Immunologic mechanisms of a short-course of Lolium perenne peptide immunotherapy: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: A 3-week short-course of adjuvant-free hydrolysates of Lolium perenne peptide (LPP) immunotherapy for rhinoconjunctivitis with or without asthma over 4 physician visits is safe, well tolerated, and effective. OBJECTIVE: We sought to investigate immunologic mechanisms of LPP immunotherapy in a subset of patients who participated in a phase III, multicenter, randomized, double-blind, placebo-controlled trial (clinical.govNCT02560948). METHODS: Participants were randomized to receive LPP (n = 21) or placebo (n = 11) for 3 weeks over 4 visits. Grass pollen-induced basophil, T-cell, and B-cell responses were evaluated before treatment (visit [V] 2), at the end of treatment (V6), and after the pollen season (V8). RESULTS: Combined symptom and rescue medication scores (CSMS) were lower during the peak pollen season (-35.1%, P = .03) and throughout the pollen season (-53.7%, P = .03) in the LPP-treated group compared with those in the placebo-treated group. Proportions of CD63+ and CD203cbrightCRTH2+ basophils were decreased following LPP treatment at V6 (10 ng/mL, P < .0001) and V8 (10 ng/mL, P < .001) compared to V2. No change in the placebo-treated group was observed. Blunting of seasonal increases in levels of grass pollen-specific IgE was observed in LPP-treated but not placebo-treated group. LPP immunotherapy, but not placebo, was associated with a reduction in proportions of IL-4+ TH2 (V6, P = .02), IL-4+ (V6, P = .003; V8, P = .004), and IL-21+ (V6, P = .003; V8, P = .002) follicular helper T cells. Induction of FoxP3+, follicular regulatory T, and IL-10+ regulatory B cells were observed at V6 (all P < .05) and V8 (all P < .05) in LPP-treated group. Induction of regulatory B cells was associated with allergen-neutralizing IgG4-blocking antibodies. CONCLUSION: For the first time, we demonstrate that the immunologic mechanisms of LPP immunotherapy are underscored by immune modulation in the T- and B-cell compartments, which is necessary for its effect.

Enhancement of transfection efficiency through rapid and noncovalent post-PEGylation of poly(dimethylaminoethyl methacrylate)/DNA complexes.

PURPOSE: The aim of this work was to develop a new strategy to introduce poly(ethylene glycol) (PEG) into methacrylate-based polymer/ DNA complexes in order to produce hemocompatible particles able to transfect cells in the presence of serum. METHODS: Atom transfer radical polymerization was used to synthesize a well-defined poly(2-(dimethylamino)ethyl methacrylate) homopolymer (PDMAEMA) and a poly(2-(dimethylamino)ethyl methacrylate-b-poly(ethylene glycol) alpha-methyl ether, omega-methacrylate) palm-tree-like copolymer (P(DMAEMA-b-MAPEG)). The complexes obtained by self assembly of the pCMVbeta plasmid and the polymers were used to transfect Cos-7 cells. Their physical properties--particle size and zeta potential--were characterized respectively by dynamic light scattering and electrophoretic mobility measurements. Ex vivo hemocompatibility was also determined. RESULTS: The PDMAEMA/pCMVbeta complexes transfected Cos-7 cells exclusively in the absence of serum. Although the P(DMAEMA-bMAPEG) copolymer had no transfection activity per se, the addition of the latter to pre-formed PDMAEMA/DNA complexes significantly enhanced the activity and allowed transfection even in the presence of serum. The presence of palm-tree-like copolymers also improved the hemocompatibility properties of the complexes. No effect on platelet counts was observed for P(DMAEMA-b-MAPEG)/pCMVbeta complexes, whereas a decrease of platelets was clearly observed when blood cells were incubated with PDMAEMA/pCMVbeta complexes. CONCLUSIONS: Such a synergistic effect of noncovalent PEGylation of poly(amino methacrylate)/DNA complexes allows a new and versatile approach to tune up transfection efficiency.