Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Aluminum adjuvant potentiates gilthead seabream immune responses but induces toxicity in splenic melanomacrophage centers Jorge Galindo-Villegas a,* , Alicia García-Alcazar b , José Meseguer a , Victoriano Mulero a,** a Department of Cell Biology and Histology, Faculty of Biology, Institute of Biomedical Research of Murcia-Arrixaca, Campus Universitario de Espinardo, University of Murcia, 30100 Murcia, Spain b Spanish Oceanographic Institute, Murcia Oceanographic Centre, Mazarrón, Spain ARTICLE INFO Keywords: Adjuvant Aluminum hydroxide Immunity Melanomacrophage centers Montanide Seabream Side-effects Vaccination ABSTRACT A key goal of a successful vaccine formulation is the strong induction of persistent protective immune responses without producing side-effects. Adjuvants have been proved to be successful in several species at inducing in- creased immune responses against poorly immunogenic antigens. Fish are not the exception and promising results of adjuvanted vaccine formulations in many species are needed. In this study, over a period of 300 days, we characterized the apparent damage and immune response in gilthead seabream immunized by in- traperitoneal injection with the model antigen keyhole limpet hemocyanin (KLH) alone or formulated with Montanide ISA water-in-oil (761 or 763), or Imject™ aluminum hydroxide (aluminium), as adjuvants. Throughout the trial, external tissue damage was examined visually, but no change was observed. Internally, severe adhesions, increased fat tissue, and hepatomegaly were recorded, but, without impairing animal health. At 120 days post priming (dpp), histopathological evaluations of head-kidney, spleen and liver revealed the presence of altered melanomacrophage centers (MMC) in HK and spleen, but not in liver. Surprisingly, in all aluminium treated fish, classical stains unmasked a toxic effect on splenic-MMC, unequivocally characterized by a strong cell depletion. Furthermore, at 170 dpp transmission electron microscopy confirmed this data. Paradoxically, at the same time powerful immune responses were recorded in most vaccinated groups, including the aluminium treatment. Whatever the case, despite the observed adhesions and MMC depletion, fish phy- siology was not affected, and most side-effects were resolved after 300 dpp. Therefore, our data support adjuvant inclusion, but strongly suggest that use of aluminium must be further explored in detail before it might benefit the rational design of new vaccination strategies in aquaculture. 1. Introduction The extensive use of vaccines on a wide range of species among vertebrates, including fish is recognized as the most effective prophy- lactic tool against specific diseases [1,2]. In any species, vaccine success relies on the ability of enhancing the immunological memory to re- spond with greater vigor towards a subsequent infection by the same antigen. To achieve the desired effect, a number of complex signals are required. However, in fish this is not a simple task, due most antigenic preparations contained in vaccines are weakly immunogenic after in- activation, requiring the addition of immunopotentiators. Among them, adjuvants are the choice required for the elicitation of immune re- sponses that may be protective against certain pathogens [3]. Several synthetic and natural substances can be used as adjuvants to improve the efficacy of animal vaccines. Some adjuvants, like many oil based emulsions already have been used in licensed products, whereas others, like Toll-like receptor ligands or cytokines are still experimentally evaluated [4,5]. Whatever the case, several considerations in selecting adjuvants for a particular species are mandatory. Consideration high- lights include a proven effectiveness and safety, induction of a long- lasting protective immunity, compliance of human food safety regula- tion, feasibility for scale-up production, and last but not the least, cost effectiveness. Therefore, finding the appropriate adjuvant or their combinations to meet the previously mentioned criteria is one of the major challenges in animal vaccine development [6]. Despite the significant effect recorded with oil-based adjuvants, still more information is required on the side-effects they produce and the short span of the immune response promoted; both elements are ham- pering the successful development of efficient animal vaccines [3,5]. Fish aquaculture is a growing industry, but it has many constrains yet, https://doi.org/10.1016/j.fsi.2018.02.047 Received 11 October 2017; Received in revised form 24 February 2018; Accepted 27 February 2018 * Corresponding author. Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, Campus Universitario de Espinardo, 30100 Murcia, Spain. ** Corresponding author. Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, Campus Universitario de Espinardo, 30100 Murcia, Spain. E-mail addresses: jorge-galindo@usa.net (J. Galindo-Villegas), vmulero@um.es (V. Mulero). Fish and Shellfish Immunology xxx (xxxx) xxx–xxx 1050-4648/ © 2018 Elsevier Ltd. All rights reserved. Please cite this article as: Galindo-Villegas, J., Fish and Shellfish Immunology (2018), https://doi.org/10.1016/j.fsi.2018.02.047