation in response to alum and compared this with CpG HLY78 adjuvants. Synthetic oligonucleotides made up of CpG motifs actviaTLR9, expressed on a number of cell types, to create a proinflammatory environment (17). Although these two adjuvants are proposed to have quite distinct mechanisms of action, no difference in the magnitude and kinetics of.
