Béatrice Heurtault

Consequences of Isostructural Main‐Chain Modifications for the Design of Antimicrobial Foldamers: Helical Mimics of Host‐Defense Peptides Based on a Heterogeneous Amide/Urea Backbone

OTHER EXPERIENCE AND PROFESSIONAL MEMBERSHIPS : > Co-founder of ImmuPharma France http://www.immupharma.com/index.html (a small drug discovery and development company focused on developing novel, largely peptide, medicines to treat serious medical conditions). > President of the « Groupe Francais des Peptides et des Proteines » (GFPP) (2007-2009) > Organizer of the 16th GFPP Meeting (May 10-15th 2009, Albe, France). http://gfpp.free.fr/ > Management Committee Substitute Member of COST action CM0803:Functional peptidomimetic foldamers (end date 2012)

Embedded silver ions-containing liposomes in polyelectrolyte multilayers: Cargos films for antibacterial agents

A new antibacterial coating made of poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films and liposome aggregates loaded with silver ions was designed. Liposomes filled with an AgNO 3 solution were first aggregated by the addition of PLL in solution. The obtained micrometer-sized aggregates were then deposited on a PLL/HA multilayer film, playing the role of a spacer with the support. Finally, HA/PLL/HA capping layers were deposited on top of the architecture to form a composite AgNO 3 coating. Release of encapsulated AgNO 3 from this composite coating was followed and triggered upon temperature increase over the transition temperature of vesicles, found to be equal to 34 degrees C. After determination of the minimal inhibitory concentration (MIC) of AgNO 3 in solution, the antibacterial activity of the AgNO 3 coating was investigated against Escherichia coli. A 4-log reduction in the number of viable E. coli cells was observed after contact for 120 min with a 120 ng/cm (2) AgNO 3 coating. In comparison, no bactericidal activity was found for PLL/HA films previously dipped in an AgNO 3 solution and for PLL/HA films with liposome aggregates containing no AgNO 3 solution. The strong bactericidal effect could be linked to the diffusion of silver ions out of the AgNO 3 coating, leading to an important bactericidal concentration close to the membrane of the bacteria. A simple method to prepare antibacterial coatings loaded with a high and controlled amount of AgNO 3 is therefore proposed. This procedure is far superior to that soaking AgNO 3 or Ag nanoparticles into a coating. In principle, other small bactericidal chemicals like antibiotics could be encapsulated by this method. This study opens a new route to modify surfaces with small solutes that are not permeating phospholipid membranes below the phase transition temperature.

Antitumor activity of liposomal ErbB2/HER2 epitope peptide-based vaccine constructs incorporating TLR agonists and mannose receptor targeting

Synthetic and molecularly defined constructs containing the minimal components to mimic and amplify the physiological immune response are able to induce an efficient cytotoxic response. In the current study this approach was applied to the development of highly versatile liposomal constructs to co-deliver peptide epitopes in combination with TLR agonists in order to induce a specific anti-tumor cellular immune response against ErbB2 protein-expressing tumor cells. Liposomes containing ErbB2 p63-71 cytotoxic T lymphocyte (CTL) and HA307-319 T- helper (Th) peptide epitopes associated to innovative synthetic TLR2/1 (Pam(3)CAG) or TLR2/6 agonists (Pam(2)CAG and Pam(2)CGD), were injected in mice bearing ErbB2 protein-expressing tumor cells. Mannosylated ligands were also incorporated into the constructs to target antigen-presenting cells. We showed that the TLR2/6 agonists were more efficient than the TLR2/1 agonists for the eradication of tumors expressing ErbB2 protein. Furthermore, mannose-targeted liposomes displayed higher therapeutic efficiency against tumor allowing treatment with decreased quantities of both TLR ligands and peptide epitopes. Our results validated that antigen-associated mannosylated liposomes combined with efficient TLR ligands are effective vectors for vaccination against tumor. In this study we developed useful tools to evaluate the vaccination efficiency of various adjuvants and/or targeting molecules and their potential synergy.

