Evaluation of the immune response following a short oral vaccination schedule with hepatitis B antigen encapsulated into alginate-coated chitosan nanoparticles

The purpose of this work was to assess the ability of recombinant hepatitis B vaccine, encapsulated in alginate-coated chitosan nanoparticles, to induce local and systemic immune responses following oral vaccination. The antigen was administered either alone or in combination with the immunopotentiator, synthetic oligodeoxynucleotide containing immunostimulatory CpG motif (CpG ODN) as adjuvant, and associated or not with the alginate-coated chitosan nanoparticles. After two immunizations the group I (HBsAg associated with nanoparticles) and the group VI (HBsAg and CpG, both associated with nanoparticles) showed enhanced immune responses. Both groups showed significant higher values of the CD69 expression in CD4+ and CD8+ T-lymphocytes and lower values of this marker in B lymphocytes. Moreover, a strongest proliferative response of the splenocytes, ex vivo stimulated with concanavalin A, was observed in the same groups. Although with a presence of non-responder mice within the groups, only mice of the groups I and VI elicited the generation of anti-HBsAg antibodies detected in serum (IgG) and in the intestinal washings (sIgA). The results demonstrated that coated chitosan nanoparticles might have potential for being used as a deliver system for oral vaccination with the recombinant hepatitis B surface antigen.http://www.sciencedirect.com/science/article/B6T25-4PF1WCM-2/1/3fe2a6633c054a684fa0fafa7bb4a8b

Regional State Aid Control in Europe: A Legal and Economic Assessment

This paper provides a legal and economic analysis of the European rules for regional State aid according to Article 107 (1) and (3) TFEU. It summarizes the historical evolution and the trends of regional aid rules and describes the economic rationale behind them. The main principles are discussed with reference to recent academic research, leading cases and the State Aid Modernization initiative ("SAM"). The current rules for the assessment of compatibility as laid down in the General Block Exemption and the Regional Aid Guidelines 2014 are critically reviewed in light of these principles

Interactions of elastic and rigid vesicles with human skin in vitro: electron microscopy and two-photon excitation microscopy

AbstractInteractions between vesicle formulations and human skin were studied, in vitro, in relation to their composition and elasticity. The skin ultrastructure was investigated using transmission electron microscopy (TEM), freeze-fracture electron microscopy (FFEM) and two-photon fluorescence microscopy (TPE). The main difference between the vesicle formulations was their elasticity. Elastic vesicle formulations contained bilayer forming surfactants/lipids and single-chain surfactant octaoxyethylenelaurate-ester (PEG-8-L), whereas rigid vesicles contained bilayer surfactants in combination with cholesterol. TEM results showed three types of interactions after non-occlusive application of elastic PEG-8-L containing vesicle formulations on human skin: (1) the presence of spherical lipid structures containing or surrounded by electron dense spots; (2) oligolamellar vesicles were observed between the corneocytes in the upper part of the stratum corneum; and (3) large areas containing lipids, surfactants and electron dense spots were observed deeper down into the stratum corneum. Furthermore, after treatment with vesicles containing PEG-8-L and a saturated C12-chain surfactant, small stacks of bilayers were found in intercellular spaces of the stratum corneum. Rigid vesicles affected only the most apical corneocytes to some extent. FFEM observations supported the TEM findings. Major morphological changes in the intercellular lipid bilayer structure were only observed after treatment with PEG-8-L containing elastic vesicles. TPE showed a distinct difference in penetration pathways after non-occlusive application of elastic or rigid vesicles. After treatment with elastic vesicles, thread-like channels were formed within the entire stratum corneum and the polygonal cell shape of corneocytes could not be distinguished. Fluorescent label incorporated in rigid vesicles was confined to the intercellular spaces of the upper 2–5 μm of the stratum corneum and the cell contours could still be distinguished

Cordeiroda-Silva A. Alginate coated chitosan nanoparticles are an effective subcutaneous adjuvant for hepatitis B surface antigen. Int Immunopharmacol. 2008; 8: 1773-80. 37

We recently described a delivery system that is composed of a chitosan core to which the hepatitis B surface antigen (HBsAg) was adsorbed and subsequently coated with sodium alginate. In this present work, alginate coated chitosan nanoparticles were evaluated as a subcutaneous adjuvant for HBsAg. HBsAg loaded, alginate coated or uncoated chitosan nanoparticles, associated or not with CpGODN were subcutaneously administered to mice and several immunological parameters were evaluated. A high anti-HBsAg IgG titer (2271 ± 120 mIU/ml), with the majority of antibodies being of Th2 type, was observed within group I, vaccinated with HBsAg loaded onto coated nanoparticles. However, regarding cellular immune response, no significant differences were observed for antigen-specific splenocyte proliferation or for the secretion of IFN-γ and IL-4, when compared to the control group. The co-delivery of antigen-loaded nanoparticles in the presence of the immunopotentiator, CpG ODN 1826, resulted in an increase of anti-HBsAg IgG titers that was not statistically different from the first group; however, an increase of the IgG2a/IgG1 ratio from 0.1 to 1.0 and an increase (p b 0.01) of the IFN-γ production by the splenocytes stimulated with the HBV antigen was observed. The enhancement of the immune response observed with the antigen-loaded nanoparticles demonstrated that chitosan is a promising platform for parenteral HBsAg delivery and, when co-administered with the CpG ODN, resulted in a mixed Th1/Th2 type immune response

