International Journal of Nanomedicine (May 2019)
Single-dose Ag85B-ESAT-6 loaded–poly(lactic-co-glycolic acid) nanoparticles confer protective immunity against tuberculosis
Abstract
Anshu Malik,* Manish Gupta,* Rajesh Mani, Rakesh Bhatnagar Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India *These authors contributed equally to this work Background: Bacillus Calmette–Guérin, the attenuated strain of Mycobacterium bovis, remains the only available vaccine against tuberculosis (TB). However, its ineffectiveness in adults against pulmonary TB and varied protective efficacy (0–80%) speak to an urgent need for the development of an improved and efficient TB vaccine. In this milieu, poly(lactic-co-glycolic acid) (PLGA), is a preferential candidate, due to such properties as biocompatibility, targeted delivery, sustained antigen release, and atoxic by-products. Methods: In this study, we formulated PLGA nanoparticles (NPs) encapsulating the bivalent H1 antigen, a fusion of Mycobacterium tuberculosis (Mtb) Ag85B and ESAT6 proteins, and investigated its role in immunomodulation and protection against Mtb challenge. Using the classical water–oil–water solvent-evaporation method, H1-NPs were prepared, with encapsulation efficiency of 86.1%±3.2%. These spherical NPs were ~244.4±32.6 nm in diameter, with a negatively charged surface (ζ-potential -4±0.6 mV). Results: Under physiological conditions, NPs degraded slowly and the encapsulated H1 antigen was released over a period of weeks. As a proof-of-concept vaccine candidate, H1 NPs were efficiently internalized by the THP-1 human macrophages. Six weeks after a single-dose vaccination, H1 NP–immunized C57BL/6J mice showed significant increase in the production of total serum IgG (P<0.0001) and its isotypes compared to H1 alone, IgG2a being the predominant one, followed by IgG1. Further, the cytokine-release profile of antigen-stimulated splenocyte-culture supernatant indicated a strong TH1-biased immunoresponse in H1 NP–vaccinated mice, with ~6.03- and ~2.8-fold increase in IFNγ and TNFα cytokine levels, and ~twofold and 1.6 fold increase in IL4 and IL10 cytokines, respectively, compared to H1 alone–immunized mice. In protection studies, H1 NP–vaccinated mice displayed significant reductions in lung and spleen bacillary load (P<0.05) at 5-week post–Mtb H37Rv challenge and prolonged survival, with a mean survival time of 177 days, compared to H1 alone–vaccinated mice (mean survival time 80 days). Conclusion: Altogether, our findings highlight the significance of the H1-PLGA nanoformulation in terms of providing long-term protection in mice with a single dose. Keywords: PLGA, nanoparticles, Ag85B, ESAT6, Mycobacterium tuberculosis, vaccine
