PhD Stéphane Hazebrouck
email: stephane.hazebrouck@cea.fr.

MSc Blanche Guillon

MD b , Evelyne Paty

MD, PhD c , Stephen C Dreskin

PhD Karine Adel-Patient

PhD Hervé Bernard

Variable IgE cross-reactivity between peanut 2S-albumins: the case for measuring IgE to both Ara h 2 and Ara h 6

Keywords: allergy, peanut, 2S-albumin, Ara h 2, Ara h 6, cross-reactivity, IgE, epitope, sensitization

Background: 2S-albumins Ara h 2 and Ara h 6 are the most potent peanut allergens and levels of specific IgE toward these proteins are good predictors of clinical reactivity. Because of structural homologies, Ara h 6 is generally considered to cross-react extensively with Ara h 2.

Objective:

We aimed to quantify the IgE cross-reactivity between Ara h 2 and Ara h 6.

Methods: Peanut 2S-albumins were purified from raw peanuts. The IgE cross-reactivity between Ara h 2 and Ara h 6 was evaluated with 32 sera from French and US peanut-allergic patients by measuring the residual IgE binding to one 2S-albumin after depletion of IgE antibodies recognizing the other 2S-albumin. The IgE cross-reactivity between Ara h 2 and Ara h 6 was further investigated by competitive inhibition of IgE binding and by a model of mast cell degranulation.

Results:

A highly variable level of IgE cross-reactivity was revealed among the patients. The mean fraction of cross-reactive IgE antibodies represented only 17.1% of 2S-albumins-specific IgE antibodies and was lower than the mean fraction of IgE specific to Ara h 2 (57.4%) or to Ara h 6 (25.5%). The higher level of Ara h 2-specific IgE was principally due to the IgE-binding capacity of an insertion containing the repeated immunodominant linear epitope DPYSP OH S. The impact of IgE cross-reactivity on diagnostic testing was illustrated with a serum displaying an Ara h 6-specific IgE response of 26 UI/mL that was not associated with the capacity of Ara h 6 to trigger mast cell degranulation.

Conclusions & Clinical Relevance: IgE antibodies specific to peanut 2S-albumins are mainly non-cross-reactive but low-affinity cross-reactivity can affect diagnostic accuracy.

INTRODUCTION

IgE-mediated peanut allergy is a potentially life-threatening disease that affects 1 to 3% of the children. While allergy to cow's milk or hen's egg are generally outgrown before adulthood, peanut allergy persists in more than 80% of cases. [START_REF] Sicherer | Food allergy: A review and update on epidemiology, pathogenesis, diagnosis, prevention, and management[END_REF] The peanut 2S-albumins, Ara h 2 and Ara h 6, are the most potent peanut allergens. While Ara h 2 was immediately recognized as a major allergen, Ara h 6 was first considered as a minor allergen because its IgE reactivity had been evaluated on a denatured recombinant form. [START_REF] Burks | Identification and characterization of a second major peanut allergen, Ara h II, with use of the sera of patients with atopic dermatitis and positive peanut challenge[END_REF][START_REF] Kleber-Janke | Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology[END_REF] Afterwards, studies with native or correctly refolded recombinant proteins demonstrated that Ara h 2 and Ara h 6 display comparable allergenic potencies. [START_REF] Koppelman | Relevance of Ara h1, Ara h2 and Ara h3 in peanut-allergic patients, as determined by immunoglobulin E Western blotting, basophilhistamine release and intracutaneous testing: Ara h2 is the most important peanut allergen[END_REF][START_REF] Mcdermott | Contribution of Ara h 2 to peanut-specific, immunoglobulin E-mediated, cell activation[END_REF][START_REF] Flinterman | Children with peanut allergy recognize predominantly Ara h2 and Ara h6, which remains stable over time[END_REF][START_REF] Porterfield | Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants[END_REF][START_REF] Blanc | Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors[END_REF][START_REF] Kulis | The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice[END_REF][START_REF] Chen | Ara h 2 and Ara h 6 have similar allergenic activity and are substantially redundant[END_REF] Accordingly, levels of specific IgE to Ara h 2 and Ara h 6 are equally good predictors of clinical reactivity to peanut. [START_REF] Klemans | The diagnostic accuracy of specific IgE to Ara h 6 in adults is as good as Ara h 2[END_REF][START_REF] Koid | Ara h 6 complements Ara h 2 as an important marker for IgE reactivity to peanut[END_REF][START_REF] Van Erp | The IgE and basophil responses to Ara h 2 and Ara h 6 are good predictors of peanut allergy in children[END_REF][START_REF] Kukkonen | Ara h 2 and Ara 6 are the best predictors of severe peanut allergy: a double-blind placebo-controlled study[END_REF] Because of a sequence identity of 59% and similar tertiary structures between peanut 2Salbumins, Ara h 6 is considered to cross-react extensively with Ara h 2. [START_REF] Koppelman | Purification and immunoglobulin E-binding properties of peanut allergen Ara h 6: evidence for cross-reactivity with Ara h 2[END_REF][START_REF] Lehmann | Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions[END_REF][START_REF] Mueller | Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity[END_REF][START_REF] Bublin | IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3[END_REF] IgE binding to Ara h 6 has even been suggested to result essentially from cross-reactivity with other peanut allergens. [START_REF] Bublin | IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3[END_REF] Such cross-reactivity would then question the usefulness of testing both 2Salbumins for the diagnosis of peanut allergy. Nevertheless patients sensitized to only one peanut 2S-albumin have been reported thus demonstrating the presence of non-cross-reactive IgE-binding epitopes on Ara h 2 and Ara h 6. [START_REF] Asarnoj | Anaphylaxis to peanut in a patient predominantly sensitized to Ara h 6[END_REF][START_REF] Van Der Valk | Mono-sensitisation to peanut component Ara h 6: a case series of five children and literature review[END_REF][START_REF] Ballmer-Weber | IgE recognition patterns in peanut allergy are age dependent: perspectives of the EuroPrevall study[END_REF] In this regard, Ara h 2 isoforms differ from Ara h 6 by an insertion of either 14 or 26 residues containing the repeated linear epitope DPYSP OH S (Fig. E1). In a previous work, we showed that proline hydroxylation of DPYSP OH S motifs was required for high affinity IgE-binding to this major linear epitope of Ara h 2. [START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF] In contrast, the presence of such an immunodominant linear epitope in Ara h 6 was not detected, thus confirming the importance of conformational IgE-binding epitopes in peanut 2S-albumins. [START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF][START_REF] Otsu | Epitope analysis of Ara h 2 and Ara h 6: characteristic patterns of IgE-binding fingerprints among individuals with similar clinical histories[END_REF][START_REF] Chen | Conformational IgE epitopes of peanut allergens Ara h 2 and Ara h 6[END_REF] Considering the structural homologies between 2S-albumins, but also their sequence dissimilarities, we then aimed to quantify the IgE cross-reactivity between Ara h 2 and Ara h 6 by two different approaches, one using solid-phase coated allergens and the other one using fluid-phase competition.

