Characterization of epitope specificities of reference antibodies used for the quantification of the birch pollen allergen Bet v 1.

BACKGROUND: Accurate allergen quantification is needed to document the consistency of allergen extracts used for immunotherapy. Herein, we characterize the epitope specificities of two monoclonal antibodies used in an ELISA for the quantification of the major birch pollen allergen Bet v 1, established as a reference by the BSP090 European project. METHODS: The ability of mAbs 5B4 and 6H4 to recognize Bet v 1 isoforms was addressed by immunochromatography. The capacity of each mAb to compete with patients' IgE for binding to Bet v 1 was measured by ELISA inhibition. Epitope mapping was performed by pepscan analysis, site-directed mutagenesis, and hydrogen/deuterium exchange-mass spectrometry. RESULTS: The 5B4 epitope corresponds to a peptide sequence (I56-K68) overlapping with the binding sites of patients' serum IgEs. Mutation of residues P59, E60, and K65 abolishes 5B4 binding to Bet v 1 and reduces the level of IgE recognition. In contrast, 6H4 recognizes a conformational epitope lying opposite to the 5B4 binding site, involving residues located in segments I44-K55 and R70-F79. Substitution of E45 reduces the binding capacity of 6H4, confirming that it is critical for the interaction. Both mAbs interact with >90% of Bet v 1 content present in the birch pollen extract, while displaying a weak cross-reactivity with other allergens of the PR-10 family. CONCLUSIONS: MAbs 5B4 and 6H4 recognize structurally distinct epitopes present in the vast majority of Bet v 1 isoforms. These results support the relevance as a reference method of the Bet v 1-specific quantitative ELISA adopted by the European Pharmacopoeia.

Molecular variability of group 1 and 5 grass pollen allergens between Pooideae species: implications for immunotherapy.

BACKGROUND: Differences between major allergens from distinct grass species remain to be investigated, both in terms of structure and antigenicity. METHODS: Group 1 and 5 allergens purified from five common Pooideae species were analysed by mass spectrometry (MS). Major histocompatibility complex (MHC) class II-restricted T cell epitopes were identified using predictive algorithms and human leucocyte antigen (HLA)-binding assays. CD4+ T cell reactivity and IgE binding were assessed based on the induction of CD154 expression in peripheral blood mononuclear cells and using competitive ELISA assays, respectively. RESULTS: MS analysis of group 5 pollen allergens reveals considerable intra- and inter-species variability in amino acid sequence, with 30-50 predominant isoforms found for each species. Differences in the amino acid sequence as well as N- and O-glycosylation contribute to the variability of group 1 allergens, yielding 5-10 main isoforms, depending on the species. Out of 14 MHC class II-restricted T cell epitopes identified within group 1, only one is conserved among the five grass species. Significant differences in binding affinities for HLA-DR molecules result in variable CD4+ T cell recognition of group 1 and 5 allergens purified from the various species. Up to 38% and 85% of patients exhibit seric IgE responses to species-restricted (or semi-restricted) epitopes associated with group 1 or 5 allergens, respectively. CONCLUSION: Major pollen allergens from distinct grass species bear both shared and species-restricted T and B cell immune epitopes. When compared with single extracts, a five grass pollen extract is thus more suitable for specific immunotherapy, as it contains a broader repertoire of the IgE epitopes to which patients are sensitized.

Contribution of plasmid DNA to hepatotoxicity after systemic administration of lipoplexes.

