Mannose functionalized silk fibroin nanoparticles as a platform for the development of innovative nanovaccines against tuberculosis
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Date
2025-07-08
Authors
Yahya, Ahmed
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Abstract
Tuberculosis (TB) remains the world’s leading cause of death from a single infectious disease, according to the 2023 WHO report. TB is caused by Mycobacterium tuberculosis (MTB), primarily affecting the lungs. While Bacillus Calmette–Guérin (BCG) is the only approved vaccine against TB and can induce CD4⁺ T cell memory and a phenomenon known as "trained immunity"—which enhances immune responses to unrelated pathogens—its protective efficacy diminishes with age. Consequently, the majority of new TB cases occur in individuals older than 15 years. Moreover, additional booster doses of BCG have shown limited to no improvement in immune protection.
To address these limitations, alternative vaccine strategies are being explored, including whole-cell vaccines, viral-vectored vaccines, and subunit vaccines. Our work focuses on developing a subunit vaccine utilizing nanocarriers to enhance antigen delivery and immune response. Specifically, we use silk fibroin nanoparticles (SFNs) due to their known biocompatibility, biodegradability, and intrinsic adjuvant properties that can help overcome the typically poor immunogenicity of subunit vaccines.
To target antigen-presenting cells (APCs) via mannose receptors (MR) and DC-SIGN, two mannose derivatives (M1 and M4) were synthesized and conjugated to SFNs using indiscriminate glycosylation (IME) and click chemistry. We aimed to investigate whether varying sugar densities and structural types affect receptor binding and cellular uptake. The lysine content in SFNs was determined to be 46.1 ± 6.70 nmol/mg, consistent with OPA assay results of 40.48 ± 1.08 nmol/mg, providing a basis for subsequent functionalization.
We employed two chemical strategies for SFN glycosylation: (1) an indiscriminate glycosylation method (IME) and (2) a click chemistry-based approach.
In both formulations, glycosylation led to an increase in nanoparticle size,possibly proportional to the amount of sugar used. Additionally, a shift in zeta potential toward a more negative charge was observed following glycosylation. This shift qualitatively supports the success of glycosylation, as it indicates the consumption of surface amine groups,reducing their positive charge, during sugar conjugation.
In the first formulation (IME), glycosylation with M1 at concentrations of 10, 20, and 40 nmol/mg yielded incorporation levels of 1.6, 1.8, and 2.2 nmol/mg, respectively. Similar glycosylation levels were achieved with M4 (1.89 nmol/mg at 20 nmol/mg). These modifications left approximately 2 nmol/mg of lysine available for peptide attachment. In the second formulation (click chemistry), triple bonds on SFNs were quantified at 3.841 nmol/mg. After glycosylation with 4 nmol/mg of M1 or M4, 2.65 nmol/mg was incorporated, leaving ~1.2 nmol/mg of lysine for antigen conjugation.
Future studies, including surface plasmon resonance (SPR) and cellular uptake assays, are planned to evaluate how sugar density and spatial configuration influence receptor binding and APC internalization.
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Keywords
Silk fibroin nanoparticles (SFNs), Tuberculosis (TB), Subunit vaccines, Mannosylation, Targeting mannose receptors (MR)