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The authors thank the patients and their families for participating in this study. They are grateful to the members of the David Sancho laboratory, especially to Carlos del Fresno, for helpful critical discussion. Rationale: Recurrent wheezing in children represents a severe public health concern. Wheezing attacks (WA), mainly associated with viral infections, lack effective preventive therapies. Objectives: To evaluate the efficacy and safety of mucosal sublingual immunotherapy based on whole inactivated bacteria (MV130) in preventing WA in children. Methods: A Phase 3 randomized, double-blind, placebo-controlled, parallel-group trial including a cohort of 120 children

BACKGROUND: Polymerized allergens conjugated to non-oxidized mannan (PM-allergoids) are novel vaccines targeting dendritic cells (DCs). Previous experimental data indicate that PM-allergoids are readily taken up by DCs and induce Treg cells. This first-in-human study was aimed to evaluate safety and to find the optimal dose of house dust mite PM-allergoid (PM-HDM) administered subcutaneously (SC) or sublingually (SL). METHODS: In a randomized, double-blind, double-dummy, placebo-controlled trial, 196 subjects received placebo or PM-HDM at 500, 1000, 3000, or 5000 mannan-conjugated therapeutic units (mTU)/mL in 9-arm groups for 4 months. All subjects received 5 SC doses (0.5 ml each) every 30 days plus 0.2 ml SL daily. The primary efficacy outcome was the improvement of titrated nasal provocation tests (NPT) with D. pteronyssinus at baseline and at the end of the study. All adverse events and reactions were recorded and assessed. Secondary outcomes were the combination of symptom and medication scores (CSMS) and serological markers. RESULTS: No moderate or severe adverse reactions were reported. Subjects improving the NPT after treatment ranged from 45% to 62% in active SC, 44% to 61% in active SL and 16% in placebo groups. Statistical differences between placebo and active groups were all significant above 500 mTU, being the highest with 3000 mTU SL (p = 0.004) and 5000 mTU SC (p = 0.011). CSMS improvement over placebo reached 70% (p < 0.001) in active 3000 mTU SC and 40% (p = 0.015) in 5000 mTU SL groups. CONCLUSIONS: PM-HDM immunotherapy was safe and successful in achieving primary and secondary clinical outcomes in SC and SL at either 3000 or 5000 mTU/ml.

Summary MV130 is an inactivated polybacterial mucosal vaccine that confers protection to patients against recurrent respiratory infections, including those of viral etiology. However, its mechanism of action remains poorly understood. Here, we find that intranasal prophylaxis with MV130 modulates the lung immune landscape and provides long-term heterologous protection against viral respiratory infections in mice. Intranasal administration of MV130 provides protection against systemic candidiasis in wild-type and Rag1-deficient mice lacking functional lymphocytes, indicative of innate immune-mediated protection. Moreover, pharmacological inhibition of trained immunity with metformin abrogates the protection conferred by MV130 against influenza A virus respiratory infection. MV130 induces reprogramming of both mouse bone marrow progenitor cells and in vitro human monocytes, promoting an enhanced cytokine production that relies on a metabolic shift. Our results unveil that the mucosal administration of a fully inactivated bacterial vaccine provides protection against viral infections by a mechanism associated with the induction of trained immunity., Graphical abstract, Highlights • MV130 reduces morbi-mortality in mouse models of viral respiratory infection • MV130 modulates mouse immunity complying with key features of trained immunity • MV130 promotes reprogramming of mouse bone marrow progenitors • MV130 induces trained immunity in human monocytes, Brandi et al. investigate the mechanism of action of an inactivated polybacterial mucosal immunomodulator, MV130, which confers protection in patients suffering from recurrent respiratory infections. They show that MV130 induces trained immunity both in mouse bone marrow progenitors, resulting in protection from viral infection, and in human monocytes in vitro.

We have recently reported that grass pollen allergoids conjugated with nonoxidized mannan of Saccharomyces cerevisae using glutaraldehyde results in a novel hypoallergenic mannan-allergen complex with improved properties for allergen vaccination. Using this approach, human dendritic cells show a better allergen uptake and cytokine profile production (higher IL-10/IL-4 ratio) for therapeutic purposes. Here we aim to address whether a similar approach can be extended to dogs using canine dendritic cells. Six healthy Spanish Greyhound dogs were used as blood donors to obtain canine dendritic cells (DC) derived from peripheral blood monocytes. Allergens from Dermatophagoides farinae mite were polymerized and conjugated with nonoxidized mannan. Nuclear magnetic resonance (NMR), gel electrophoresis (SDS-PAGE), immunoblotting and IgE-ELISA inhibition studies were conducted to evaluate the main characteristics of the allergoid obtained. Mannan-allergen conjugate and controls were assayed in vitro for canine DC uptake and production of IL-4 and IL-10. The results indicate that the conjugation of D. farinae allergens with nonoxidized mannan was feasible using glutaraldehyde. The resulting product was a polymerized structure showing a high molecular weight as detected by NMR and SDS-PAGE analysis. The mannan-allergen conjugate was hypoallergenic with a reduced reactivity with specific dog IgE. An increase in both allergen uptake and IL-10/IL-4 ratio was obtained when canine DCs were incubated with the mannan-allergen conjugate, as compared with the control allergen preparations (unmodified D. farinae allergens and oxidized mannan-allergen conjugate). We conclude that hypoallergenic D. farinae allergens coupled to nonoxidized mannan is a novel allergen preparation suitable for canine allergy immunotherapy targeting dendritic cells.