Influence of Ligand Valency on the Targeting of Immature Human Dendritic Cells by Mannosylated Liposomes

An important challenge for the development of new generations of vaccines is the efficient delivery of antigens to antigen presenting cells such as dendritic cells. In the present study we compare the interaction of plain and targeted liposomes, containing mono-, di-, and tetraantennary mannosyl lipid derivatives, with human monocyte-derived immature dendritic cells (iDCs). Whereas efficient mannose receptor-mediated endocytosis by iDCs was observed for the mannosylated liposomes, in contrast, only nonspecific interaction with little uptake was observed with plain liposomes. In accordance with the clustering effect, liposomes prepared with multibranched mannosylated lipids displayed higher binding affinity for the mannose receptor than vesicles containing the monomannosylated analogs. Importantly, we have found that dimannosylated ligands present at the surface of the liposomes were as efficient as tetramannosylated ones to engage in multidentate interactions with the mannose receptor of iDCs, resulting in both cases in an effective uptake/endocytosis. This result will greatly facilitate, from a practical standpoint, the design of mannose-targeted vaccination constructs. Moreover, we showed that mannose-mediated uptake of liposomes did not result in an activation of iDCs. Altogether, our results suggest that antigen-associated targeted liposomes containing diantennary mannosylated lipids could be effective vectors for vaccines when combined with additional DC activation signals.

Liposomes as delivery systems for nasal vaccination: strategies and outcomes

Importance of the field: Among the particulate systems that have been envisaged in vaccine delivery, liposomes are very attractive. These phospholipid vesicles can indeed deliver a wide range of molecules. They have been shown to enhance considerably the immunogenicity of weak protein antigens or synthetic peptides. Also, they offer a wide range of pharmaceutical options for the design of vaccines. In the past decade, the nasal mucosa has emerged as an effective route for vaccine delivery, together with the opportunity to develop non-invasive approaches in vaccination. Areas covered in this review: This review focuses on the recent strategies and outcomes that have been developed around the use of liposomes in nasal vaccination. What the reader will gain: The various formulation parameters, including lipid composition, size, charge and mucoadhesiveness, that have been investigated in the design of liposomal vaccine candidates dedicated to nasal vaccination are outlined. Also, an overview of the immunological and protective responses obtained with the developed formulations is presented. Take home message: This review illustrates the high potential of liposomes as nasal vaccine delivery systems.

Extensive study of human insulin immunoassays: promises and pitfalls for insulin analogue detection and quantification.

Abstract Background: Over the last few decades, new synthetic insulin analogues have been developed. Their measurement is of prime importance in the investigation of hypoglycaemia, but their quantification is hampered by variable cross-reactivity with many insulin assays. For clinical analysis, it has now become essential to know the potential cross-reactivity of analogues of interest. Methods: In this work, we performed an extensive study of insulin analogue cross-reactivity using numerous human insulin immunoassays. We investigated the cross-reactivity of five analogues (lispro, aspart, glulisine, glargine, detemir) and two glargine metabolites (M1 and M2) with 16 commercial human insulin immunoassays as a function of concentration. Results: The cross-reactivity values for insulin analogues or glargine metabolites ranged from 0% to 264%. Four assays were more specific to human insulin, resulting in negligible cross-reactivity with the analogues. However, none of the 16 assays was completely free of cross-reactivity with analogues or metabolites. The results show that analogue cross-reactivity, which varies to a large degree, is far from negligible, and should not be overlooked in clinical investigations. Conclusions: This study has established the cross-reactivity of five insulin analogues and two glargine metabolites using 16 immunoassays to facilitate the choice of the immunoassay(s) and to provide sensitive and specific analyses in clinical routine or investigation.

