A Fusion Protein Consisting of the Vaccine Adjuvant Monophosphoryl Lipid A and the Allergen Ovalbumin Boosts Allergen-Specific Th1, Th2, and Th17 Responses In Vitro

Abstract

Background. The detoxified TLR4-ligand Monophosphoryl Lipid A (MPLA) is the first approved TLR-agonist used as adjuvant in licensed vaccines but has not yet been explored as part of conjugated vaccines. Objective. To investigate the immune-modulating properties of a fusion protein consisting of MPLA and Ovalbumin (MPLA : Ova). Results. MPLA and Ova were chemically coupled by stable carbamate linkage. MPLA : Ova was highly pure without detectable product-related impurities by either noncoupled MPLA or Ova. Light scattering analysis revealed MPLA : Ova to be aggregated. Stimulation of mDC and mDC : DO11.10 CD4(+) TC cocultures showed a stronger activation of both mDC and Ova-specific DO11.10 CD4(+) TC by MPLA : Ova compared to the mixture of both components. MPLA : Ova induced both strong proinflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokine responses from mDCs while also boosting allergen-specific Th1, Th2, and Th17 cytokine secretion. Conclusion. Conjugation of MPLA and antigen enhanced the immune response compared to the mixture of both components. Due to the nonbiased boost of Ova-specific Th2 and Th17 responses while also inducing Th1 responses, this fusion protein may not be a suitable vaccine candidate for allergy treatment but may hold potential for the treatment of other diseases that require a strong stimulation of the host's immune system (e.g., cancer).

Figure 1

Generation of MPLA : Ova fusion protein. Applied chemical coupling strategy (a). Analysis of MPLA : Ova by reducing SDS-PAGE (b, M: molecular weight marker, 1: EndoGrade Ova, and 2: MPLA : Ova) and dynamic light scattering (c). For light scattering analysis, 70 μL of MPLA (1 mg/mL), MPLA : Ova (0.6 mg/mL), LPS (1 mg/mL), or Ova (1 mg/mL) in PBS was analyzed at room temperature. Three individual measurements per sample were performed and the mean frequencies (calculated as relative % in class) of hydrodynamic radii (r _H) in nm were plotted.

Figure 2

The MPLA : Ova fusion protein boosts mDC-derived cytokine secretion compared to the mixture of both components. Cytokine secretion determined from either BALB/c mDC (3.2 × 10⁵ cells/mL, a) or BALB/c mDC (3.2 × 10⁵ cells/mL) plus DO11.10 CD4⁺ T cell (8.0 × 10⁵ cells/mL, >95% purity) cocultures (b) stimulated with equimolar amounts of Ova (white bars), MPLA (light grey bars), MPLA + Ova (dark grey bars), and the MPLA : Ova fusion protein (black bars) for 72 h and analyzed by ELISA. ELISAs were performed using either BD OptEIA™ ELISA (BD Biosciences) or Ready-SET-Go! ELISA Sets (eBiosciences). Data are mean results of two independent experiments ± SD. The hypothesis of a significant higher cytokine secretion among all three concentrations used for stimulation was tested with a two-factorial analysis of variance (ANOVA) with factors stimulus (0.2, 1.0, and 5.0) and group (“MPLA + OVA” or “MPLA : OVA”). For statistical significant results the following convention was used: ^∗ p value < 0.05, ^∗∗ p value < 0.01, and ^∗∗∗ p value < 0.001. The statistical analysis was performed with SAS®/STAT software, version 9.4, SAS System for Windows.

Figure 3

The MPLA : Ova fusion protein nonspecifically boosts Th1, Th2, and Th17 cytokine secretion from Ova-specific T cells. Cytokine secretion from BALB/c mDC (3.2 × 10⁵ cells/mL) and DO11.10 CD4⁺ T cell (8.0 × 10⁵ cells/mL, >95% purity) cocultures stimulated with Ova (white bars), MPLA (light grey bars), MPLA + Ova (dark grey bars), or MPLA : Ova (black bars) for either 24 h (IL-2) or 72 h (all other cytokines). ELISAs were performed using either BD OptEIA ELISA (BD Biosciences) or Ready-SET-Go! ELISA Sets (eBiosciences). Data are mean results of two independent experiments ± SD. Statistical analysis was performed according to Figure 2.