Background: Recurrent wheezing affects one third of children being a global health problem with considerable expenditure and impact on quality of life. Virtually all wheezing attacks (WA) in young children are of viral etiology. Their prevention is a major concern as effective therapies are still lacking. Aims: To evaluate the efficacy and safety of a mucosal whole-cell inactivated polybacterial preparation (MV130) in the prevention of WA, and to investigate the underlying mechanism of action. Methods: A randomized, double-blind, placebo-controlled, parallel-group study, including 120 children Results: A reduction of 40% in the number of WA in the active vs the placebo group (p Administration of MV130 conferred protection to intranasal viral infection in mice. This non-specific resistance was mediated by trained immunity. Conclusion: We provide evidence for the first time that a mucosal (sublingual) polybacterial formulation induces trained immunity, confers broad protection beyond the pathogens included in the preparation and shows clinical efficacy against a virus-related condition such as WA, with an excelent safety profile.

9 p.-8 fig., Immunotherapy for treating IgE-mediated allergies requires high doses of the corresponding allergen. This may result in undesired side effects and, to avoid them, hypoallergenic allergens (allergoids) polymerized with glutaraldehyde are commonly used. Targeting allergoids to dendritic cells to enhance cell uptake may result in a more effective immunotherapy.Allergoids coupled to yeast mannan, as source of polymannoses, would be suitable for this purpose, since mannose-binding receptors are expressed on these cells.Conventional conjugation procedures of mannan to proteins use oxidized mannan to release reactive aldehydes able to bind to free amino groups in the protein; yet, allergoids lack these latter because their previous treatment with glutaraldehyde.The aim of this study was to obtain allergoids conjugated to mannan by an alternative approach based on just glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone. Allergoid-mannan glycoconjugates were produced in a single step by treating with glutaraldehyde a defined mixture of allergens derived from Phleum pratense grass pollen and native mannan (nonoxidized) from Saccharomyces cerevisae. Analytical and structural studies, including 2D-DOSY and 1H-13C HSQC nuclear magnetic resonance spectra, demonstrated the feasibility of such an approach. The glycoconjugates obtained were polymers of high molecular weight showing a higher stability than the native allergen or the conventional allergoid without mannan. The allergoid-mannan glycoconjugates were hypoallergenic as detected by the IgE reactivity with sera from grass allergic patients, even with lower reactivity than conventional allergoid without mannan. Thus, stable hypoallergenic allergoids conjugated to mannan suitable for using in immunotherapy can be achieved using glutaraldehyde. In contrast to mannan oxidation, the glutaraldehyde approach allows to preserve mannoses with their native geometry, which may be functionally important for its receptor-mediated recognition., This work was supported by grant IDI-20110410 from the Centre for the Development of Industrial Technology (CDTIMINECO, Spain)

Background Polymerized allergoids coupled to nonoxidized mannan (PM‐allergoids) may represent novel vaccines targeting dendritic cells (DCs). PM‐allergoids are better captured by DCs than native allergens and favor Th1/Treg cell responses upon subcutaneous injection. Herein we have studied in mice the in vivo immunogenicity of PM‐allergoids administered sublingually in comparison with native allergens. Methods Three immunization protocols (4‐8 weeks long) were used in Balb/c mice. Serum antibody levels were tested by ELISA. Cell responses (proliferation, cytokines, and Tregs) were assayed by flow cytometry in spleen and lymph nodes (LNs). Allergen uptake was measured by flow cytometry in myeloid sublingual cells. Results A quick antibody response and higher IgG2a/IgE ratio were observed with PM‐allergoids. Moreover, stronger specific proliferative responses were seen in both submandibular LNs and spleen cells assayed in vitro. This was accompanied by a higher IFNγ/IL‐4 ratio with a quick IL‐10 production by submandibular LN cells. An increase in CD4+ CD25high FOXP3+ Treg cells was detected in LNs and spleen of mice treated with PM‐allergoids. These allergoids were better captured than native allergens by antigen‐presenting (CD45+ MHC‐II +) cells obtained from the sublingual mucosa, including DCs (CD11b+) and macrophages (CD64+). Importantly, all the differential effects induced by PM‐allergoids were abolished when using oxidized instead of nonoxidized PM‐allergoids. Conclusion Our results demonstrate for the first time that PM‐allergoids administered through the sublingual route promote the generation of Th1 and FOXP3+ Treg cells in a greater extent than native allergens by mechanisms that might well involve their better uptake by oral antigen‐presenting cells.