N-Trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: Biological properties and immunogenicity in a mouse model

In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of a monovalent influenza subunit vaccine was investigated. The antigen-loaded nanoparticles were prepared by mixing a solution containing TMC and monovalent influenza A subunit H3N2 with a tripolyphosphate (TPP) solution, at ambient temperature and pH 7.4 while stirring. The nanoparticles had an average size of about 800 nm with a narrow size distribution and a positive surface charge. The nanoparticles showed a loading efficiency of 78% and a loading capacity of 13% (w/w). It was shown that more than 75% of the protein remained associated with the TMC nanoparticles upon incubation of the particles in PBS for 3 h. The molecular weight and antigenicity of the entrapped hemagglutinin was maintained as shown by polyacrylamide gel electrophoresis and Western blotting, respectively. Single i.n. or i.m. immunization with antigen-loaded TMC nanoparticles resulted in strong hemagglutination inhibition and total IgG responses. These responses were significantly higher than those achieved after i.m. administration of the subunit antigen, whereas the IgG1/IgG2a profile did not change substantially. The i.n. administered antigen-TMC nanoparticles induced higher immune responses compared to the other i.n. antigen formulations, and these responses were enhanced by i.n. booster vaccinations. Moreover, among the tested formulations only i.n. administered antigen-containing TMC nanoparticles induced significant IgA levels in nasal washes of all mice. In conclusion, these findings demonstrate that TMC nanoparticles are a potent new delivery system for i.n. administered influenza antigens. (c) 2006 Elsevier Ltd. All rights reserved

Biowaiver monographs for immediate-release solid oral dosage forms: Stavudine

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate-release (IR) solid oral dosage forms containing stavudine (d4T) are reviewed. According to Biopharmaceutics Classification System (BCS), d4T can be assigned to BCS class I. No problems with BE of IR d4T formulations containing different excipients and produced by different manufacturing methods have been reported and, hence, the risk of bioinequivalence caused by these factors appears to be low. Furthermore, d4T has a wide therapeutic index. It is concluded that a biowaiver is appropriate for IR solid oral dosage forms containing d4T as the single active pharmaceutical ingredient (API) provided that (a) the test product contains only excipients present in the IR d4T drug products that have been approved in a number of countries for the same dosage form, and (b) both test product and its comparator are either very rapidly dissolving or rapidly dissolving with similarity of dissolution profiles demonstrated at pH 1.2, 4.5, and 6.8. (c) 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:1016, 201

Pulmonary delivery of DNA encoding Mycobacterium tuberculosis latency antigen Rv1733c associated to PLGA–PEI nanoparticles enhances T cell responses in a DNA prime/protein boost vaccination regimen in mice

During persistent infection and hypoxic-stress, Mycobacterium tuberculosis (Mtb) expresses a series of Mtb latency antigens. The aim of this study was to evaluate the immunogenicity of a DNA vaccine encoding the Mtb latency antigen Rv1733c and to explore the effect of pulmonary delivery and co-formulation with poly (d,l-lactide-co-glycolide) (PLGA)-polyethyleneimine (PEI) nanoparticles (np) on host immunity. Characterization studies indicated that PLGA-PEI np kept their nanometer size after concentration and were positively charged. The np were able to mature human dendritic cells and stimulated them to secrete IL-12 and TNF-alpha comparable to levels observed after lipopolysaccharide (LPS) stimulation. Mtb latency antigen Rv1733c DNA prime combined with Rv1733c protein boost enhanced T cell proliferation and IFN-gamma secretion in mice in response to Rv1733c and Mtb hypoxic lysate. Rv1733c DNA adsorbed to PLGA-PEI np and applied to the lungs increased T cell proliferation and IFN-gamma production more potently compared to the same vaccinations given intramuscularly. The strongest immunogenicity was obtained by pulmonary priming with np-adsorbed Rv1733c DNA followed by boosting with Rv1733c protein. These results confirm that PLGA-PEI np are an efficient DNA vaccine delivery system to enhance T cell responses through pulmonary delivery in a DNA prime/protein boost vaccine regimen