Several studies have demonstrated that intravenous administration of DNA complexed with cationic lipid vectors induces the production of large quantities of proinflammatory cytokines. In this study we confirm these observations, using cationic lipid DOTAP and cationic phospholipid compounds. Moreover, we demonstrate that although intravenous administration of lipid-DNA complexes does not induce toxic effects in the lung, high transgene expression in lung correlates with histopathological lesions in liver, this fact being documented by high transaminase levels in serum of treated mice. We examine the contribution of various components of the lipoplexes in this observed liver toxicity, as well as in the increasing level of transaminases, and more particularly the role of nonmethylated CpG sequences of plasmid DNA. We show that blood samples from animals treated either with cationic lipid alone, or with cationic lipid complexed with methylated plasmid DNA, contain low levels of transaminases. The significant decrease in transaminase levels after injection of cationic lipid-methylated pDNA complexes leads us to believe that nonmethylated CpG sequences could play a major role in this hepatoxicity. Similar results were observed when using a vector that did not encode a transgene, demonstrating that the expression of luciferase in lung was not responsible for this liver toxicity. All these observations suggest that significant work should be devoted to understand more clearly the mechanism of cationic lipid-DNA complex toxicity, and to overcome the problems subsequent to administration of non-methylated CpG sequences of plasmid DNA.

Factors influencing the efficiency of lipoplexes mediated gene transfer in lung after intravenous administration 1 *.

The objectives of this study were to test the influence of different parameters on the in vivo cationic lipid mediated gene transfer in lung after intravenous administration. Luciferase activity was evaluated in lung tissue 24 hours after intravenous administration of different types of lipoplexes. These included lipoplexes prepared using cationic phosphonolipids or DOTAP and various amounts of plasmid DNA. Using two different plasmids we tested the influence of plasmid size on transfection efficiency in vivo. In a last series of experiments, lipoplexes were prepared using different excipients (water, NaCl or 5% glucose solution) and three injection volumes were tested. We demonstrate that chemical structure modifications such as cation substitution and increment of the aliphatic chain length significantly improve transfection efficiency. High luciferase levels are obtained by increasing lipid to DNA charge ratio and plasmid DNA dose and decreasing plasmid size. Lipoplexes prepared in physiological NaCl solution and injected using a volume of 800mul are significantly the most effective. Cationic lipid mediated gene transfer in lung tissue after intravenous administration is influenced by factors including cationic lipid chemical structure, lipid to DNA ratio and plasmid dose. Nevertheless, plasmid size, injection volume and the excipient, used for the lipoplexes preparation, are also important factors and must be considered for an optimization of in vivo gene delivery using intravenous administration.

Cation substitution in cationic phosphonolipids: a new concept to improve transfection activity and decrease cellular toxicity.

Cationic lipids have been shown to be an interesting alternative to viral vector-mediated gene delivery into in vitro and in vivo model applications. Prior studies have demonstrated that even minor structural modifications of the lipid hydrophobic domain or of the lipid polar domain result in significant changes in gene delivery efficiency. Previously, we developed a novel class of cationic lipids called cationic phosphonolipids and described the ability of these vectors to transfer DNA into different cell lines and in vivo. Up until now, in all new cationic lipids, nitrogen atoms have always carried the cationic or polycationic charge. Recently we have developed a new series of cationic phosphonolipids characterized by a cationic charge carried by a phosphorus or arsenic atom. In a second step, we have also examined the effects of the linker length between the cation and the hydrophobic domain as regards transfection activity. Transfection activities of this library of new cationic phosphonolipids were studied in vitro in different cell lines (HeLa, CFT1, K562) and in vivo using a luciferase reporter gene. A luminescent assay was carried out to assess luciferase expression. We demonstrated that cation substitution on the polar domain of cationic phosphonolipids (N --> P or As) results in significant increase in transfection activity for both in vitro and in vivo assays and decrease of cellular toxicity.

Cationic phosphonolipids as nonviral vectors: in vitro and in vivo applications.

Since the development of the concept of gene therapy using cationic lipids as nonviral vectors by Felgner's group in 1987, numerous molecules have been synthesized. Such vectors were first proposed to avoid viral vector-induced drawbacks. But, it quickly became clear that a thorough knowledge of their physical and chemical characteristics was fundamental to use them under optima conditions. Over the last years our laboratory has developed a family of cationic lipids called phosphonolipids whose structure is based on that of natural phosphonolipids; compared with other vectors, these compounds had to be well-tolerated by biologic membranes. Some of our synthesized molecules exhibited an interesting potential for gene transfer, both in vitro and in vivo. Structural changes in the different parts (hydrophobic, hydrophilic, and intermediary domains) of these vectors were evaluated in vitro on different cell-lines; these studies led us to select some of these molecules to carry out in vivo tests. So, the plasmid/phosphonolipid complexes were first administered to mice by intratracheal and aerosol routes with a beta-galactosidase plasmid as reporter gene. In a second set of experiments, we explored the possibilities offered by intravenous injection; in these studies, we used a luciferase plasmid as reporter gene because of its high sensibility. These experiments revealed a transgene expression essentially localized in the lungs. In a further study, we compared systemic administration with local ones; we, then, observed that the optimum formulation of a given molecule depended on its route of administration.