Background: Little information is available on the clinical efficacy of sublingual immunotherapy (SLIT) using extracts derived from mammalian epithelia. Objectives: To assess clinical efficacy of cat SLIT based on natural exposure challenge test (NCT). Material and methods: Fifty cat allergic patients with rhinoconjunctivitis with or without asthma were included in a randomized double blind placebo controlled clinical trial of cat SLIT during 1 year. Twenty-five patients received active treatment and 25 placebo. Sublingual immunotherapy efficacy was assessed by natural exposure challenge to a cat in a cat-room and by skin tests. Airborne Fel d 1 levels, symptom scores and peak expiratory flow (PEF) values were monitored. Results: Thirty-three (66%) out of 50 patients completed the treatment. Fel d 1 content of the maximum concentration was 0.51 μg per ml. During the build up phase, the accumulated dose was 1.7 μg of Fel d 1 and during the entire length of the study was 17.1. No adverse reports were reported. The active group showed a marked reduction (62%) in symptoms during the NCT (P

Background Numerous varieties of Olea europaea have been described in Mediterranean countries. Objective To investigate the immunochemical characteristics of 6 varieties of Olea europaea collected during 5 consecutive years. Methods The varieties Carrasqueno, Manzanillo, Acebuche (wild olive), Hojiblanco, Picual, and Nevado were analyzed. Pollen samples from each variety were collected for 5 consecutive years from the same cultivars by trained personnel. The antigenic and allergenic profiles of these extracts were evaluated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using the serum of 29 O europaea -allergic individuals. Ole e 1 was measured using enzyme-linked immunosorbent assay and purified Ole e 1 and rabbit polyclonal antibodies. Allergenic potency was determined using enzyme-linked immunosorbent assay inhibition and is expressed in histamine equivalent prick units per gram of raw material. Results Hojiblanco and Acebuche had the lowest mean ± SD Ole e 1 content in the 5 years (0.045 ± 0.029 and 0.059 ± 0.031 μg/μg of freeze-dried material, respectively). The variety with the highest mean ± SD Ole e 1 content was Picual (0.19 ± 0.075 μg/μg). Hojiblanco had the lowest total biological potency throughout the study. A positive correlation was obtained between rainfall in the winter months and total allergenicity of the 6 varieties. Conclusions The different varieties of O europaea pollen demonstrated great differences in allergenic potency and Ole e 1 content. These differences were maintained throughout the study, suggesting that they are due to genetic differences intrinsic to the varieties, although certain climatic effects may also play a role.

Background Mite sensitivity is highly prevalent in tropical and subtropical regions, such as the Canary Islands. Objectives To evaluate the clinical efficacy and safety of a depigmented polymerized allergen vaccine containing a 50% mixture of Dermatophagoides pteronyssinus and Dermatophagoides farinae . Methods Sixty-four patients participated in the study. It was prospective, double-blind, and placebo-controlled, with random allocation of patients to receive active treatment or placebo. The active group received a mixture of modified allergen extracts containing 50% D pteronyssinus and 50% D farinae ; the control group received placebo. All individuals were diagnosed with mild/moderate asthma and rhinoconjunctivitis caused by sensitization to D pteronyssinus and D farinae. Bronchial provocation test (BPT) was considered the main outcome to document clinical efficacy. Results Fifty-four patients completed the study. The active group experienced a significant improvement in BPT ( P P = .648). At the end of the study, 20 patients in the active versus 9 in the placebo group ( P = .013; odds ratio, 5.71 [1.76, 18.51]) needed more than twice the amount of allergen to obtain a positive BPT. The median improvement in the active group over placebo was 53.76% in total symptom and 58.09% in medication scores. Conclusion Immunotherapy with a mixture of modified allergen extracts of D pteronyssinus and D farinae is safe and efficacious to treat mite-allergic asthma. Clinical implications This immunotherapy modifies the natural course of the illness because it improves all clinical outcomes measured and prevents the worsening of specific bronchial hyperreactivity.

Summary Background The physicochemical modification of allergen extracts provides a chance for administering higher doses of allergen vaccines. Objective To evaluate the safety of a chemically modified (depigmented-glutaraldehyde polymerized) therapeutic vaccine of Phleum pratense administered at doses that are 10 times higher than those used in clinical practice, in comparison with conventional doses of the corresponding non-modified alum-adsorbed vaccine. Materials and methods The design of the study was randomized, double-blind, parallel and included two groups of patients. Twenty-three patients were treated weekly during nine visits for the build-up phase, followed by two weekly maintenance doses (a total of 11 injections per patient). Twelve patients received a vaccine containing the standardized unmodified extract, at a maximum concentration of 308.5 mcg of freeze dried material/mL (Group A). Eleven patients received a standardized modified allergen extract (Group B). The maximum dose used was 2400 mcg/mL. Safety was evaluated recording all adverse events. Skin test results and specific antibody levels were evaluated at the beginning and at the end of the study. Results Group A patients experienced three local immediate (two clinically irrelevant and one with a diameter > 5 cm) and 18 delayed reactions (15 irrelevant and three with a diameter > 10 cm), while Group B experienced six local immediate and 12 delayed reactions (all clinically irrelevant). Nine Group A patients experienced 12 systemic reactions (one immediate of grade 1, one of grade 2; and one delayed of grade 1; four of grade 2 and three of grade 3), while Group B patients experienced one immediate systemic reaction of grades 1, and 1 delayed reaction of grade 1. Conclusions The modified extract of P. pratense is safe to treat sensitive patients, even at concentrations that are 10 times higher than those regularly administered in clinical practice. The majority of the local reactions were clinically irrelevant. No systemic reactions of grade 2, 3 or 4 were reported using the modified extract.