Novel glycolipid TLR2 ligands of the type Pam2Cys-α-Gal: Synthesis and biological properties

A more complete understanding of the mechanism of action of TLR agonists has fueled the investigation of new synthetic immunoadjuvants. In this context, we designed and synthesized glycolipids of the type Pam(2)Cys-α-Galactose as novel immunoadjuvants. Their synthesis required modifying a hydrophobic tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety, i.e. the minimal structure required for TLR2 agonist activity, by addition of a hydrophilic head, either an α-Galactosylpyranose or an α-Galactosylfuranose to gain respectively Pam(2)CGalp and Pam(2)CGalf. While preparing a carbohydrate building block, an unexpected stereoselectivity was observed during a halide ion-catalytic process on a protected galactofuranose: the alpha anomer was obtained with surprisingly high selectivity (α/β ratio>9) and with good isolated yield (51%). The TLR2 binding properties of Pam(2)CGalp and Pam(2)CGalf were then fully evaluated. Their efficiency in triggering the proliferation of BALB/c mouse splenocytes was also compared to that of Pam(2)CAG and Pam(3)CAG, two well-established ligands of TLRs. Moreover, the maturation state of murine dendritic cells previously incubated with either Pam(2)CGalp or Pam(2)CGalf was monitored by flow cytometry and compared to that induced by lipopolysaccharide. Pam(2)CGalp and Pam(2)CGalf were found to be equivalent TLR2 agonists, and induced splenocyte proliferation and DC maturation. With very similar activity, Pam(2)CGalp and Pam(2)CGalf were also 10-fold to 100-fold better than Pam(2)CAG and Pam(3)CAG at inducing B cell proliferation. This represents the first time a glucidic head has been added to the tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety whilst maintaining the immunomodulating activity. This should greatly enrich the data available on Pam(2)C structure/activity relationships.

Thyroid-stimulating hormone and free thyroxine on the ADVIA Centaur immunoassay system: A multicenter assessment of analytical performance.

OBJECTIVES We assessed the analytical performance of the TSH and FT4 assays on ADVIA Centaur in a multicenter national evaluation. DESIGN AND METHODS A precision study and a method comparison were performed. Reference values stated by the manufacturer were checked from 379 normal subjects. RESULTS For TSH and FT4, the intra-assay CVs were below 2.3 and 5.2%, respectively, and the inter-assay CVs below 4.4% and 7.2%, respectively. Therefore, the precision and reproducibility were acceptable. Bland-Altman bias plots revealed good correlation and agreement with Cobas assays. TSH and FT4 data yielded reference ranges of 0.64-3.24 mIU/L and 10.5-18.9 pmol/L, respectively. CONCLUSION These assays demonstrate reliable characteristics. The reference ranges obtained can be used for interpretation of thyroid function.

Novel powerful water-soluble lipid immunoadjuvants inducing mouse dendritic cell maturation and B cell proliferation using TLR2 pathway

Four novel water-soluble lipid immunoadjuvants were designed, synthesized and characterized by MS and NMR. They all induce mouse dendritic cell maturation and B cell proliferation. We demonstrate that in spite of the chemical modification, the four compounds remain TLR2 agonists.

Liposomal constructs for antitumoral vaccination by the nasal route

Mucosal surfaces are promising routes for vaccination. Among mucosa, airway mucosa provides the opportunity to develop non-invasive approaches for vaccine delivery. In the current study, nasal route was used to investigate the potency of highly versatile di-epitopic liposomal constructs of different size, structure and composition to exhibit antitumor efficiency after prophylactic vaccination in mice. Well-characterized small unilamellar (SUV), multilamellar (MLV), reverse-phase evaporation (REV) and ultraflexible small unilamellar vesicles (Uf-SUV), containing the ErbB2 T-cytotoxic epitope, the influenza-derived HA T-helper epitope and the lipopeptide adjuvant Pam2CAG, were formulated. These vaccines were administered into the nasal cavity of BALB/c mice, followed by i.v. or s.c. implantation of ErbB2-surexpressing cancer cells. Nasal vaccination with the SUV vaccine resulted in an efficient antitumor activity against lung tumors and a non-significant protection against s.c. tumors. Size, structure and flexibility of liposomes did not impact vaccine immunity and antitumoral efficiency against lung tumors, in contrast to total dose of vaccine or dose of adjuvant. These results showed an undeniable interest of liposomes as lipid-based carriers for antitumor vaccine delivery by the nasal route, opening new perspectives for cancer treatment.