METHODS

Human sera

Sera were collected from 20 French peanut-allergic children recruited at the Paediatric Allergy Clinic of Hospital Necker-Enfants Malades after informed consent from patient's parents (Table E1, sera 1 to 20). [START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF][START_REF] Bernard | Serological characteristics of peanut allergy in children[END_REF] All samples were collected during routine clinical practice and were studied in accordance with the purpose of the initial study. Based on their medical history, symptoms of the IgE-mediated peanut allergy involved skin, respiratory tract, gastrointestinal tract and cardiovascular system. Twelve sera from US peanut-allergic patients with a strong history of peanut-induced immediate hypersensitivity and peanutspecific IgE ≥ 13 KAU/L (ImmunoCap, Thermo Fischer Scientific) in serum were also included (Table E1, sera 21 to 32). All adult patients and the parents or guardians of minors signed informed consent. Minors who were >6 years of age, signed an assent. The University of Colorado Denver Institutional Review Board approved this study.

Allergens and peptide preparation

The 2S-albumins Ara h 2 and Ara h 6 were purified from whole peanut protein extract prepared with raw peanuts of a typical blend consumed in Europe (50% Chinese Hsuji's and Red Skins varieties, 50% Argentinian Runners variety), as previously described. [START_REF] Bernard | Identification of a new natural Ara h 6 isoform and of its proteolytic product as major allergens in peanut[END_REF][START_REF] Wavrin | Cutaneous or respiratory exposures to peanut allergens in mice and their impacts on subsequent oral exposure[END_REF] Recombinant 2S-albumins were obtained by expression of genes optimized for bacterial expression (Genscript USA Inc., Piscataway, NJ, USA) and inserted into the E.coli expression plasmid pET9c (Novagen-Merck, Damstadt, Germany). [START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF][START_REF] Clement | Expression in Escherichia coli and disulfide bridge mapping of PSC33, an allergenic 2S albumin from peanut[END_REF] The variant rAra h 2.Δ was obtained by replacing the domain GRDPYSPSQDPYSPSP of rAra h 2.01 by the dipeptide DS occurring naturally in Ara h 6. Expression, purification and refolding of recombinant proteins were performed as previously described. [START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF][START_REF] Hazebrouck | Trypsin resistance of the major peanut allergen Ara h 6 and allergenicity of the digestion products are abolished after selective disruption of disulfide bonds[END_REF] Correct refolding of the recombinant proteins was verified by circular dichroism spectroscopy. [START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF] The peptide containing three hydroxyprolines (pep 3P OH , DPYSP OH SQDPYSP OH SQDPDRRDPYSP OH SPY) corresponds to the major linear epitope found in Ara h 2.02 isoform. The peptide was synthesized using a standard solid phase synthesis by the Fmoc (9-fluorenyl-methoxycarbonyl) continuous-flow method (peptide synthesizer 433A, Applied Biosystems, Foster City, CA). After standard procedure including TFA cleavage and ether precipitation, crude peptides were purified by RP-HPLC and characterized by MALDI-TOF. [START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF]

IgE-binding measurement by solid-phase assay

IgE levels to individual native 2S-albumins were quantified using a direct enzyme allergosorbent test in which purified antigens (2.5 μg/mL) were passively adsorbed on microtiter plates as previously described (Fig. 1A; Ara h 2, red triangle; Ara h 6, blue triangle). [START_REF] Bernard | Serological characteristics of peanut allergy in children[END_REF] After overnight incubation with 2 or 3 dilutions of sera (50 μL/well, Fig. 1A, step 1), IgE binding was revealed by the addition of a labeled anti-human IgE monoclonal antibody (clone BS17). Tracer was prepared by covalent linkage of the monoclonal antibody to the tetrameric form of acetylcholinesterase. [START_REF] Bernard | Serological characteristics of peanut allergy in children[END_REF] After washing, Ellman's reagent was used as the enzyme substrate and absorbance was measured at 414 nm.

Cross-reactivity between Ara h 2 and Ara h 6 was evaluated by measuring the residual IgE binding to one 2S-albumin after depletion of IgE antibodies recognizing the other 2Salbumin. After overnight incubation of 50 μL of diluted sera in microtiter plate coated with one 2S-albumin (Fig. 1A, step 1), 45 μL of the resulting depleted serum was transferred into another microtiter plate (Fig. 1A, step 2) coated either with the same 2S-albumin (in order to measure the efficacy of IgE depletion) or with the other 2S-albumin (in order to evaluate the decrease of IgE binding to that 2S-albumin, Fig. 1A, step 3). Depletion efficacy of Ara h 2-specific IgE antibodies was 96.5 ± 6.2% and depletion efficacy of Ara h 6-specific IgE antibodies was 98.3 ± 3.8% (n=32, data not shown).

IgE levels to both 2S-albumins (Ara h 2/6 Calc ) were calculated by adding the level of Ara h 2-specific IgE (after IgE-depletion on Ara h 6, in red, Fig. 2), the level to Ara h 6-specific IgE (after IgE-depletion on Ara h 2, in blue) and the level of cross-reactive IgE (corresponding to the mean decrease of IgE-binding induced by IgE-depletion on Ara h 2 and on Ara h 6, in green).

In addition, IgE binding to a mixture of Ara h 2 and Ara h 6 was assessed by binding both proteins (each at 1 μg/mL) on the solid-phase and IgE binding was measured as previously described. The experimental IgE level toward the mixture, Ara h 2/6 Meas was compared to the sum of IgE levels measured separately against Ara h 2 and Ara h 6 (Ʃ) and to Ara h 2/6 Calc (see above).