Background: The physiochemical modification of allergens to reduce allergenicity, while retaining immunogenicity, provides a chance for the administration of higher doses of immunotherapy, with a decreased risk of systemic reactions. Objective: To evaluate the safety of doses of depigmented glutaraldehyde-polymerized vaccine of Dermatophagoides pteronyssinus increasing those used in normal clinical conditions in comparison with regular doses of a non-modified vaccine. Materials and Methods: The study was double-blind, parallel and included two patient groups. Twenty-three patients were treated weekly during 9 visits for the build-up phase, followed by 2-weekly maintenance doses (a total of 11 injections per patient). Eleven patients (mean age 22 years) received immunotherapy with the standardized modified vaccine. The maximum dose used was the result of depigmenting and polymerizing 100 times the maximum dose used with the native extract. The maximum concentration used was 990 µg/ml of freeze-dried material. Twelve patients (mean age 24 years) received a standardized, unmodified allergenic extract. The maximum concentration used was 70 µg of freeze-dried material/ml, with a potency of 10 HEPL/ml. The tolerance was evaluated recording all the side reactions related to immunotherapy and graded following the recommendations of the European Academy of Allergology and Clinical Immunology. Results: In both groups, all immediate local reactions were clinically not relevant. Patients treated with the native extract experienced 42 local immediate and 24 delayed reactions (1 of them had a diameter >10 cm), whereas patients treated with the modified extract experienced 30 local immediate and 21 delayed reactions (2 of them had a diameter >10 cm). Four systemic reactions in 2 patients, 1 immediate of grade 1 and 3 delayed of grade 2 were reported in the group treated with native extract and 1 delayed of grade 2 in the group treated with the modified preparation. Conclusions: The modified extract of D. pteronyssinus, used at concentrations which are 10 times higher than those regularly administered in clinical practice, demonstrated to be safe to treat D. pteronyssinus-sensitive patients. The major ity of the local reactions (immediate and delayed) were clinically not relevant (diameter

Background: Solubility is an important characteristic of allergenic molecules. The aim of this study was to investigate the solubility of Ole e 1, a major allergen of Olea europaea, using different solvents. Material and methods: Olea europaea pollen was placed in a glass column and extracted using three different solvents: deionized water, phosphate buffer 0.01 M (PBS) and normal saline (NaCl 0.9%). Several fractions were collected after extraction with each solvent and pooled based on individual protein content. Each fraction corresponded to a different elution profile, as determined by linear regression analysis. After 130 min of extraction, the pollen that remained in the column was further extracted overnight. A control olive pollen extract was also prepared with each solvent. The antigenic and allergenic profiles of all the eluted and pooled fractions were analysed by SDS-PAGE and inmmunoblots. Protein and Ole e 1 content and the amount of protein needed to produce 50% inhibition were also calculated. Ten patients were skin prick tested with the fractions obtained with deionized water. Results: Four elution profiles were obtained using deionized water as the extracting solution and three with the two other solvents. The three solvents produced different kinetics of allergen release. Ole e 1 was rapidly released when water was used, obtaining a total of 256 lg of Ole e 1/ml after only 7 min of extraction (fraction EC1). Using PBS, or NaCl 0.9%, the release of Ole e 1 started after 4 and 9 min of extraction, respectively. The highest amount of Ole e 1 was eluted after 44 and 26 min, with a total concentration of 162 and 203 lg of Ole e 1/ml, respectively. The presence of Ole e 1 in each phase was verified by SDS-PAGE and immunoblot analyses. Conclusions: The extracting solution seems to determine the antigenic profile of olive pollen extracts. Ole e 1 is rapidly released from the pollen grain after extraction in deionized water. The solubility seems to be affected by the use of other solvents. These techniques could be used to manipulate the Ole e 1 content in O. europaea extracts.

Background: The modulation of T cell responses by natural peptides is of potential use in the treatment of allergic diseases and reduces the risk of IgE-mediated reactions. The objectives of this study were to evaluate the allergenicity of depigmented, pepsin-digested fragments of Oela europaea and Phleum pratense extracts and their capacity to induce T cell proliferation. Methods: Lyophilized O. europaea and P. pratense extracts were depigmented following standardized methods, digested with pepsin and fractionated by ultrafiltration; a fraction containing components in the molecular weight range of 1–10 kD was selected. The blood of 20 allergic individuals was used to conduct T cell proliferation studies. Results: Pepsin-digested fragments of both pollen extracts lost more than 95% of their IgE-binding capacity by ELISA inhibition. Greater proliferation indexes than their respective native extracts were obtained in 55% of O. europaea and in 95% of P. pratense allergic patients. Conclusions: This study demonstrates that pepsin-digested allergenic fragments of depigmented extracts can be produced in a standardized, reproducible manner. These fragments are significantly less allergenic and retain, or even increase, their capacity to induce T cell proliferations.

