In vitro evaluations on canine monocyte-derived dendritic cells of a nanoparticles delivery system for vaccine antigen against Echinococcus granulosus

Abstract

Since dogs play a central role in the contamination of humans and livestock with Echinococcus granulosus, the development of an effective vaccine for dogs is essential to control the disease caused by this parasite. For this purpose, a formulation based on biodegradable polymeric nanoparticles (NPs) as delivery system of recombinant Echinococcus granulosus antigen (tropomyosin EgTrp) adjuved with monophosphoryl lipid A (MPLA) has been developed. The obtained nanoparticles had a size of approximately 200 nm in diameter into which the antigen was correctly preserved and encapsulated. The efficiency of this system to deliver the antigen was evaluated in vitro on canine monocyte-derived dendritic cells (cMoDCs) generated from peripheral blood monocytes. After 48 h of contact between the formulations and cMoDCs, we observed no toxic effect on the cells but a strong internalization of the NPs, probably through different pathways depending on the presence or not of MPLA. An evaluation of cMoDCs activation by flow cytometry showed a stronger expression of CD80, CD86, CD40 and MHCII by cells treated with any of the tested formulations or with LPS (positive control) in comparison to cells treated with PBS (negative control). A higher activation was observed for cells challenged with EgTrp-NPs-MPLA compared to EgTrp alone. Formulations with MPLA, even at low ratio of MPLA, give better results than formulations without MPLA, proving the importance of the adjuvant in the nanoparticles structure. Moreover, autologous T CD4+ cell proliferation observed in presence of cMoDCs challenged with EgTrp-NPs-MPLA was higher than those observed after challenged with EgTrp alone (p<0.05). These first results suggest that our formulation could be used as an antigen delivery system to targeting canine dendritic cells in the course of Echinococcus granulosus vaccine development.

Fig 1. Nanoparticles characterization.

(A) Transmission electronic microscopy image of the EgTrp-NPs-MPLA formulation. (B) SDS page analysis after Coomassie Blue staining of EgTrp extracted from NPs. line 1: molecular weight marker), line 2: EgTrp (positive control), line 3: EgTrp treated with acetonitrile (control of extraction step), line 4: total EgTrp-NPs-MPLA suspension after treatment with acetonitrile, line 5: supernatant, line 6: pellet of NPs after treatment with acetonitrile. (C) Western blot analysis of EgTrp extracted from NPs and revealed with an anti- EgTrp rabbit serum. line 1: EgTrp treated with acetonitrile (control), lines 2, 3 and 4: EgTrp obtained after treatment with acetonitrile of 3 different preparations of EgTrp-NPs-MPLA.

Fig 2

Nanoparticles internalization in cMoDCs observed with Perls staining (A) to (C) and MGG staining (D). (A) after 2h of incubation (A1) with NPs-MPLA 0.1% or (A2) Blank-NPs. (B) after 24h of incubation (B1) with Blank-NPs, (B2) NPs-MPLA 0.1%, (B3) NPs-MPLA 0.025%. (C) after 48h of incubation (C1) with NPs-MPLA 0.025%, (C2) NPs-MPLA 0.05%, (C3) NPs-MPLA 0.1%, (C4) NPs-MPLA 0.2%. (D) after 48 of incubation with (D1) with NPs-MPLA 0.025%, (D2) NPs-MPLA 0.05%, (D3) NPs-MPLA 0.1%, (D4) NPs-MPLA 0.2%.

Fig 3. Flow cytometry profiles of cell viability obtained after Annexin V and IP staining of cMoDCs 48 h after incubation with Blank-NPs, NPs-MPLA 0.025%, NPs-MPLA 0.05%, NPs-MPLA 0.1% and NPs-MPLA 0.2%.

Annexin V is on FL1-A channel and propidium iodide is on FL2-A channel.

Fig 4

(A) Percentages of positive cells and (B) Fold Increase of MFI compared to PBS for CD80, CD86, MHC II and CD40 expression on the four dogs tested. cMoDCs were challenged with PBS, LPS, Blank-NPs, EgTrp alone, EgTrp-NPs, NPs-MPLA, EgTrp-NPs-MPLA. * Significant difference p ≤ 0.05.

Fig 5. Measure of autologous TCD4+ proliferation by optical density at 450nm after incubation with CCK8.

cMoDCs were previously challenged 24 h with PBS (negative control), LPS (positive control), EgTrp alone, Blank-NPs, NPs-MPLA or EgTrp-NPs-MPLA. * Significant difference p ≤ 0.05.