Systemic administration of cationic phosphonolipids/DNA complexes and the relationship between formulation and lung transfection efficiency.

Performances of cationic lipid formulations for intravenous gene delivery to mouse lungs have been previously reported. We report in this study that cationic phosphonolipids, when appropriately formulated, can be good synthetic vectors for gene delivery to lung after intravenous administration. One of our reagents, GLB43, was capable of mediating a 500-fold higher expression in the lungs of mice than could be obtained with free pDNA alone (P=0.018). We demonstrate that the most important parameters for cationic phosphonolipid transfection activity after systemic administration are the chemical structure of the cationic phosphonolipid, the lipid to DNA charge ratio and the inclusion of co-lipid in the formulation. We report using a luciferase reporter gene that transfection activity in vivo 24 h after cationic phosphonolipid systemic administration could not be predicted from in vitro analysis. In contrast to in vitro studies, cationic phosphonolipids including the oleyl acyl chains (GLB43) were more effective than its analogue with the myristyl acyl chains (GLB73). Using pathological analysis of animal livers, we demonstrate that the toxicity level was correlated with the lipoplex formulation and the lipid to DNA ratio.

Cationic Phosphonolipids Containing Quaternary Phosphonium and Arsonium Groups for DNA Transfection with Good Efficiency and Low Cellular Toxicity**

Replacing the ammonium polar head in cationic lipids 1 (A=N) by a phosphonium or an arsonium group (A=P, As) improves their properties as synthetic vectors for DNA transfection. The increased volume of the cationic head is supposed to modify the interactions of the vector with the solvent and DNA.

Cationic phosphonolipids as nonviral gene transfer agents in the lungs of mice.

With the aim of developing new gene transfer tools for treating CF with gene therapy, we have synthesized a novel family of molecules named cationic phosphonolipids. The most efficient among them were selected by in vitro screening to compare their activities in vivo in mouse lungs. We used a reporter gene whose activity was measured cytofluorimetrically (FACS-Gal assay) and by means of a chemiluminescence technique. These tests allowed us to identify the percentage of transfected cells and to quantify total beta-galactosidase in the lungs. This enabled us to identify two molecules, significantly efficient in comparison with DNA alone: GLB73 (p = 0.0015) and GLB253 (p = 0.007). Their use resulted in a time lag between transfection and maximum efficiency: maximum efficiency was observed 4 days after transfection with GLB73, whereas it was noticeable only on day 7 with GLB253. Moreover, from toxicity studies carried out in vivo, GLB73 seems to be nontoxic. In vivo results were correlated with in vitro results obtained with CF epithelial cell lines. Consequently, GLB73 is a potential candidate for phase I clinical trials in humans.

New biocompatible cationic amphiphiles derivative from glycine betaine: a novel family of efficient nonviral gene transfer agents.

With the aim of developing new efficient agents for transfecting of eukaryotic cells we have designed and synthesized a novel family of cationic lipid vectors derived from glycine betaine. In this study we present three novel molecules differing by the length of their aliphatic chains (R=12,R=14,R=16). The lyotropic properties of these cationic lipids have been determined, and their transfection efficiency on different cell lines evaluated, using a luminescent assay. Two of these compounds, GB14 and GB12 are efficient in vitro experiments. Cytoxicity evaluation of these new molecules showed promising results with a low cytotoxicity, especially when co-lipids were included in the formulation. These compounds represent a new family of gene transfer vectors which display good transfection efficiency and low toxicity, possibly due to the natural properties of glycine betaine. These compounds have great potential for the future development of in vivo gene transfer protocols.