Background Allergen immunotherapy (AIT) is the only curative treatment for allergy. AIT faces pitfalls related to efficacy, security, duration, and patient compliance. Novel vaccines overcoming such inconveniences are in demand. Objectives We sought to study the immunologic mechanisms of action for novel vaccines targeting dendritic cells (DCs) generated by coupling glutaraldehyde-polymerized grass pollen allergoids to nonoxidized mannan (PM) compared with glutaraldehyde-polymerized allergoids (P) or native grass pollen extracts (N). Methods Skin prick tests and basophil activation tests with N, P, or PM were performed in patients with grass pollen allergy. IgE-blocking experiments, flow cytometry, confocal microscopy, cocultures, suppression assays, real-time quantitative PCR, ELISAs, and ELISpot assays were performed to assess allergen capture by human DCs and T-cell responses. BALB/c mice were immunized with PM, N, or P. Antibody levels, cytokine production by splenocytes, and splenic forkhead box P3 (FOXP3) + regulatory T (Treg) cells were quantified. Experiments with oxidized PM were also performed. Results PM displays in vivo hypoallergenicity, induces potent blocking antibodies, and is captured by human DCs much more efficiently than N or P by mechanisms depending on mannose receptor– and dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin–mediated internalization. PM endorses human DCs to generate functional FOXP3 + Treg cells through programmed death ligand 1. Immunization of mice with PM induces a shift to nonallergic responses and increases the frequency of splenic FOXP3 + Treg cells. Mild oxidation impairs these effects in human subjects and mice, demonstrating the essential role of preserving the carbohydrate structure of mannan. Conclusions Allergoids conjugated to nonoxidized mannan represent suitable vaccines for AIT. Our findings might also be of the utmost relevance to development of therapeutic interventions in other immune tolerance–related diseases.

An in vitro technique for measuring the relative potency of allergenic extracts has been developed and compared with the dose-response skin-prick test. The in vitro technique is a modification of an ELISA based on the method of antigen capture using polystyrene microtitre wells as solid phase (REINA-competition) and is also based on a dose-response. Both methods have been applied to allergen extracts of Dermatophagoides pteronyssinus, Phleum pratense, Secale cereale, Lolium perenne, Plantago lanceolata, Artemisia vulgaris, Salsola kali and Parietaria judaica. For each allergen, the slope of the regression log-log dose-response lines displayed by skin-prick test and REINA-C does not show a statistically significant difference, being parallel (P < 0.001), and indicating that the allergen-ligand binding kinetics are identical in both methods.

There has been an increasing interest on pricing power plants. The main reasons are making power investment decisions, related to the risks derived from managing or investing in combined cycle power plants. A stochastic model for the electricity futures curve has been applied for studying, using the option pricing theory framework, the pricing and hedging of a combined cycle power plant. The solution is obtained by means of solving a stochastic control problem over any feasible future load strategy. We discuss the practical case of hedging against market futures the plant risks. Finally we test against market historical values, the validity of the hedging strategy.

Summary Background The major complication of allergen immunotherapy is a severe reaction. Objective To evaluate the safety of depigmented and glutaraldehyde-modified allergen extracts in a large group of patients undergoing immunotherapy treatment. Material and methods Seven hundred sixty-six patients, having rhinoconjunctivitis and/or asthma, were entered in a prospective, multi-centre, observational cohort study, to evaluate the safety of immunotherapy with modified allergen vaccines. Patients were sensitized to mites and/or pollen and received a therapeutic vaccine containing depigmented and polymerized allergen extracts of mites and/or pollens adsorbed onto aluminium hydroxide. The schedule of administration consisted of a build-up phase of 4- to 6-weekly injections, followed by 12-monthly injections of the maintenance dose. Tolerance was assessed by recording all side reactions related to immunotherapy. Results All patients completed the study. Fifty-four clinically relevant local reactions (43 immediate and 11 delayed) were observed (0.4% of injections). The systemic reactions were 34 in 12 patients. Six reactions were immediate (all of grade 2) and 28 delayed (18 of grade 1 in two patients, nine of grade 2 and one of grade 3). The systemic reactions of grade 2 or 3 occurred in 0.12% of the injections. All systemic reactions were mild and resolved spontaneously without the need for medication. Conclusion Specific immunotherapy using modified allergen vaccines is safe to treat allergic patients. The percentage of adverse reactions detected is lower than those reported in the literature with native-unmodified allergen extracts.

