Preparation and Characterization of PLGA Encapsulated Protective Antigen Domain 4 Nanoformulation
Poly (lactic-co-glycolic acid) (PLGA) based particulate systems have been widely explored for the development of subunit based vaccines owing to its biodegradability, biocompatibility, controlled release of entrapped antigen, targeted delivery potential,
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Introduction PLGA based nanoparticle vaccine formulations have been extensively explored for the controlled antigen release and achieve single dose immunization schedule [1–4]. However, the major challenges encountered in such endeavors are the exposure of antigen to aqueous/organic phase interface during particle formulation process and the acidic pH environment arising as a result of the PLGA degradation that can alter the native conformation of antigen and consequently affect the quality of immune response [5–7]. Hence, the search for an immunogenic moiety which can maintain the native structure even in such harsh environments remains a major challenge for designing an effective PLGA based vaccine. Protective antigen (PA) of Bacillus anthracis is a dominant antigen that elicits protective immunity. The recombinant protective antigen (PA) domain 4 (PAD4) molecule has been shown to maintain its native structural conformation at acidic conditions [8]. The crystal structure of PAD4 alone (pdb id: 3INO) shows conformation similar to that of domain 4 in native PA molecule (pdb id: 1ACC). Domain 4 of the PA molecule (PAD4) is responsible for the binding of PA molecule with the host anthrax toxin receptors— tumor endothelial marker 8 (TEM-8) and capillary morphogenesis gene 2 (CMG-2) [9]. Furthermore, PAD4 has been extensively studied as a potential vaccine candidate against anthrax [10]. We evaluated the recombinant PAD4 for the suitability in PLGA based nanoformulation and assessed the enhancement in protective immune response generated by this PLGA encapsulated PAD4 nanoformulation (PAD4-NP) [11]. The recombinant PAD4 was purified from PAD4 expressing Escherichia coli cell using Ni-NTA affinity chromatography and employing urea
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_43, © Springer Science+Business Media New York 2016
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denaturation lysis followed by on-column renaturation. Water/ oil/water (w/o/w) solvent evaporation method was employed for the preparation of PAD4-NP (Fig. 1). W/o/w solvent evaporation method is a widely used method for ease of preparation without the need of high end equipment. In this method, a prospective antigen is first dissolved in aqueous environment or phase making it suitable for encapsulation of proteins. This aqueous phase constitutes “internal aqueous phase” in the final formulation. It is dispersed in a PLGA polymer containing “organic phase” followed by the dispersion of this PLGA (organic phase) encapsulated protein antigen containing aqueous internal phase in another aqueous phase termed as “external aqueous phase” that normally contains a surfactant such as poly vinyl alcohol (PVA). Later, the evaporation of volatile organic solvent employed as organic phase is effected causing the hardening of PLGA matrices (hence the term “solvent evaporation”). This method has been extensively studied by various investigators, he
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