IgE-binding analysis by fluid-phase assay

We performed a second set of experiments by using a reverse enzyme allergo-sorbent test that measures the binding of labeled allergens by patients' IgE antibodies captured by an anti-human IgE monoclonal antibody immobilized on the solid phase. [START_REF] Bernard | Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays[END_REF] Here, plates were first coated with an anti-human IgE monoclonal antibody (clone LE27). Fifty μL/well of serum from each patient at adequate dilutions were incubated overnight at 4°C. After washing, 25 μL of inhibitors (i.e. increasing concentrations of native 2S-albumins) and 25 μL of biotinylated native 2S-albumins were added and incubated for 4 h at room temperature. After washing, neutravidin labeled with acetylcholinesterase was added for 15 min at room temperature and acethylcholinesterase enzymatic activity was revealed after extensive washing and addition of Ellman's reagent. [START_REF] Bernard | Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays[END_REF][START_REF] Guillon | Heat processing of peanut seed enhances the sensitization potential of the major peanut allergen Ara h 6[END_REF] Results were expressed as B/B0: B0 and B represent the amount of labelled 2S-albumin bound to immobilized IgE antibodies in the absence or presence of a known concentration of inhibitor, respectively.

Mediator release assay

Degranulation assay was performed with rat basophilic leukemia (RBL) SX -38 cells as previously described. [START_REF] Blanc | Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors[END_REF] Cells were passively sensitized with IgE antibodies immunopurified from individual serum. [START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF] Mediator release was induced by incubation with different concentrations of native 2S-albumins and was measured by assaying the β-hexosaminidase activity in cell culture supernatant. Results were expressed as the percentage of the reference release, determined for each serum and induced with anti-human IgE (LE27 clone; 100 ng/ mL).

Statistical analysis

Data were analyzed using the non-parametric Friedman's test with Dun's multiple comparison test and correlation was assessed with the non-parametric Spearman correlation test. Statistical analyses were performed with GraphPad Prism 5.04 software. A p<0.05 was considered as significant.

RESULTS

IgE cross-reactivity between Ara h 2 and Ara h 6 investigated with a solid-phase assay

Using allergens purified from raw peanut and coated on the solid-phase (Table E1), the levels of Ara h 2-specific IgE (median 33.7 IU/mL, interquartile range (IQR) 14.1-109.2) were on average higher than the levels of Ara h 6-specific IgE (median 23.2 IU/mL, IQR 7.7-63.4) but IgE responses to the two 2S-albumins remained highly correlated (Spearman r value = 0.89, p<0.0001, data not shown). We then measured the relative decrease of IgE binding to one 2S-albumin after depletion of IgE antibodies recognizing the other 2Salbumin (Fig 1A ). The IgE level against a mixture of Ara h 2 and Ara h 6 was also measured in order to validate our calculation of the 2S-albumin total IgE levels (Table 1). As expected, IgE levels to the mixture (Ara h 2/6 Meas ) were lower than the mere sum of Ara h 2 and Ara h 6-IgE levels since these latter encompassed the binding of common cross-reactive IgE antibodies. In contrast, the calculated IgE levels to 2Salbumins, Ara h 2/6 Calc , were in excellent correlation with the experimental IgE levels to Ara h 2/6 mixture (Spearman r value = 0.998, linear regression slope = 1.018, Fig. 3).

Contribution of the DPYSP OH S motifs to the higher IgE-binding capacity of Ara h 2

The fraction of Ara h 2-specific IgE was strikingly two-fold higher than that of Ara h 6. Since a major structural difference between Ara h 2 and Ara h 6 is the presence of the repeated DPYSP OH S motif inserted on a surface loop of Ara h 2 (Fig. E1), we further investigated the IgE-reactivity of a synthetic peptide containing three DPYSP OH S motifs (pep3P OH ) that overlapped the natural insert found in the isoform 2.02. The solid-phase assay revealed that IgE-binding to pep3P OH accounted for about half of the overall IgEbinding capacity of Ara h 2 (Fig. 4). The IgE level to Ara h 2 was then fully recapitulated by adding the IgE level to pep3P OH to that of a complementary construct, rAra h 2.Δ, which was generated by deleting the DPYSP OH S motifs (Fig 4). The difference of IgE reactivity between Ara h 2 and Ara h 6 was thus largely due to the IgE-binding capacity of DPYSP OH S motifs.

IgE cross-reactivity between Ara h 2 and Ara h 6 investigated with a competitive fluid-phase assay

Solid-phase assays are performed in excess of coated allergens, which allows maximum binding of IgE (and IgG) antibodies but without distinction between low-and high-affinity bindings. This absence of distinction is illustrated in Fig 4 with nAra h 2 and rAra h 2 displaying similar IgE-binding capacities although the absence of hydroxyproline in rAra h 2 has been shown to reduce the binding affinity of IgE recognizing the DPYSP OH S motifs in nAra h 2. In contrast, assays such as IgE-binding competitive inhibition or in vitro mast cell degranulation can evidence differences of binding affinity and could thus demonstrate the higher allergenic activity of nAra h 2 compared to rAra h 2. [START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF][START_REF] Deak | Determination of Crucial Immunogenic Epitopes in Major Peanut Allergy Protein, Ara h2, via Novel Nanoallergen Platform[END_REF] We therefore investigated cross-reactivity between 2S-albumins with a competitive fluid-phase assay (Fig 5A). This assay confirmed that IgE reactivity of Ara h 2 and Ara h 6 was mainly mediated by noncross-reactive antibodies. Indeed, some sera, such as serum 18, exhibited a very low crossreactivity between the two 2S-albumins (Fig 5B ) and in most cases, half-inhibition of IgE binding to one 2S-albumin was not achieved with the other 2S-albumin at the concentration sufficient for complete self-inhibition (Figs E2 andE3). Otherwise, about 10-to 100-fold higher concentrations of Ara h 6 than of Ara h 2 were required for half-inhibition of IgE binding to Ara h 2. Nevertheless, we observed that for some sera, such as sera 11, 17 or 26 (Fig 5C and Fig. E3), Ara h 2 and Ara h 6 inhibition curves of IgE-binding to Ara h 6 were partially overlapping, probably because of a fraction of cross-reactive IgE antibodies primarily directed toward Ara h 2. However the presence of non-cross-reactive epitopes was almost always established for both allergens since one 2S-albumin could not totally inhibit IgE binding to the other one (Figs E2 andE3). Complete inhibition of IgE binding to Ara h by Ara h 2 was observed only for serum 8 (Fig 5D). In this case, Ara h 2 was even a better inhibitor of IgE binding to Ara h 6 than Ara h 6 itself, thus suggesting that IgE binding to Ara h 6 was only due to cross-reactive IgE antibodies. Indeed, conversely to Ara h 2, Ara h was unable to induce an efficient degranulation of mast cells passively sensitized with IgE antibodies immunopurified from serum 8 (Fig 5D) . Therefore, although an IgE response higher than 20 IU/mL pointed to Ara h 6 as a harmful allergen for patients 18 and 8, the mast cell assay suggested that patient 8 was sensitized to Ara h 6 only because of low-affinity crossreactivity of anti-Ara h 2 specific IgE.

