A novel nanoemulsion vaccine induces mucosal Interleukin-17 responses and confers protection upon Mycobacterium tuberculosis challenge in mice

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is contracted via aerosol infection, typically affecting the lungs. Mycobacterium bovis bacillus Calmette-Guerin (BCG) is the only licensed vaccine and has variable efficacy in protecting against pulmonary TB. Additionally, chemotherapy is associated with low compliance contributing to development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Thus, there is an urgent need for the design of more effective vaccines against TB. Experimental vaccines delivered through the mucosal route induce robust T helper type 17 (Th17)/ Interleukin (IL) -17 responses and provide superior protection against Mtb infection. Thus, the development of safe mucosal adjuvants for human use is critical. In this study, we demonstrate that nanoemulsion (NE)-based adjuvants when delivered intranasally along with Mtb specific immunodominant antigens (NE-TB vaccine) induce potent mucosal IL-17T-cell responses. Additionally, the NE-TB vaccine confers significant protection against Mtb infection, and when delivered along with BCG, is associated with decreased disease severity. These findings strongly support the development of a NE-TB vaccine as a novel, safe and effective, first-of-kind IL-17 inducing mucosal vaccine for potential use in humans.

Keywords: IL-17 Responses; Mucosal vaccines; Nanoemulsion.

Figure 1. NE-TB vaccine induced IL-17 mucosal responses

(A-D) B6 mice were mucosally vaccinated I.N. three times with three week intervals with Ag85B or ESAT-6 protein (25 μg each) along with NE adjuvant (20%), or NE adjuvant alone (20%), or with BCG S.C (1×10⁶ CFU). Two weeks after last booster, lungs and spleens were harvested and IL-17 and IFNγ responses were determined by antigen-driven ELISpot assays. The data points represent the means (±SD) of values from 4-5 mice. ***P< 0.0001, **P<0.01, *P<0.05 by one way ANOVA.

Figure 2. NE-TB vaccination conferred vaccine-induced protection and decreased TB disease upon

Mtb challenge. B6 mice either left unvaccinated (PBS group) or vaccinated S.C. with BCG(BCG group, 1×10⁶ CFU). B6 mice were also vaccinated I.N. three times with three week intervals with NE-TB vaccine (Ag85B and ESAT-6 proteins (25 μg each) with 20% NE, (NE:NE group). B6 mice received BCG S.C. (1×10⁶ CFU) and boosted sequentially with NE-TB vaccine twice (BCG:NE group) or vaccinated concurrently both BCG (S.C.) (1×10⁶ CFU) and mucosally with NE-TB vaccine, followed by two boosts with NE-TB vaccine (BCG+NE:NE group). All groups of B6 mice were rested for 4 weeks after which mice were challenged with Mtb HN878 (100 CFU). (A) Mtb CFU was determined on 30 days post-infection. (B, C) Pulmonary inflammation was quantitated on formalin-fixed, paraffin-embedded lung sections from mice on H&E-stained sections. The data points represent the means (±SD) of values from 10 mice per group. ***P< 0.0001, **P<0.01, *P<0.05 by one way ANOVA.

Figure 3. Decreased chemokine induction and improved B cell lymphoid follicle formation is associated with mucosal delivery of NE-TB vaccine in previously BCG vaccinated mice

(A) B cell lymphoid follicle formation was determined by CD3 and B220 staining on formalin-fixed, paraffin-embedded sections by immunofluorescent staining. The total area occupied by B cell follicles per lobe was quantitated using the morphogenetic Axiovision Rel 4.8 software tool of the Zeiss Axioplan microscope. Representative images of B cell follicles from the different groups are shown, at a magnification of 20×. (B-D) Lung homogenates from mice groups in Figure 2 were used to measure chemokine levels by Milliplex assay. The data points represent the means (±SD) of values from 10 mice per group. (Student t-test) *P<0.05, **P<0.005, ***P<0.0005, ns- not significant.