Vol.:(0123456789) 1 3 Molecular Biotechnology https://doi.org/10.1007/s12033-019-00180-8 ORIGINAL PAPER A Novel Approach to Deliver a Mycobacterium avium subsp. paratuberculosis Antigen in Eukaryotic Cells Azar Motamedi Boroojeni 1  · Abdollah Derakhshandeh 1  · Masoud Haghkhah 1  · Seyed Amir Hossein Jalali 2  · Vida Eraghi 1 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract This study was aimed to express and deliver a Mycobacterium avium subsp. paratuberculosis antigen to macrophages using salmonella as carrier. The coding sequence of a fbronectin attachment protein which is expressed by Mycobacterium avium subsp. paratuberculosis was cloned into pcDNA3.1 (+) plasmid. The construct was introduced into the attenuated Salmonella typhimurium strain SL7207 (ΔhisG, ΔaroA) as carrier. In order to evaluate the delivery capacity of Salmonella and gene expression by antigen-presenting cells, the THP-1 derived macrophages were infected with the salmonella carrier. SDS-PAGE and western blot analysis showed the successful delivery and expression of targeted gene in THP-1 cell line. Although, in vitro stimulation of peripheral blood mononuclear cells with Salmonella containing plasmid did not trigger IFNγ production signifcantly. But it seems that this carrier can increase plasmid uptake and antigen expression by host intestinal antigen-presenting cells after mucosal administration. So, the construct can be used for further in vivo studies on the Salmonella carrier’s efciency in mycobacterial DNA vaccines. Keywords DNA vaccine · FAP-P · IFNγ · Mycobacterium avium subsp. paratuberculosis · Salmonella carrier Introduction Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic, progres- sive enteric disease in ruminants [1]. Paratuberculosis has a worldwide distribution and results in serious economic losses for the dairy and cattle industries [2, 3]. Moreover, studies have suggested a link between MAP and human Crohn’s disease [4] that increases the infection control importance. Researchers have evaluated a number of killed or live-attenuated vaccines against this infection. In addi- tion to being unable to provide complete protection against infection, whole cell based vaccines interfere with the diagnostic tests used for paratuberculosis and bovine tuber- culosis [5, 6]. So, recent studies have focused on subunit vaccines including recombinant antigens and plasmid DNA encoding immunogenic proteins of MAP to overcome these drawbacks. MAP is an intracellular bacterium and Th1 cell-medi- ated immune responses have a pivotal role in the protec- tion against intracellular pathogens [7]. Macrophages and dendritic cells (DC) may be the frst immunological cells to interact with a pathogen [8]. DCs induce Th1 diferen- tiation by secreting Th1-driving cytokines such as IL-12 and increasing expression of surface markers [9, 10]. Th1 responses play a critical role in the host defense against MAP through IFN-γ production that activates macrophages to kill intracellular MAP [11]. Based on these facts, MAP anti- gens that have the capacity to elicit cell-mediated immune response through DCs activation ofer attractive vaccine potential. Fibronectin (FN) attachment proteins (FAPs) are a family of FN-binding proteins expressed in several species of mycobacteria that are critical for attachment and inter- nalization of bacteria to host cells via fbronectin. MAP also expresses FAP known as FAP-P [12]. This antigen induces cell-mediated immune responses through DCs activation by increasing the expression of CD80, CD86, MHC class I and II and inducing Th1-promoting IL-12 secretion [13]. * Abdollah Derakhshandeh drkhshnd77@gmail.com 1 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz 71345-1731, Iran 2 Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Esfahān, Iran