Improved proliferation of antigen-specific cytolytic T lymphocytes using a multimodal nanovaccine

Abstract

The present study investigated the immunoenhancing property of our newly designed nanovaccine, that is, its ability to induce antigen-specific immunity. This study also evaluated the synergistic effect of a novel compound PBS-44, an α-galactosylceramide analog, in boosting the immune response induced by our nanovaccine. The nanovaccine was prepared by encapsulating ovalbumin (ova) and an adjuvant within the poly(lactic-co-glycolic acid) nanoparticles. Quantitative analysis of our study data showed that the encapsulated vaccine was physically and biologically stable; the core content of our nanovaccine was found to be released steadily and slowly, and nearly 90% of the core content was slowly released over the course of 25 days. The in vivo immunization studies exhibited that the nanovaccine induced stronger and longer immune responses compared to its soluble counterpart. Similarly, intranasal inhalation of the nanovaccine induced more robust antigen-specific CD8⁺ T cell response than intraperitoneal injection of nanovaccine.

Keywords:

• nanovaccine
• dendritic cells
• GalCer
• inhalable vaccine

Supplementary materials

Characterization data for PBS-44 molecule

Proton and ¹³C-nuclear magnetic resonance (NMR) of PBS-44

¹H-NMR (500 MHz, CDCl₃) δ 7.42 (d, J=8.5 Hz, 1H), 5.35 (t, J=4.5 Hz, 2H), 4.91 (d, J=3.5 Hz, 2H), 4.19 (q, J=4.5 Hz, 2H), 3.94 (d, J=3 Hz, 1H), 3.89 (d, J=3 Hz, 3H), 3.87 (t, J=4.5 Hz, 1H), 3.8–3.6 (m, 6H), 3.6–3.5 (m, 1H), 3.38 (m, 1H), 2.2 (t, 6 Hz, 2H), 2.1 (q, J=4.5 Hz, 8H), 1.6 (m, 8H), 1.3 (m, 44H), 0.9 (t, J=6 Hz, 6H); ¹³C-NMR (CDCl₃, 500 MHz) δ 174.86, 130.17, 100.05, 77.69, 77.43, 74.96, 72.33, 71.14, 70.59, 70.09, 69.25, 67.70, 62.14, 50.78, 49.52, 49.35, 49.17, 49.02, 48.85, 36.78, 32.75, 32.23, 32.21, 30.05, 29.80, 29.59, 27.48, 27.46, 26.20, 26.18, 22.96, 22.95, 14.23; ESI-MS calcd. (M+H)⁺ (C₄₈H₉₃NO₉) 828.6850, found 828.6955 and (M+Na)⁺ 850.6756.

Figure S1 ESI mass spectrometry.

Abbreviation: ESI, electrospray ionization.

Figure S2 Proton NMR.

Abbreviation: NMR, nuclear magnetic resonance.

Figure S3 ¹³C NMR.

Abbreviation: NMR, nuclear magnetic resonance.

Figure S4 2D-NMRs.

Abbreviations: 2D, two-dimensional; NMR, nuclear magnetic resonance.

Figure S5 COSY NMR.

Abbreviation: NMR, nuclear magnetic resonance.

Figure S6 (A) DLS and (B) zeta potential data of the OvaVac.

Abbreviation: DLS, dynamic light scattering.

Figure S7 Time-course release of ova from OvaVac nanoparticles.

Note: The assay was performed in physiological pH over a 25-day period. Each value represents the mean from three experiments.

Figure S8 In vitro assessment of IFN-γ and IL-4 secreted from splenocytes after they are treated with NanoVac and soluble Vac.

Notes: One million splenocytes from wt C57BL/6 mouse were distributed in a round bottom 96-well plate in 100 μL of RPMI640 medium, the equivalent amount of vaccine components (ova and PBS-44) of either NanoVac or soluble Vac were then added to corresponding wells, and media were collected by centrifugation for ELISA analysis at the indicated time points. (A) IFN-γ levels and (B) IL-4 levels were indicated by two different y-axes, left axis (red) is for soluble Vac and right axis (black) is for nanovac treatment.

Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; IL, interleukin; wt, wild type.

Acknowledgments

The authors appreciate Dr Pavlo Gilchuk for the technical support during the course of the work. This work was partially funded by a grant from NIH, RO1CA16700 (WP), RO1AI042284 (SJ), the National Center for Research Resources, UL1 RR024975-01, which is now the National Center for Advancing Translational Sciences, 2UL1 TR000445-06, under the VICTR CTSA grant, and the Vanderbilt-Ingram Cancer Center Thoracic Program Initiative, all funded to WP. It was also partially supported by a VA Merit Award BX001444 (SJ). The authors would like to thank VMC Flow Cytometry Shared Resource for performing the flow cytometry experiments, which was supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404).

Disclosure

The authors report no conflicts of interest in this work.