Dermatophagoides pteronyssinus and D. farinae are the most common house dust mites and are among the most common sources of indoor allergens worldwide. These species are very common in humid regions, where most allergic individuals are sensitized to house dust mites. Specific immunotherapy with mite extracts has demonstrated clinical benefits in several double-blind, placebo-controlled trials that are included in recent reviews of subcutaneous immunotherapy, including pediatric and adult patients with rhinoconjunctivitis and or asthma. Most successful studies of mite immunotherapy have used native allergen extracts adsorbed onto aluminum hydroxide, or chemically modified mite-allergen extracts. Several studies have also shown efficacy using sublingual immunotherapy in pediatric and adult patients with asthma and/or rhinitis. Additionally, the efficacy of subcutaneous immunotherapy has been demonstrated in patients with atopic dermatitis, although more double-blind, placebo-controlled studies are needed. Based on several studies, it cannot be concluded that mite immunotherapy is more dangerous or safer than immunotherapy with grasses, epithelia, or animal epithelia. Because the delivery of high doses of allergen carries with it the risk for immunoglobulin E (IgE)-mediated events, several methods have been developed to reduce specific IgE binding to mite-allergen extracts. An important challenge for future mite immunotherapy modalities is the delivery of relatively high doses without a significant risk for severe reactions.

Background: Four administration schedules of immunotherapy have been reported: conventional, cluster, rush and ultra-rush. Objectives: To evaluate the safety and the clinical advantage of using standardized modified allergen extracts in an ultra-rush protocol without premedication and/or hospitalization. Material and Methods: One thousand and sixty-eight patients with rhinoconjunctivitis and/or asthma sensitized to mites and/or pollen were included in a prospective observational study. Patients received a therapeutic vaccine containing depigmented and glutaraldehyde-modified extracts (mites and/or pollens) adsorbed onto alum prescribed by a specialist. The schedule of administration consisted of injecting 0.2 and 0.3 ml of the vial of maximum concentration during the first day of immunotherapy, separated by a time interval of 30 min. All patients reached the maximum dose (0.5 ml) after 2 injections. Tolerance was assessed by recording all side reactions related to immunotherapy, classified according to the criteria of the EAACI. Results: The total number of injections was 2,136. All patients reached the maximum established dose on the 1st day. No premedication was used. Seven clinically relevant local reactions were recorded. The systemic reactions were 5 grade-1 (2 immediate and 3 delayed) and 3 delayed grade-2 reactions. Conclusions: The therapeutic vaccines containing chemically modified extracts can be administered using an alternative ultra-rush schedule in an immunotherapy unit, reaching the maximum dose on the 1st day with 2 injections, without the need of premedication and/or hospitalization.

The physiochemical modification of allergens to reduce allergenicity, while retaining immunogenicity, provides a chance for the administration of higher doses of immunotherapy, with a decreased risk of systemic reactions.To evaluate the safety of doses of depigmented glutaraldehyde-polymerized vaccine of Dermatophagoides pteronyssinus increasing those used in normal clinical conditions in comparison with regular doses of a non-modified vaccine.The study was double-blind, parallel and included two patient groups. Twenty-three patients were treated weekly during 9 visits for the build-up phase, followed by 2-weekly maintenance doses (a total of 11 injections per patient). Eleven patients (mean age 22 years) received immunotherapy with the standardized modified vaccine. The maximum dose used was the result of depigmenting and polymerizing 100 times the maximum dose used with the native extract. The maximum concentration used was 990 mug/ml of freeze-dried material. Twelve patients (mean age 24 years) received a standardized, unmodified allergenic extract. The maximum concentration used was 70 mug of freeze-dried material/ml, with a potency of 10 HEP(L)/ml. The tolerance was evaluated recording all the side reactions related to immunotherapy and graded following the recommendations of the European Academy of Allergology and Clinical Immunology.In both groups, all immediate local reactions were clinically not relevant. Patients treated with the native extract experienced 42 local immediate and 24 delayed reactions (1 of them had a diameter10 cm), whereas patients treated with the modified extract experienced 30 local immediate and 21 delayed reactions (2 of them had a diameter10 cm). Four systemic reactions in 2 patients, 1 immediate of grade 1 and 3 delayed of grade 2 were reported in the group treated with native extract and 1 delayed of grade 2 in the group treated with the modified preparation.The modified extract of D. pteronyssinus, used at concentrations which are 10 times higher than those regularly administered in clinical practice, demonstrated to be safe to treat D. pteronyssinus-sensitive patients. The majority of the local reactions (immediate and delayed) were clinically not relevant (diameter5 cm). No systemic reactions of grade 3 or 4 were noted.

