Evaluation of protective efficacy using a nonstructural protein NS1 in DNA vaccine–loaded microspheres against dengue 2 virus

Shih-shiung Huang,¹ I-Hsun Li,^2,3 Po-da Hong,¹ Ming-kung Yeh<sup>1,2

1</sup>Biomedical Engineering Program, Graduate Institute of Applied Science and Technology, and Department of Materials Science and Engineering, National Taiwan University of Science and Technology, Taiwan; ²School of Pharmacy, National Defence Medical Center and Bureau of Pharmaceutical Affairs, Military of National Defence Medical Affairs Bureau, Taipei, Taiwan; ³Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan

Abstract: Dengue virus results in dengue fever or severe dengue hemorrhagic fever/dengue shock syndrome in humans. The purpose of this work was to develop an effective antidengue virus delivery system, by designing poly (dl-lactic-co-glycolic) acid/polyethylene glycol (PLGA/PEG) microspheres using a double-emulsion solvent extraction method, for vaccination therapy based on locally and continuously sustained biological activity. Nonstructural protein 1 (NS1) in deoxyribonucleic acid (DNA) vaccine–loaded PLGA/PEG microspheres exhibited a high loading capacity (4.5% w/w), yield (85.2%), and entrapment efficiency (39%), the mean particle size 4.8 µm, and a controlled in vitro release profile with a low initial burst (18.5%), lag time (4 days), and continued released protein over 70 days. The distribution of protein on the microspheres surface, outer layer, and core were 3.0%, 28.5%, and 60.7%, respectively. A release rate was noticed to be 1.07 µg protein/mg microspheres/day of protein release, maintained for 42 days. The cumulative release amount at Days 1, 28, and 42 was 18.5, 53.7, and 62.66 µg protein/mg microspheres, respectively. The dengue virus challenge in mice test, in which mice received one dose of 20 µg NS1 protein content of microspheres, in comparison with NS1 protein in Al(OH)₃ or PBS solution, was evaluated after intramuscular immunization of BALB/c mice. The study results show that the greatest survival was observed in the group of mice immunized with NS1 protein–loaded PLGA/PEG microspheres (100%). In vivo vaccination studies also demonstrated that NS1 protein–loaded PLGA/PEG microspheres had a protective ability; its steady-state immune protection in rat plasma changed from 4,443 ± 1,384 pg/mL to 10,697 ± 3,197 pg/mL, which was 2.5-fold higher than that observed for dengue virus in Al(OH)₃ at 21 days. These findings strongly suggest that NS1 protein–loaded PLGA/PEG microspheres offer a new therapeutic strategy in optimizing the vaccine incorporation and delivery properties of these potential vaccine targeting carriers.

Keywords: dengue virus, adjuvant, NS1 protein–loaded PLGA/PEG microspheres, vaccination