Abstract
Biodegradable polymeric nanoparticles are vehicles of choice for drug delivery and have the ability to encapsulate and present at their surface different molecules of interest. Among these bio-nanocarriers, poly(lactic acid) (PLA) nanoparticles have been used as adjuvant and vehicle for enhanced vaccine efficacy. In order to develop an approach to efficient vaccine delivery, we developed nanoparticles to target α5β1 positive cells. We first overproduced, in bacteria, human fibronectin FNIII9/10 recombinant proteins possessing an integrin α5β1 binding site, the RGDS sequence, or a mutated form of this site. After having confirmed the integrin binding properties of these recombinant proteins in cell culture assays, we were able to formulate PLA nanoparticles with these FNIII9/10 proteins at their surface. We then confirmed, by fluorescence and confocal microscopy, an enhanced cellular uptake by α5β1+ cells of RGDS-FNIII9/10 coated PLA nanoparticles, in comparison to KGES-FNIII9/10 coated or non-coated controls. As a first vaccination approach, we prepared PLA nanoparticles co-coated with p24 (an HIV antigen), and RGDS- or KGES-FNIII9/10 proteins, followed by subcutaneous vaccine administration, in mice. Although we did not detect improvements in the apparent humoral response to p24 antigen in the serum of RGDS/p24 nanoparticle-treated mice, the presence of the FNIII proteins increased significantly the avidity index of anti-p24 antibodies compared to p24-nanoparticle-injected control mice. Future developments of this innovative targeted vaccine are discussed.
Highlights
During recent decades, attempts to develop cheap, efficient, easy-to-use, and non-toxic vaccines with less side effects have included the use of new adjuvants, new supporting materials, and new targeting strategies [1]
Wild-type recombinant protein used in this study consisted of FNIII domains 9 and 10 of the human fibronectin that are involved in RGD-dependent binding to α5β1 integrin
The tripeptide RGD sequence is located in the FNIII-10 domain and its interaction with the α5β1 integrin strongly reinforced by the synergy sequence PHSRN present in the FNIII-9 repeat [32,33,34]
Summary
Cell lines and reagentsThe human fibrosarcoma cell line HT-1080 (ATCC1 CCL-121TM) was maintained as recommended by the ATCC. HIV-1 p24 protein was produced and purified by PX’Therapeutics (Protein’eXpert, Grenoble, France). DNA matrices used to generate, by PCR, the sequence encoding FNIII domains 9 and 10 of human fibronectin were a gift from Dr Helen J Mardon ([12]; nucleotides 4599–5162, NCBI reference sequence NM_212482.2). PCR was carried out with the proofreading pfu DNA polymerase, and the Eco RI-Pst I fragments were cloned into a pT7/7 derivative that allows the production in bacteria of recombinant proteins with a 6-His tag at their C-terminus. Recombinant protein was expressed in the BL21(DE3) Escherichia coli strain, and soluble peptides were purified, as previously described [23], except that the final dialysis was done against 20 mM phosphate buffer pH7. Endotoxins were removed from purified proteins using an affinity column (PierceTM high capacity endotoxin removal resin; Thermo Fisher Scientific, Villebon sur Yvette, France) following the manufacturer’s instructions. Proteins were dialyzed against PBS (pH 7.4), and the endotoxin level was determined using Endosafe1-PTSTM (Charles River, Ecully, France), which consists of a spectrophotometer and a FDA licensed reagent cartridge based on the limulus amebocyte lysate (LAL)
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