Background: The purposes of this study were: to determine the prevalence of sensitization and immunochemical characterization of Eleagnus angustifolia pollen (Russian olive) that belongs to the family Eleagnaceae. Methods: A total of 134 patients with rhinoconjunctivitis and/or asthma were studied. Its allergenicity, cross-reactivity with olive pollen and the presence of Ole e 1 and Ole e 4-like molecules were evaluated. Results: Eleagnus angustifolia pollen was detected from May to June. Seventythree of 134 (30.5%) had positive skin test to E. angustifolia, all of them were positive to olive. There was a good correlation between specific immunoglobulin (Ig)E levels to E. angustifolia and Olea europaea (r ¼ 0.77, P ¼ 0.002). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots revealed major IgE-binding bands in the E. angustifolia extract of 43 and 63.7 kDa. The E. angustifolia extract was not able to inhibit olive, whereas O. europaea inhibited E. angustifolia up to 41%. The presence of Ole e 1- and Ole e 4-like allergens in E. angustifolia extract was confirmed by enzyme-linked immunosorbent (ELISA) inhibition assays. Nasal challenge with E. angustifolia was positive in three of six patients with positive skin test to both pollens and negative in five patients with positive skin test only to O. europaea. Conclusions: This study confirms that E. angustifolia is capable of sensitizing individuals in Madrid. A minimal-to-moderate cross-reactivity with olive pollen was established, suggesting some cross-reactivity but not excluding co-sensitization.

Background Allergoids were first used in the decades of the 60s and 70s of the last century as an effective treatment of allergic respiratory diseases. Allergoids can be modified with formaldehyde or glutaraldehyde. Modified allergens, or allergoids, decrease the risk of adverse reactions while administering higher allergen doses. The objective of this study was to analyse specific IgE and IgG binding to glutaraldehyde modified and non-modified allergen extracts of Phleum pratense. Methods The sera of 69 patients sensitized to P. pratense were tested. All these patients had signs and symptoms of rhinoconjunctivitis with, or without, asthma in May and June of 2011. All these patients had positive skin prick tests to a standardized extract of P. pratense, and other grass species. Most patients were also sensitized to olive pollen. Specific IgE and IgG binding were analysed by direct ELISA against P. pratense native (non-modified) and allergoid extracts. Relative potencies were evaluated through ELISA inhibition assays, and the protein composition of non-modified and allergoid samples was determined by Mass Spectrometry (MS/MS). Results Mean Specific IgE levels against the native extract was 16.68 ± 11.65 Units (U) and against the allergoid: 7.26 ± 8.24 U (P < 0.0001; Mann-Whitney). On the other hand, mean specific IgG binding against the non-modified extract was 90.34 ± 75.57 U versus 76.19 ± 70.31 U against the allergoid (P = 0.16; Mann-Whitney). Linear regression coefficients obtained between immunoglobulin reactivity against both extracts were: r2 = 0.51 for specific IgE and r2 = 0.83 for specific IgG. An important decrease in the allergenic activity, measured by inhibition ELISA, was clearly observed. The MS/MS assay revealed the presence of the mayor allergen, and some isoforms, in non-modified and allergoid extracts. Conclusions Results obtained demonstrate that the glutaraldehyde polymerization process induces an important decrease in specific IgE binding to allergoids of P. pratense while there are no significant differences in specific IgG binding. The allergenic composition of the P. pratense allergoid was equivalent to the non-modified pollen extract.

Ole e 1 is an important allergen in Olea europaea pollen extracts. This study describes the development of two new methods that can be used to estimate the Ole e 1 content in olive tree pollen extracts. They are based on (1) an enzyme immunoassay that uses rabbit polyclonal, monospecific antibodies and purified Ole e 1, and (2) scanning densitometry of SDS-PAGE gels. Twelve extracts were evaluated by in vivo and in vitro methods. The in vivo biological potency was estimated by prick skin testing 17 allergic individuals; the in vitro allergenic potency by direct IgE and IgE inhibition assays. The enzyme immunoassay showed an operative range of 0.03-100 microg/ml and demonstrated to be specific for Ole e 1. The Ole e 1 content ranged from 1% to 5% of the total protein in the 12 extracts. The amount of Ole e 1, assessed by gel scanning densitometry significantly correlated with the Ole e 1 content obtained by the immunoassay (r = 0.92; p < 0.001). The Ole e 1 content showed a significant correlation with the total allergenic potency of the extracts, evaluated by direct IgE, specific IgE inhibition and skin-prick testing. These two methods can be used to determine the Ole e 1 content in olive pollen extracts. The content of Ole e 1 can vary from 1% to 5% of the total protein in the extracts.

Background The most common allergenic pollens in patients with pollinosis in Central Spain are grasses and olive pollen, with a prevalence of positive skin prick test results of 94 and 61%, respectively. Shared proteins have been described in olive and grasses, such as profilins, polcalcin and trypsin inhibitors, but there are few in vitro studies analyzing the potential cross reactivity between both species. The aim of the present study was to analyze the protein composition of both allergen extracts, and the allergenic cross-reactivity between O. europaea and different grass pollen species. Methods Seventy-two (72) patients (mean age 10.4 years) were included in the study. All of them suffered rhinoconjuntivitis and/or asthma and were sensitized to olive and/or grass pollen. Specific IgE of the individual patients against O. europaea, and to the grass species: Dactylis glomerata, Phleum pratense and Trisetum paniceum were determined by ELISA. Inhibition assays were performed to verify allergenic crossreactivity between grass species and olive. Mass spectrometry analysis was performed to characterize the extracts and establish if there are common proteins in both, grass and olive pollens, that could act as cross reactive proteins. Results Three different sensitization patterns were observed: 1) sensitization to olive and grass pollen, 2) sensitization to olive and not to grasses and 3) sensitization to grasses and not to olive. Different pools of sera were mixed according to this classification and used for the different assays. Correlation coefficients found for the 3 grass species were significant (P < 0.0001; Spearman), but not for olive pollen (P = 0.14; Spearman). Proteomic analysis revealed the presence of more than 40 common proteins in grasses and olive pollens, but inhibition assays demonstrated no allergenic cross-reactivity between both families. Conclusions There is no in vitro crossreactivity between O. europaea and Grass pollen extracts, in spite of the allergens and the large number of common proteins shared by these pollens. We can conclude that sensitization to olive and grasses is species specific.