DISCUSSION

Diagnostic testing of food allergies have been significantly improved by the use of purified or recombinant allergenic components for identification of the offending food allergens. [START_REF] Kim | Diagnostic accuracy, risk assessment, and cost-effectiveness of component-resolved diagnostics for food allergy: A systematic review[END_REF] However, diagnostics based on serum IgE measurements are still hampered by their inability to discriminate clinically relevant IgE binding from clinically irrelevant sensitization. [START_REF] Ehlers | Can alternative epitope mapping approaches increase the impact of B-cell epitopes in food allergy diagnostics?[END_REF] Evaluation of IgE cross-reactivity between two homologous proteins also required to consider the IgE epitope specificity, critical epitopes and affinity relevant for the capacity of a cross-reactive allergen to induce or not mast cell degranulation. [START_REF] Chen | Conformational IgE epitopes of peanut allergens Ara h 2 and Ara h 6[END_REF][START_REF] Ehlers | Can alternative epitope mapping approaches increase the impact of B-cell epitopes in food allergy diagnostics?[END_REF][START_REF] Hazebrouck | Goat's milk allergy without cow's milk allergy: suppression of non-cross-reactive epitopes on caprine beta-casein[END_REF] In this regard, peanut allergens have been extensively studied and structural homologies between Ara h 2 and Ara h 6 seemed to ensure a high level of IgE-cross-reactivity between these two proteins. Nevertheless, the report of peanut-allergic patients sensitized only to Ara h 6 or with highly variable ratio between Ara h 2-specific IgE and Ara h 6-specific IgE suggested that cross-reactivity between peanut 2S-albumins was not as consistent as previously considered. [START_REF] Koid | Ara h 6 complements Ara h 2 as an important marker for IgE reactivity to peanut[END_REF][START_REF] Magnusdottir | Ara h 1 and Ara h 6 Sensitization Causes Clinical Peanut Allergy in Ara h 2-Negative Individuals[END_REF] We thus aimed to provide a quantification of the IgE-cross-reactivity between these two homologous allergens.

For this purpose, we used two different assays to characterize the IgE-cross-reactivity between Ara h 2 and Ara h 6. Solid-phase and fluid-phase assays showed both that the IgEcross-reactivity was not as exhaustive as initially expected and that the IgE-reactivity of Ara h 2 and Ara h 6 was mainly due to the binding of non-cross-reactive antibodies. Moreover, when a significant cross-reactivity was evidenced, the 2S-albumin that was not primarily targeted by the cross-reactive IgE antibodies generally exhibited a 10-to 100-fold lower inhibitory capacity than the other 2S-albumin. A strong correlation between IgE levels to Ara h 6 and to Ara h 2 has been nevertheless consistently observed (here and [START_REF] Klemans | The diagnostic accuracy of specific IgE to Ara h 6 in adults is as good as Ara h 2[END_REF][START_REF] Koid | Ara h 6 complements Ara h 2 as an important marker for IgE reactivity to peanut[END_REF][START_REF] Van Erp | The IgE and basophil responses to Ara h 2 and Ara h 6 are good predictors of peanut allergy in children[END_REF][START_REF] Otsu | Epitope analysis of Ara h 2 and Ara h 6: characteristic patterns of IgE-binding fingerprints among individuals with similar clinical histories[END_REF] ). This was usually attributed to an exhaustive cross-reactivity between 2S-albumins, with Ara h 2 generally considered as the most allergenic protein. However, our results suggest that this strong correlation was rather due to the similar intrinsic allergenic properties of Ara h 2 and Ara h 6 that resulted in comparable levels of co-sensitization. In this regard, the two-fold higher fraction of Ara h 2-specific IgE was unexpected. However, as previously reported, around half of the Ara h 2 IgE-reactivity corresponded to the IgE-binding to DPYSP OH S motifs which was not associated or correlated with that to a corresponding epitope in Ara h 6. [START_REF] Bublin | IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3[END_REF][START_REF] Bernard | Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes[END_REF][START_REF] Otsu | Epitope analysis of Ara h 2 and Ara h 6: characteristic patterns of IgE-binding fingerprints among individuals with similar clinical histories[END_REF] Thereby, when considering only the IgE-binding to the remaining folded parts of Ara h 2 that also contains the potentially cross-reactive epitopes, the IgE-reactivity of Ara h 2 was then comparable to that of Ara h 6. Why the natural insertions containing the DPYSP OH S motifs are such an immunodominant linear epitope in Ara h 2 remains an important question whose answer would certainly provide critical insights in predicting IgEbinding epitopes in food allergens. It would be also interesting to determine whether the level of sensitization to this particular epitope could be associated with the severity of the allergic reaction or with a pattern of peanut processing and/or consumption. We also found an example of sensitization to Ara h 6 only due to cross-reactive IgE antibodies primarily directed against Ara h 2. In this case, Ara h 6 was unable to induce an efficient mast cell degranulation. This result sheds some light on the sensitization to a food allergen without clinical reactivity. Since even an IgE response higher than 20 UI/mL could be clinically irrelevant, it confirmed the difficulty to establish an accurate risk estimation of the IgE-cross-reactivity between allergenic proteins. This question is particularly critical for the assessment of potential cross-reactivity between "novel" food proteins and already known allergenic food.