Transgene expression kinetics after transfection with cationic phosphonolipids in hematopoietic non adherent cells.

Cationic lipids are considered to be capable of efficiently and safely mediating DNA transfer into cells, although expression is transient. A new family of cationic lipids, called phosphonolipids, has been developed, with the relationship between the hydrophobic domain of the lipid molecules and the significant enhancement of transduction efficiency in a non-adherent cell line characterised in the present study. The kinetics of transfection efficiency were also investigated. Our results demonstrate that the peak of the transient expression of these reporter genes mediated by cationic lipids occurred within 3 to 14 days, depending on the aliphatic chain length of the complex used and on its formulation in the presence or absence of DOPE. Furthermore, the kinetics of transgene expression were found to differ in adherent and non-adherent cells. These results were obtained using three different techniques: CPRG, luminescence, and FACS-gal, and were in agreement with electron microscopy studies. We thus hypothesized that the plasma membrane composition of cells could affect the efficiency of transfection with cationic lipids. Our results suggest that phosphonolipids constitute a promising class of compounds for gene transfer protocols, and that galenic optimization should improve and modify the transfection efficiency of these DNA-lipid complexes.

Cationic phosphonolipids as non viral vectors for DNA transfection in hematopoietic cell lines and CD34+ cells.

The ability to transfer genes into a hematopoietic stem cell and to achieve regulation of their expression in lymphoid or myeloid lineages should open many new therapeutic opportunities. Besides gene transfer mediated by virus vectors like retrovirus or adenovirus, non viral systems have the theoretical advantage of being safe and easy to manage. We developed a new family of cationic lipids called phosphonolipids, synthesized 24 new molecules, and then in a first step we tested their potential to transfer genes in human hematopoietic cell lines (K562 and TF1). A LacZ plasmid under the control of a strong viral promoter was used as a reporter gene and a FACS-Gal assay and a quantitative test CPRG assay evaluated the beta gal expression. The targeted cells were analyzed 48 hours after transfection. The present work shows that seven novel molecules display a high transfer efficiency. One of them is nine-fold more efficient than the commercially available cationic lipids. The results obtained ex vivo on CD34 cells with the FACS-Gal assay show that at day 10 after transfection, 45 percent of cells are expressing gal.

Enhanced glomerular procoagulant activity and fibrin deposition in rats with mercuric chloride-induced autoimmune nephritis.

The mechanism involved in glomerular fibrin deposition was investigated during mercuric chloride (HgCl2)-induced autoimmune glomerulonephritis in the Brown Norway rat. To ascertain whether the local hemostatic system was activated secondarily to the immunological conflict, the ability of glomerular lysates to induce coagulation in vitro was assessed in treated and control rats. Glomerular procoagulant activity (PCA) of HgCl2-injected rats was measured on day 12 (latent phase of the disease), day 20 (acme), and days 32 and 42 (recovery phase) after the first mercury injection. PCA rose 3-fold (p less than 0.02) at day 20 and then almost returned to control values. Proteinuria, PCA, and the incidence of glomerular fibrin deposits peaked concomitantly at day 20. Glomerular PCA was characterized as thromboplastin. The number of Ia positive cells detected by monoclonal OX-6 antibody was not different from the control number at any phase of the disease; the number of macrophages per glomerular section detected by electron microscopy at day 20 in HgCl2-injected rats was 1.80 +/- 0.60, versus 0.30 +/- 0.11 in the controls. No correlation was found between glomerular PCA and either the number of monocytes/macrophages or of Ia-positive cells present in the glomeruli. Since glomerular PCA was maximal at the onset of fibrin formation in the glomeruli and then decreased toward its basal level, and since the fibrin disappeared, it is concluded that increased production of thromboplastin by glomeruli, with activation of the extrinsic coagulation pathway, may contribute to intraglomerular fibrin deposition in HgCl2-induced glomerulonephritis.