Vaccine 32 (2014) 2420–2427 Contents lists available at ScienceDirect Vaccine j o ur na l ho me page: www.elsevier.com/locate/vaccine The adjuvant PCEP induces recruitment of myeloid and lymphoid cells at the injection site and draining lymph node Sunita Awate a,b , Heather L. Wilson b , Baljit Singh c , Lorne A. Babiuk d , George Mutwiri a,b,∗ a Vaccinology and Immunotherapeutics, School of Public Health, 107 Wiggins Road University of Saskatchewan, Canada b Vaccine and Infectious Disease Organization-International Vaccine Centre, 120 Veterinary Road, University of Saskatchewan, Saskatoon, S7N 5E3, Canada c Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine University of Saskatchewan, S7N 5E5, Canada d University of Alberta, 3-7 University Hall, Edmonton, AB, T6G 2J9, Canada a r t i c l e i n f o Article history: Received 16 May 2013 Received in revised form 7 February 2014 Accepted 7 March 2014 Available online 21 March 2014 Keywords: Adjuvants Mechanisms Polyphosphazenes PCEP Myeloid cells Recruitment Draining lymph nodes a b s t r a c t Poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) has shown great potential as a vaccine adjuvant, but the mechanisms that mediate its adjuvant activity have not been investigated. Previously, we had reported the potential of PCEP to induce cytokines and chemokines at the site of injection. Hence, we hypothesized that PCEP creates strong immuno-competent environment leading to recruit- ment of immune cells at the injection site. Intramuscular injection of mice with PCEP induced significant recruitment of neutrophils, macrophages, monocytes, dendritic cells (DCs), and lymphocytes at the site of injection as well as in the draining lymph nodes. Flow cytometric analysis showed that the majority of the recruited immune cells took up and/or were associated with PCEP at the injection site, with lympho- cytes taking up PCEP in lesser quantity. Further, confocal analysis revealed intracytoplasmic lysosomal localization of PCEP in recruited immune cells. These observations suggest that recruitment of distinct immune cells to the site of injection site may be an important mechanism by which PCEP potentiates immune responses to antigens. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Vaccination continues to be an important public health tool to decrease the mortality and morbidity caused by infectious diseases. Modern vaccines containing highly purified antigens are poorly immunogenic and require addition of adjuvants to induce effective immune responses. Despite their critical role in vaccines, the mech- anisms of action of many adjuvants remain poorly understood. Understanding the mechanisms of action of adjuvants is important for development of safe and effective vaccines. Polyphosphazenes are high-molecular weight, water-soluble polymers and promote enhanced and long lasting immune responses with a variety of viral and bacterial antigens [1–5]. Intranasal administration of influenza X:31 antigen with poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) resulted in significantly higher antibody titers in nasal, lung and vaginal mucosal secretions in mice, suggesting that PCEP is an effective parenteral and mucosal adjuvant [1,6]. Although detailed mechanisms of action of polyphosphazenes are not known, they have been shown to form water-soluble, non-covalent complex ∗ Corresponding author. E-mail address: george.mutwiri@usask.ca (G. Mutwiri). with protein antigens and this physical association with antigen might help to deliver antigens to antigen-presenting cells (APCs) [7]. The potent adjuvant activity of PCEP may be a consequence of activation of innate immunity. In vitro studies have demonstrated that polyphosphazenes stimulated the production of innate cytokines in splenocytes [3]. We recently reported that PCEP is a potent modulator of “adjuvant core response genes” at the site of injection that includes cytokines, chemokines, innate immune receptors and interferon-induced genes resulting in significant production of local cytokines and chemokines [8]. Several adjuvants including alum and MF59 induce recruitment of innate immune cells, increase antigen uptake by APCs and trans- port antigen to draining lymph nodes to initiate immune responses [9–11]. We hypothesized that PCEP induced potent cytokines and chemokines at the site of injection influences local recruitment of various immune cells, which are involved in activation of immunity. 2. Materials and methods 2.1. Animal experiments Four to six week old female BALB/c mice (purchased from Charles River Laboratories, North Franklin, CT, USA) were used in this experiment. The animal experiments were approved by the http://dx.doi.org/10.1016/j.vaccine.2014.03.014 0264-410X/© 2014 Elsevier Ltd. All rights reserved.