In conclusion, since each 2S-albumins display non-cross-reactive IgE-binding epitopes, Ara h 6 should be considered as allergenic as Ara h 2 and thereby, using both allergens remains the optimal strategy for diagnostic testing. Furthermore, we suggest that IgE responses to peanut 2S-albumins could be also considered collectively instead of individually and that measurement of IgE specific to a mixture of peanut 2S-albumins could offer significant advantages. First, as shown in Table 1, the sensitivity of IgE-binding detection would be improved since the IgE level to a mixture of Ara h 2 and Ara h 6 was higher than those against only one 2S-albumin. Even if a positive IgE response to Ara h 2 is usually conclusive [START_REF] Klemans | The diagnostic accuracy of specific IgE to Ara h 6 in adults is as good as Ara h 2[END_REF][START_REF] Van Erp | The IgE and basophil responses to Ara h 2 and Ara h 6 are good predictors of peanut allergy in children[END_REF] , further research should also evaluate whether the level of sensitization to the mixture of 2S-albumins can be more insightful than that to Ara h 2 only, in predicting the severity of the allergic symptoms. Accordingly, van Erp et al. proposed to assess the clinical relevance of the sensitization to peanut 2S-albumins in a stepwise approach including a basophil activation test to a mixture of Ara h 2 and Ara h 6. [START_REF] Van Erp | The IgE and basophil responses to Ara h 2 and Ara h 6 are good predictors of peanut allergy in children[END_REF] Secondly, patients sensitized to only one 2S-albumin could be all identified with one test, which should then limit the production of false negative. Finally, IgE cross-reactivity would not affect IgE measurement since cross-reactive IgE antibodies would be detected regardless of their primary specificity. Moreover, Ara h 7, another peanut 2S-albumin which is present in about 10-to 30-fold lower amounts than Ara h 2 and Ara h 6, has been reported to exhibit some non-crossreactive IgE-binding epitopes and could then be also included in the mixture of 2S-albumins. [START_REF] Blankestijn | Specific IgE to peanut 2S albumin Ara h 7 has a discriminative ability comparable to Ara h 2 and 6[END_REF] Thus, in addition to being less expensive by reducing the number of tests, using a mixture of peanut 2S-albumins may result in improved diagnostic performance. Future clinical studies should then assess whether increasing the sensitivity of IgE measurements by using a mixture of 2S-albumins, in detriment of the specificity of the IgE response toward only one representative member of the 2S-albumin family, could improve the accuracy of peanut allergy diagnosis. IgE cross-reactivity between Ara h 2 and Ara h 6 investigated with a solid-phase assay. A, schematic principle of the solid-phase assay (Ara h 2, red triangle; Ara h 6, blue triangle, see Methods); B, relative decrease of IgE binding to one 2S-albumin after depletion of IgE antibodies recognizing the other 2S-albumin (n=32, mean is indicated). Fractions of 2S-albumin-specific IgE antibodies recognizing nAra h 2 (red), nAra h 6 (blue) or both (green), n=32. Correlation between the IgE levels Ara h 2/6 Calc (calculated as described in Methods) and Ara h 2/6 Meas (determined experimentally against a mixture of Ara h 2 and Ara h 6). IgE-binding capacity of rAra h 2, rAra h 2Δ and pep 3P OH relatively to that of nAra h 2. The sum of IgE binding to rAra h 2.Δ and pep 3P OH is shown (n=12, median with interquartile range). Data were analyzed using the non-parametric Friedman test with Dunn's multiple comparison test. Clin Exp Allergy. Author manuscript; available in PMC 2020 August 01.

  . The same difference of potency between Ara h 2 and Ara h 6 was also observed with serum 15 (Fig E4), in which nearly 99% of the 2S-albumins-specific IgE antibodies recognized Ara h 2 (Fig 2). Comparatively, both 2Salbumins triggered an efficient degranulation of mast cells sensitized with IgE antibodies from serum 18 (Fig 5B) or from other sera exhibiting various levels of IgE cross-reactivity between Ara h 2 and Ara h 6 (Fig E4)

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5. IgE cross-reactivity between Ara h 2 and Ara h 6 investigated with a competitive fluid-phase assay. A, schematic principle of fluid-phase assay (see Methods); B, an example of serum displaying co-sensitization with very low IgE-cross-reactivity to Ara h 2 and Ara h 6 and the corresponding capacity of 2S-albumins to trigger mast cell degranulation; C, an example of serum displaying high IgE-cross-reactivity between Ara h 2 and Ara h 6; D, an example of serum displaying IgE reactivity to Ara h 6 (as detected by solid-phase assay) only due to

  ). As shown in Fig 1B, a highly variable level of IgE cross-reactivity was revealed among patients. Overall, depletion of Ara h 6-specific IgE antibodies induced a mean decrease of IgE binding to Ara h 2 of 24% (median 19%) thus indicating that about 76% of IgE binding to Ara h 2 was mediated by non-cross-reactive antibodies. Depletion of Ara h 2-specific IgE antibodies induced a higher mean decrease of IgE binding to Ara h 6 of 46% (median 47%). Subsequently, IgE levels to both 2S-albumins were calculated for each patient by summing levels of IgE recognizing Ara h 2, Ara h 6 and both (see Methods). As shown in Fig 2, the mean fraction of cross-reactive antibodies represented only 17.1% of 2Salbumins-specific IgE antibodies and was thereby lower than the mean fraction of IgE specific to Ara h 2 (57.4%) or to Ara h 6 (25.5%

Table 1 .

 1 Serum IgE concentrations to a mixture of Ara h 2 and Ara h 6

Specific IgE levels to 2S-albumins (IU/mL) Patient no. Ara h 2 Meas Ara h 6 Meas Ʃ (Ara h 2 Meas + Ara h 6 Meas ) Ara h 2/6 Meas Ara h 2/6 Calc

  Ʃ, sum of IgE levels to Ara h 2 and Ara h 6; Ara h 2/6 Meas , measured IgE levels to a mixture of Ara h 2 and Ara h 6; Ara h 2/6 Calc , calculated IgE levels to Ara h 2 and Ara h 6 by taking into account IgE cross-reactivity (see supplementary Methods).