Background D. pteronyssinus and B. tropicalis are the most common house dust mite species worldwide. D. pteronyssinus and B. tropicalis are the most prevalent species in the tropics. Twelve allergens have been described in B. tropicalis and fifteen in D. pteronyssinus. The objectives of this study were: 1) to analyze the prevalence of sensitization to B. tropicalis and D. pteronyssinus on the tropical Island of Martinique; 2) to evaluate the allergenic crossreactivity between D. pteronyssinus and B. tropicalis and 3) to identify common, and species specific allergens, using mass spectrometry (MS). The serum of patients sensitized to a single, or to both species, was further analyzed by western blots. Methods The sera of 1,243 consecutive patients evaluated for allergic rhinitis and/or asthma were evaluated for specific IgE using direct ELISA. All patients resided on the tropical island of Martinique. Crossreactivity between both species was analyzed by ELISA Inhibitions. Proteins of both species were digested with trypsin and analyzed by MS/MS (MALDI TOF/TOF). Different databases were used for the identification of the proteins. Results Sensitization to B. tropicalis was detected in 868 sera and to D. pteronyssinus in 919 sera; 135 were sensitized exclusively to B. tropicalis and 177 to D. pteronyssinus. Specific IgE levels to D. pteronyssinus were significantly higher than to B. tropicalis (Mean 0.67 OD ± 0.89 vs. 0.86 ± 1.04; P < 0.001). The correlation coefficient between specific IgE levels to both species was 0.135. ELISA Inhibition studies demonstrated minimal crossreactivity between both species. Western blots done with the serum of poly or monosensitized patients revealed specific IgE binding to similar proteins. Proteomic analysis revealed the presence of the following allergens: B. tropicalis: groups 2, 3, 4, 6, 9, 14 and groups 1, 2, 3, 4, 8, 11, 14, 15, 16 and 18 in D. pteronyssinus. Species specific, as well as common proteins were detected. Conclusions There is limited crossreactivity between B. tropicalis and D. pteronyssinus. Sensitization to their allergens seems to be a parallel phenomenon. Allergen extracts of both species are needed for the correct diagnosis and treatment of mite allergic individuals in the Caribbean.

Background Allergoids have been successfully used in the treatment of respiratory allergic diseases. They are modified allergen extracts that allow the administration of high allergen doses, due to their reduced IgE binding capacity.They maintain allergen-specific T-cell recognition. Since they are native allergen extracts that have been polymerized with glutaraldehyde, identification of the allergenic molecules requires more complicated methods. The aim of the study was to determine the qualitative composition of different polymerized extracts and investigate the presence of defined allergenic molecules using Mass spectrometry. Methods Proteomic analysis was carried out at the Proteomics Facility of the Hospital Nacional de Parapléjicos (Toledo, Spain). After reduction and alkylation, proteins were digested with trypsin and the resulting peptides were cleaned using C18 SpinTips Sample Prep Kit; peptides were separated on an Ultimate nano-LC system using a Monolithic C18 column in combination with a precolumn for salt removal. Fractionation of the peptides was performed with a Probot microfraction collector and MS and MS/MS analysis of offline spotted peptide samples were performed using the Applied Biosystems 4800 plus MALDI TOF/TOF Analyzer mass spectrometer. ProteinPilot Software V 2.0.1 and the Paragon algorithm were used for the identification of the proteins. Each MS/MS spectrum was searched against the SwissProt 2010_10 database, Uniprot-Viridiplantae database and Uniprot_Betula database. Results Analysis of the peptides revealed the presence of native allergens in the polymerized extracts: Der p 1, Der p 2, Der p 3, Der p 8 and Der p 11 in D. pteronyssinus; Bet v 2, Bet v 6, Bet v 7 and several Bet v 1 isoforms in B. verrucosa and Phl p 1, Phl p 3, Phl p 5, Phl p 11 and Phl p 12 in P. pratense allergoids. In all cases, potential allergenic proteins were also identified, including ubiquitin, actin, Eenolase, fructose-bisphosphate aldolase, luminal-binding protein (Heat shock protein 70), calmodulin, among others. Conclusions The characterization of the allergenic composition of allergoids is possible using MS/MS analysis. The analysis confirms the presence of native allergens in the allergoids. Mayor allergens are preserved during polymerization.