Vaccine 28 (2010) 8288–8299 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine A novel triple adjuvant formulation promotes strong, Th1-biased immune responses and significant antigen retention at the site of injection Heather L. Wilson a , Jennifer Kovacs-Nolan b , Laura Latimer a , Rachelle Buchanan a , Susantha Gomis c , Lorne Babiuk d , Sylvia van Drunen Littel-van den Hurk a,e,∗ a Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada b Department of Food Science, University of Guelph, Guelph, ON N1G 2W1, Canada c Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada d Office of the Vice-President of Research, University of Alberta, Edmonton, AB T6G 2J9, Canada e Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada article info Article history: Received 21 June 2010 Received in revised form 25 September 2010 Accepted 5 October 2010 Available online 16 October 2010 Keywords: Adjuvants Fluorescent imaging Polyphosphazenes Antigen retention abstract Ovalbumin (OVA) was labeled with a near infra-red dye (*OVA) and formulated with the host defense peptide indolicidin (Indol), CpG oligodeoxynucleotide (ODN) 1826 (CpG) and/or poly(p- dicarboxylatophenoxy)-phosphazene (PP4). The immunogenicity of these *OVA formulations was evaluated in mice. All double and triple adjuvant combinations elicited strong antibody responses. *OVA formulated with CpG ODN in combination with indolicidin, PP4 or both induced only IFN-, while formu- lations with indolicidin and/or PP4 promoted predominantly IL-5 production. Overall, both IgG and IFN- production was superior when *OVA was combined with CpG/Indol/PP4. Furthermore, mice injected with *OVA formulated with CpG/Indol/PP4 contained detectable *OVA in the injection site two months post immunization. These results indicate that the CpG/Indol/PP4 combination promotes prolonged antigen retention and strong, antigen-specific Th1-biased immune responses. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Adjuvants are vaccine components which are added to pro- mote or direct an immune response through one of several distinct mechanisms including: (1) promoting uptake and presentation of antigen to T cells in lymph nodes, (2) enhancing cellular recruitment to sites of injection, (3) protecting antigen against degradation, and (4) augmenting the innate immune response [1]. We propose that by combining three select adjuvants, i.e, CpG ODN, host defense peptide (HDPs) and polyphosphazene, which are thought to mediate their effects via complementary mech- anisms, a vaccine formulation can be developed that induces a strong, antigen-specific humoral and cell-mediated immune response. Host defense peptides are small, naturally occurring peptides that are typically less than 50 amino acids long and tend to have an over-representation of positively charged and/or hydrophobic amino acids [2–4]. Host defense peptides (HDPs) are present in plants, mammals, amphibians, fish, and insects, and it has proven difficult for pathogens to develop resistance to them. Some HDPs ∗ Corresponding author. Tel.: +1 306 966 1559; fax: +1 306 966 7478. E-mail address: sylvia.vandenhurk@usask.ca (S. van Drunen Littel-van den Hurk). are expressed as precursor proteins in a variety of cells (i.e. myeloid cells, epithelial cells, neutrophils) and many organs (i.e. skin, oral mucosa, gastrointestinal tract), and they are found in their prote- olytically processed, mature form at mucosal surfaces and in most body secretions (i.e. sweat, breast milk and saliva) [5–9]. Most HDPs have inherent antimicrobial activity, but this property may be sub- stantially suppressed under physiological salt conditions found at mucosal surfaces [10]. HDPs promote cell recruitment and activa- tion of antigen presenting cells (APCs) [4], and therefore HDPs in vaccines may have immunomodulatory properties [10–12] mak- ing them ideal adjuvant candidates. Due to their highly cationic properties, HDPs are also known to form complexes with immunos- timulatory oligodeoxynucleotides (ODNs), which may promote a depot effect at the site of injection [13,14]. Hypomethylated CpG motifs are characteristic of bacterial, viral and protozoal DNA, and their presence can stimulate var- ious myeloid cells to express proinflammatory cytokines and induction of B cell mitogenesis [15–18]. CpG DNA can be mim- icked by synthetic, hypomethylated CpG oligonucleotides (ODNs) [19]. Upon receptor-ligand interaction, CpG ODN triggers mono- cytes/macrophages to produce IL-12, TNF-, IL-6 and IL-1, and dendritic cells to produce type I IFN [20–29]. As adjuvants, synthetic CpG ODNs have been found to increase the production of antigen- specific humoral immunity and to promote balanced or Th1-biased immune responses [12,13,30–36]. 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.10.006