	1	77.8	45.0	122.8	89.6	98.8
	2	3.7	0.4	4.1	3.8	4.0
	3	114.0	66.4	180.4	120.0	129.8
	4	90.7	43.3	134.0	109.9	122.3
	6	30.3	25.6	55.9	39.4	45.3
	7	94.6	81.1	175.7	106.7	110.6
	8	52.2	26.4	78.6	64.7	65.1
		29.8	29.7	59.5	33.0	40.1
		35.1	23.4	58.5	36.4	41.6
		32.4	20.7	53.1	43.6	47.3
		12.6	0.9	13.5	12.2	12.9
		18.8	15.1	33.9	21.7	26.1
		15.6	2.2	17.8	15.2	17.0
		4.7	19.0	23.7	16.9	18.7
		122.8	88.9	211.7	180.7	193.0
		122.0	23.0	145.0	144.9	144.9
		293.0	124.0	417.0	390.0	396.9
		24.1	14.5	38.6	28.1	31.8
		40.2	54.2	94.4	58.9	73.5
		5.5	3.6	9.1	6.8	7.6
		5.2	3.8	9.0	7.2	7.8
		3.4	1,0	4.4	4.3	4.2
		19.2	17.2	36.4	31.5	35.6
		48.7	18.7	67.4	55.4	55.3
		140.7	146.8	287.5	249.2	239.8
		213.3	66.3	279.6	225.2	222.1
		30.5	6.1	36.6	36.2	34.6
		119.8	75.0	194.8	135.0	132.8
		13.6	12.5	26.1	22.4	21.2
		35.8	25.4	61.2	53.8	53.1

Clin Exp Allergy. Author manuscript; available in PMC 2020 August 01.

Acknowledgments

Funding: Supported by AlimH department of INRA and grants R01-AI099029 and R21-AI135397 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Bethesda MD, USA to Dr. Dreskin

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Abbreviations used:

IgE

Immunoglobulin E

RBL

Rat Basophilic leukemia

P OH Hydroxyproline


Jean Vernin
email: vernin@unice.fr

Hervé Trinquet

George Jumper

Edmund Murphy

Anthony Ratkowski

H Trinquet Luan

OHP02 gravity wave campaign in relation to optical turbulence

Keywords: Gravity waves, Atmospheric optics, Atmospheric turbulence effects

Herein we present a campaign dedicated to the detection and the characterization of Gravity Waves (GW) in the Earth's atmosphere in relation to the generation of Optical Turbulence (OT). The observations took place in France from 17 to 24 July 2002 at the Haute Provence Observatory (OHP) and simultaneously at the Sirene Observatory, some 20 km apart. From both sites, several balloons were launched that measured the classical PTU-Wind profiles and additionally the structure constant of the temperature field C 2 T vertical profiles. A Generalized Scidar (GS) technique was implemented at the 1.93 m-diameter OHP telescope, providing C 2 N (h) profiles every minute. From our observations, a significant amount of GW activity was observed at both sites, but without clear evidence of correlation between the two sites. It seems from our observations that a wide spectrum of GW is present at a given altitude and that this could result in a lack of correlation between observations made from two sites 20 km apart. Most GW are non-stationary with long horizontal wavelengths (λ ∼ 100-200 km), kilometric vertical wavelengths (λ ∼ 0.5-2 km) and long intrinsic period (T ∼ 2-15 h). They belong in the category of "hydrostatic rotating or non-rotating waves". Layers of optical turbulence detected by balloons and the Scidar technique correlate well with regions of GW activity.

Introduction

The three goals of this paper are [START_REF] Roddier | Progress in optics[END_REF] to detect GW, [START_REF] Vernin | Mechanism of formation of optical turbulence[END_REF] to characterize them and (3) to relate them with OT. To achieve these goals, teams from the Air Force Research Laboratory (AFRL) and the Laboratoire Universitaire d'Astrophysique de Nice (LUAN) planned a joint observation campaign under the auspices of the Air Force Office of Scientific Research (AFOSR). The OHP site was chosen because it complies with a mountainous orography, well suited for GW activity and because telescopes were available to implement the GS technique.

The prediction of the occurrence of OT is very challenging since it degrades the performance of High Angular Resolution Astronomy [START_REF] Roddier | Progress in optics[END_REF] and also of optical communication through our atmosphere in both uplink and downlink directions, with implications for both commercial and military applications. OT appears only when two conditions are satisfied: when the flow is turbulent and when the medium is stratified [START_REF] Vernin | Mechanism of formation of optical turbulence[END_REF]. The first condition is governed by a constraint on the Richardson number, the ratio between the potential energy, E pot , and the kinetic energy, E kin :

R i = E pot E kin = g θ dθ dz ( dU dz ) 2 < 1 4 (1)
and by a second constraint on the gradient of the potential temperature, θ :

dθ dz = 0( 2 )
where g is the gravity constant, z is the altitude and U is the horizontal wind speed. The paradox is that most of the time R i > 1/4, impeding the growth of turbulence. In our opinion, GW locally forces the wind speed gradient to increase untill constraint (1) is achieved and dynamical turbulence is triggered, mixing air parcels of different temperature, as expected from inequality (2).

Instrumental setup

In order to detect and characterize the GW, our strategy was to make simultaneous measurements of PTU-Wind profiles employing balloon-borne sondes at two locations, close enough to detect the spatio-temporal coherence of an eventual GW but not so close as to be dominated by profile variations.

In order to detect OT, our balloons were instrumented with micro-thermal sensors to measure the structure function of the temperature in two points, r , apart according to:

D T (r ) = (T (ρ) -T (ρ + r )) 2 .
(

) 3
Knowing r (in our sondes we use r = 0.95 m and r = 0.30 m), we can deduce the temperature structure constant. Assuming a Kolmogorov spectrum, the structure function can be written as follows:

D T (r ) = C 2 T r 2/3 with l 0 < r < L 0 ( 4
)
where l 0 and L 0 are the inner and outer scale of turbulence, respectively. From Eq. 4 and using Gladestone formulae, temperature and index structure constants are related through: Only a few balloons could be launched during a night, resulting in poor temporal resolution. We decided to implement the GS [START_REF] Avila | Whole atmospheric-turbulence profiling with generalized scidar[END_REF] which is able to remotely retrieve vertical C 2 N (h) profiles, as well as V(h) profiles. This technique analyzes, in real time, the scintillation at the entrance pupil of the telescope and provides such profiles every minute, during the many hours taken by a star to remain within a ±30 deg cone centered on the zenith.

C 2 N = C 2 T 80 10 -6 P(z) T 2 (z) 2 . (5)
Forty-two balloons were launched from both sites and some 6000 C 2 N (h) profiles were obtained with the GS.

