Activation of Antigen-Specific T Cell-Responses by Mannan-Decorated PLGA Nanoparticles
Summary
Mannosylation of vaccine formulations is a promising strategy to target antigens to the mannose receptor (MR) on dendritic cells (DCs), potentially improving antigen uptake, processing, and presentation. While PLGA nanoparticles (NPs) are established vaccine delivery vehicles, the effect of chemical conjugation of mannan (MN) to antigen-loaded PLGA-NPs on antigen-specific T-cell responses was not fully characterized. This study addresses whether. DC Uptake: - OVA/FITC-Mannan-NPs: 64% FITC⁺ cells vs. 58% for OVA/FITC-NPs; MFI 305 vs. 295. - Double-positive FITC⁺/CD11c⁺ cells: 55.1% (MN-decorated) vs. 50.4% (non-decorated). In Vitro T-Cell Activation: -.
Keywords
- Secondary aim 1: Assess the ability of MN-decorated OVA-NPs to stimulate antigen-specific CD4⁺ and CD8⁺ T-cell responses in vitro using DCs and transgenic OT-I/OT-II T cells.
- Secondary aim 2: Evaluate in vivo vaccination efficacy by measuring antigen-specific T-cell proliferation and cytokine secretion in lymph nodes and spleens of immunized C57BL/6 mice.
- Secondary aim 3: Compare MN-decorated NPs against non-decorated OVA-NPs, empty NPs, and Mannan-NPs alone.
Component: Polymer; Description: COOH-terminated PLGA (50:50 lactide:glycolide), MW ≈ 7,000 Da
Component: Nanoparticle Type; Description: Double-emulsion (W/O/W) solvent evaporation NPs
Component: Targeting Ligand; Description: Mannan (MN), polymannose from Saccharomyces cerevisiae, chemically conjugated to PLGA surface carboxyl groups via EDC/sulfo-NHS chemistry
Component: Payload; Description: Ovalbumin (OVA) as model antigen; OVA/FITC used for uptake tracking
Component: Formulations; Description: Empty-NPs, Mannan-NPs, OVA-NPs, OVA/Mannan-NPs
Component: Size; Description: Empty-NPs: 328 ± 30 nm; MN-NPs: 430 ± 45 nm; OVA-NPs: 372 ± 5 nm; OVA/MN-NPs: 406 ± 14 nm
Component: Zeta Potential; Description: Empty-NPs: −36.2 ± 3 mV; MN-NPs: −46.8 ± 2 mV; OVA-NPs: −16.8 ± 4 mV; OVA/MN-NPs: −29.8 ± 7 mV
Component: MN Content; Description: MN-NPs: 365 ± 36 µg/mg; OVA/MN-NPs: 346 ± 52 µg/mg
Component: OVA Content; Description: OVA-NPs: 9.04 ± 0.916 µg/mg; OVA/MN-NPs: 10.12 ± 0.86 µg/mg
In Vivo Model: - Female C57BL/6 mice, 6–12 weeks old, 4 groups (n = 4 mice/group). - Immunization: subcutaneous, right flank, twice (2 weeks apart) with 10 mg of Empty-NPs, Mannan-NPs, OVA-NPs, or OVA/Mannan-NPs. - One week after last immunization: draining inguinal lymph nodes and spleens harvested. - Ex vivo recall: lymphocytes or purified CD4⁺/CD8⁺ T cells co-cultured with irradiated APCs and stimulated with OVA₃₂₃₋₃₃₉ (CD4 test), OVA₂₅₇₋₂₆₄ (SIINFEKL; CD8 test), or irrelevant peptides (MUC1 lipopeptide, TRP₁₈₀₋₁₈₈). - Proliferation by ³H-thymidine; IL-2 and IFN-γ by ELISA; stimulation index (S.I.) calculated relative to Empty-NP-immunized mice.
Technique: Dynamic light scattering (DLS); Purpose: Particle size and zeta potential
Technique: Phenol-sulfuric acid assay; Purpose: Quantify MN incorporation
Technique: BCA protein assay; Purpose: Quantify OVA loading
Technique: Flow cytometry; Purpose: DC uptake (OVA/FITC; CD11c marker)
Technique: ³H-thymidine incorporation; Purpose: T-cell proliferation (in vitro and ex vivo)
Technique: ELISA; Purpose: IL-2 and IFN-γ secretion
Technique: Ex vivo recall stimulation; Purpose: Antigen-specific T-cell responses in lymph nodes and spleens
Technique: Stimulation Index (S.I.); Purpose: Normalize proliferation to Empty-NP controls
In Vitro T-Cell Activation: - OVA/Mannan-NPs efficiently primed both CD4⁺ and CD8⁺ T cells; CD4⁺ responses were greater than CD8⁺ at all DC:T ratios. - At DC:T ratio 1:20, CD4⁺ proliferation was ~20-fold higher than CD8⁺: 53,915 ± 25,987 cpm vs. 2,727 ± 1,146 cpm (p < 0.05). - OVA-NPs alone induced only marginal CD4⁺ proliferation at high DC numbers and almost no CD8⁺ activation.
IL-2 Secretion (In Vitro): - CD4⁺ T cells with OVA/Mannan-NPs: 1,900 ± 13.7 pg/mL (1:5), 1,866 ± 13.7 pg/mL (1:10), 1,546 ± 50.45 pg/mL (1:20). - CD4⁺ T cells with OVA-NPs: 184.5 ± 1.5 pg/mL at 1:5; undetectable at 1:10 and 1:20. - CD8⁺ T cells with OVA/Mannan-NPs: maximum ~1,006.9 ± 21.7 pg/mL at 1:5; lower than CD4⁺.
In Vivo Lymph Node Responses: - Only OVA/Mannan-NPs induced detectable antigen-specific CD4⁺ and CD8⁺ T-cell proliferation. - S.I. > 8-fold: 8.3 for CD4⁺ and 8.5 for CD8⁺ compared with Empty-NPs. - OVA-NPs alone: marginal responses similar to controls.
In Vivo Spleen Responses: - CD4⁺ T cells from OVA/Mannan-NP-immunized mice: S.I. > 150 for OVA₃₂₃₋₃₃₉; IL-2 = 22.4 ± 3.5 pg/mL; IFN-γ = 417 ± 5 pg/mL. - OVA-NPs: no detectable cytokine secretion. - CD8⁺ T cells from OVA/Mannan-NP-immunized mice: S.I. = 5.3 ± 0.9; IL-2 = 7.5 ± 0.1 pg/mL; no detectable IFN-γ. - OVA-NPs induced slight non-specific CD8⁺ proliferation not dependent on recall antigen.
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