Nesane, MashuduPretorius, AlriVan Wyngaardt, WouterTshilwane, Selaelo IvyFaber, Frederika E.Steyn, H.Lemmer, Y.Van Kleef, MirindaThema, Nontobeko2025-11-062025-08Nesane, M., Pretorius, A., Van Wyngaardt, W. et al. 2025, 'In vitro characterization of the E. ruminantium pLAMP multi-epitope DNA poly (lactic-co-glycolic acid) nanoparticle vaccine in sheep peripheral blood mononuclear cells', Veterinary Immunology and Immunopathology, vol. 286, art. 110977, pp. 1-12, doi : 10.1016/j.vetimm.2025.110977.0165-2427 (print)1873-2534 (online)10.1016/j.vetimm.2025.110977http://hdl.handle.net/2263/105140Heartwater is a tick-borne disease caused by Ehrlichia ruminantium, that has a major economic impact on smallholder farmers. This study investigates the potential of Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles (NPs) as a delivery system for the pLAMP multi-epitope DNA vaccine against heartwater. The vaccine was designed to enhance antigen presentation and activation of specific immune responses, including CD4 + and CD8 + T cell activation. Spherical microspheres with smooth surfaces ranging from 180 nm to 5 µm in diameter were produced, with an adsorption efficiency of 83 %. The in vitro release kinetics demonstrated an initial release of adsorbed pLAMP DNA from PLGA NPs peaking at day 7 and again at day 35. Cellular uptake and gene expression were confirmed using the Vitality hrGFP II plasmid that was adsorbed onto PLGA NPs. High throughput transcriptome sequencing was utilized to determine the immune response activated by the vaccine in vitro in immune sheep peripheral blood mononuclear cells (PBMCs). The pLAMP plasmid transcripts were shown to be present, and key immune pathways, including DNA sensing pathways, MHC presentation and CD4 + T cell and CD8 + T cell pathways were activated that corresponded to those identified and used for the vaccine design previously. This is an indication of the capability of the pLAMP-NP vaccine to induce the desired immune responses, demonstrating potential for in vivo studies. HIGHLIGHTS • The pLAMP DNA vaccine was adsorbed to PLGA nano particles showing controlled release kinetics. • RNA-seq data confirmed treatment-specific immune pathway activation in heartwater immune sheep PBMC. • The pLAMP-nano vaccine induced a similar vaccine immune response as was previously reported in vivo.en© 2025 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies. Notice : this is the author’s version of a work that was accepted for publication in Veterinary Immunology and Immunopathology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. A definitive version was subsequently published in Veterinary Immunology and Immunopathology, vol. 286, art. 110977, pp. 1-12, doi : 10.1016/j.vetimm.2025.110977.Ehrlichia ruminantiumHeartwaterTick-borne diseasePoly Lactic-co-Glycolic Acid (PLGA)PLGA nanoparticlesMulti-epitope DNA vaccineImmune responseTranscriptome sequencingPeripheral blood mononuclear cells (PBMCs)Postprint Article