The campaign took place at OHP (Long.: 5 42 45 E, Lat.: +43 55 46) and Sirene (Long.: 05 29 13 E, Lat.: +44 00 00) as shown in Fig. 1, from July 17 to 24, 2002.

Gravity wave detection

3.1GWinarotatingfluid

As explained by Gill [START_REF] Gill | Atmosphere Ocean dynamics[END_REF], 1982, we considered a gravity adjustment under the small-disturbance hypothesis, in a continuously stratified incompressible fluid and with rotation effects. For small pressure perturbations, p ′ , and density perturbations, ρ ′ , and for a vertical angular velocity = 1/2 f , the basic equations are:

-conservation of density:

∂ρ ′ /∂t + wdρ 0 /dz = 0( 6 )

-momentum equations:

∂u/∂t -f v =-ρ -1 0 ∂ p ′ /∂ x (7) ∂v/∂t -fu =-ρ -1 0 ∂ p ′ /∂ y (8) ∂w/∂t =-ρ -1 0 ∂ p ′ /∂z -ρ ′ ρ -1 0 g. ( 9
)
If the vertical scale of the waves is small compared with the scale height, then the Boussinesq approximation yelds:

∂ 2 ∂t 2 ∂ 2 ∂ x 2 + ∂ 2 ∂ y 2 + ∂ 2 ∂z 2 w + f 2 ∂ 2 w ∂z 2 + N 2 ∂ 2 ∂ x 2 + ∂ 2 ∂ y 2 w = 0( 1 0 )
where N is the Brunt-Väiäslä frequency, or buoyancy frequency, defined by:

N 2 =- g ρ 0 dρ 0 dz . ( 11
)
If we consider a wave-form as follows:

w = w 0 cos(kx + ly + mz -ωt) (12)
the dispersion relation for a gravity wave becomes:

ω 2 = ( f 2 m 2 + N 2 (k 2 + l 2 ))/(k 2 + l 2 + m 2 ). (13)
When the wave-number is given in polar coordinates as defined by Fig. 2, the dispersion relation becomes:

ω 2 = f 2 sin 2 ϕ + N 2 cos 2 ϕ. ( 14
)
For a plane wave, where the motion is confined within the plane perpendicular to the wave-number vector, and, if the x ′ axis is chosen to be in the direction of the horizontal East Fig. 3 Hodograph of (u,v) wind velocity GW perturbation component of the wave-number vector (Fig. 3), the polarization relations are:

u ′ =-w tan ϕ v ′ =-if ω u ′ = if ω w tan ϕ (15)
and thus

⎧ ⎪ ⎪ ⎨ ⎪ ⎪ ⎩ u ′ v ′ = i ω f u ′ w =-tan ϕ =-k m . (16)
The velocity vector rotates anti-cyclonically in space as one moves in a direction opposite to the phase velocity. From an hodograph of wind velocity (in the (u,v)plane), an anti-cyclonic rotation with height, (i.e., in clockwise for north hemisphere) corresponds to a downward phase propagation and, hence to an upward component of group velocity.The frequency of internal waves does not depend on the magnitude of the wave-number but depends only on the angle ϕ that the wave-number vector makes with the horizontal plane. The phase velocity is directed along the wave-number vector, with its magnitude given by:

V φ = ω K = N K cos ϕ. ( 17
)
The group velocity is defined as the gradient of ω in the wave-number space and is, therefore, perpendicular to the conical surfaces of constant ω. Its magnitude is given by:

V g = ∂ω ∂ K = N 2 -f 2 ωK . cos ϕ sin ϕ (18)

GW characterization

During the sounding, the balloon measures the speed and direction of the horizontal wind in the atmosphere according to a local reference frame (u,v,w), respectively along the East-North direction, with w being the vertical speed of the balloon. In order to fully characterize the GW, we implemented an interactive software tool, based on a graphical interface, as follows:.

-First one selects a portion of the atmosphere in which wind speed oscillations are visible (Fig. 4). -Then, one measures the local vertical wavelength (λ z ) and adapts the bandwidth of the filter [GWmin, GWmax] in order to have λ z in its center, leading to an estimate of the vertical wave-number component, m, using:

m = 2π λ z . ( 19
)
-When the ellipse in the hodograph is well defined (Fig. 5), one can measure the best parameters that fits the ellipse. From (16), we deduce K H , the direction θ and the intrinsic pulsation ω of the gravity wave:

ω = f major axes minor axes ( 20
)
where f = 2 sin is the inertial pulsation at latitude . Knowing ω, fandN,weevaluate the amplitude of K H from the dispersion relation (13):

K H = f 2 -ω 2 ω 2 -N 2 |m|. (21)
The module of the gravity wave-number is:

K = K 2 H + m 2 . ( 22
)
From the ellipse angle (θ), one can deduce the horizontal wavenumber components:

k = K H cos θ l = K H sin θ (23)
and the horizontal wavelengths:

λ x = 2π/k λ y = 2π/l. ( 24
)
One can now evaluate the wavenumber direction with respect to the horizontal plane:

ϕ = tan -1 m K H . (25)
At this step, the user can select the direction of propagation (up for a clockwise hodograph, down for an anti-clockwise one), and all the wave parameters are finally recorded in a file.

Case study

Here we analyze in detail flight 258 (launched from Sirene at 00:55 (UT) on July 18, 2002) following an approach described by Cot and Barat [START_REF] Cot | Wave-turbulence interaction in the stratosphere-A case study[END_REF], 1986. In Fig. 4, one can see a résumé of the vertical profiles of some parameters of interest. Then, as an example, we selected a slab between 4100 and 6000 m in which we applied a filtering around 1850 m (GW min = 1200 m, GW max = 2500 m), in which an elliptical trend is visible in the hodograph of the horizontal velocity plotted in Fig. 5. The GW detected with our method from 2.4 km altitude up to 16.5 km are shown in Table 1. The line that begins with h min = 4.1 refers to the above-discussed GW. One can notice that, as in most cases, this GW has a very extended horizontal wavelength and a short intrinsic ω pulsation. Table 1 Gravity wave parameters for flight 258 The GS [START_REF] Avila | Whole atmospheric-turbulence profiling with generalized scidar[END_REF] is an improved version of the "classical" scidar [START_REF] Rocca | Detection of atmospheric turbulent layers by spatiotemporal and spatioangular correlation measurements of stellar-light scintillation[END_REF] which is able to retrieve both vertical profiles of C 2 n (h) and wind speed profiles from the analysis of the scintillation produced by a double star on the entrance pupil of a telescope. Profiles begin at ground level and end at around 20-30 km altitude. Figure 6 shows the temporal evolution of the distribution of the optical turbulent layers in the atmosphere during the night of 17-18 July at OHP between 0:47 and 2:09 UT, close to flight 258.

h min h max OG min OG max λ x λ y λ z K * 10 3 ϕθ ω [km] [km] [km] [km] [km] [km] [km] [m -1 ] [deg] [deg] [mrad. s -
Several layers are visible, at ground level, at 2.5, 4.5, 11-12 and 15 km, lasting for hours. Our balloons are equipped to measure the microstructure of the temperature field [START_REF] Vernin | Optical seeing at La Palma Observatory II. Intensive site testing campaign at the Nordic Optical Telescope[END_REF], giving C 2 T or C 2 N profiles with a vertical resolution of about 6 m. Figure 7 shows a comparison between C 2 N profiles taken by the GS and flight 271, at approximately the same time. Taking into account that both techniques are not simultaneously scrutinizing the same part of the atmosphere, the agreement is very good in terms of the absolute values and the altitudes of the layers.

5G W / O Tr e l a t i o n

Figure 8 shows, on the left, the vertical gradient of the horizontal wind and, on the right, the C 2 N distribution. In this plot, we selected a zone, between 4 and 6 km, corresponding to the same slab as in Fig. 5. As seen in this last figure, the wind speed gradient might be maximal twice within one vertical wavelength of the GW, i.e., around 4.6 and 5.6 km. From Table 1, we deduced that the vertical wavelength was 1.9 km, leading to a half-wavelength of ∼1 km. But, as one can see in Fig. 8, the turbulent layers appear clearly at 4.6 km, which was expected, and at 5.3 km, 300 m below our expectation.

In Fig. 9, we detail the GW/OT correlation from the analysis of flight 267. Considerable GW activity is visible in the hodograph. Again, in the upper part of the plot, optical turbulence layers clearly appear where the vertical gradient is at a maximum.

The two above-mentioned examples show the good correspondence between GW and OT at high vertical resolution, as can be achieved with balloon sounding. But, as a test, we N profiles obtained from flight 258, corresponding with the ellipse in Fig. 5. C 2 n layers at 4.6 and 5.4 km appear where the wind speed gradient is maximum, i.e., where the velocity vector changes rapidly in the hodograph tried to compare a whole C 2 N profile, from ground level up to 30 km, with a local Fourier analysis employing a sliding window on the vertical profile of the horizontal wind. At a given altitude, and within a small vertical window, we computed the spatial Fourier spectrum of the horizontal speed to assess:

W V (h, K ) = FT[V(h), h -h < h <, h + h]. ( 26
)
where V represents the horizontal wind speed and FT is the Fourier Transform operator. In both representations, it appears that most of the OT is concentrated within the first 2 km and above 11 km, with a 6 km "hole" empty of turbulence. Again, GW activity is related to large variance of the horizontal wind speed variations, and it coincides well with OT.

6C o n c l u s i o n

Our first analyses of the data from OHP02 measurement campaign are described herein. OHP02 was intended to detect gravity waves, to characterize them and to explore their relation to optical turbulence. Data from many balloonsondes launched from two sites, OHP and Sirene Observatories in Southern France, and from the Generalized Scidar at OHP, allowed us to fully characterize numerous GW. We also showed that, in many cases, GW activity is related to optical turbulence confined within thin turbulent layers. However, while waves were active over both sites, no clear correlation was evident between the 2 sites, some 20 km apart. This lack of coherence might indicate that packets of GW were developing within slabs of the atmosphere, instead of assuming monochromatic GW. In most cases, GW have long horizontal wavelengths (∼100-200 km), kilometric vertical wavelengths (λ ∼0.5-2 km) and long intrinsic periods (T ∼ 2-15 h).

Fig. 1

 1 Fig. 1 Map showing the position of the OHP and Sirene observatories (circled in red), ∼20 km apart in a NW-SE alignment

Fig. 2

 2 Fig.2Notations and conventions: K is the wave-number (k,l,m), K H is the projection of K on the horizontal plane (k, l)

Fig. 4 Fig. 5

 45 Fig.[START_REF] Gill | Atmosphere Ocean dynamics[END_REF] Some vertical profiles deduced from flight 258: Cn 2 , Brunt-Väisälä frequency N , Richardson number R i , hodograph, horizontal wind speed profiles V x , V y , vertical ascent speed V z and potential temperature T pot .FromV x sounding data we clearly see an oscillation with a period of about 1700 m

Fig. 6

 6 Fig. 6 Temporal evolution of the optical turbulence remote sensed by the G-Scidar, on 17/18 July 2004 above OHP

Fig. 7 Fig. 8

 78 Fig. 7 Comparison between C 2 N (h) profiles obtained with the GS and the flight 271, on July 19, at OHP, approximately at the same hour

Fig. 9

 9 Fig. 9 Analysis of flight 267, on July 18. The C 2 N (h), vertical gradient of horizontal wind, wind speed hodograph and balloon ascent speed are plotted from top left to bottom right

Fig. 10

 10 Fig. 10 On the same plot are superimposed a C 2 N profile and W V (h, K ) (see text), extracted from flight 258

Acknowledgements

This work was made possible thanks to AFOSR IRI grant number F61775-02-C0002. We are indebted to Max Azouit, Karim Agabi, Lyu Abe, Zouhair Benkhaldoun, El Fettah Habib, Aziz Ziad, Mohamed Lazrek, Eric Donnadei and Tony Travouillon who all contributed their precious assistance to the smooth running of this campaign. A special mention is given to Mr Petitpas (OHP) who helped us to manage the processing of tens of crates shipped from several countries. We also gratefully acknowledge the generosity, open-ness and outstanding support provided by M. Frédéric Bardin and Solange Fouvet of the Observatoire SIRENE (and their local volunteers) and M. Velghe of the CNRS Service d'Aéronomie (CNRS/SA) at OHP who made his calibration laboratory available to us for the preparation of AFRL ozonesondes.