Comparison of Intact Allergen Extracts and Allergoids For Subcutaneous Immunotherapy: The Effect of Chemical Modification Differs Both Between Species and Between Individual Allergen Molecules

Background: Allergen products for subcutaneous immunotherapy (SCIT) contain intact allergen extracts or chemically modified allergoids. Chemical modification was introduced to reduce allergenicity while retaining immunogenicity and thereby enable safer and more efficient allergy immunotherapy. Methods: Experimental allergoids were produced from intact allergen extract for birch, grass, and house dust mite (HDM) to evaluate the effects of chemical modification. Preparations were compared with commercial allergoids and analyzed using SDS-PAGE/immunoblotting, IgE-inhibition assays, and crossed immunoelectrophoresis (CIE). Dermatophagoides pteronyssinus (Der p) vaccines were also tested for protease activity and immunizing capacity in a mouse model. Results: The composition of IgE-binding epitopes in allergoids differed from that of intact allergen vaccines. Birch and grass allergoids produced smears of protein aggregates on SDS-PAGE, whereas intact allergen preparations showed distinct protein bands as expected. Der p allergoid vaccines, however, showed a distinct protein band corresponding to major allergen Der p 1 in both SDS-PAGE and CIE analysis, and commercial Der p allergoid vaccines showed Der p 1–related cysteine protease activity. Conclusion: Allergoids and intact allergen preparations differ with respect to the composition of IgE-binding epitopes. However, chemical cross-linking does not affect every allergen molecule to the same degree. Der p 1, for example, remains largely unmodified. Furthermore, the investigational HDM allergoid vaccines showed reduced and delayed immune responses when used for immunization of mice (AU)

Antecedentes: Los productos de alérgenos para inmunoterapia subcutánea (SCIT) contienen extractos de alérgenos intactos o alergoides modificados químicamente. En este trabajo se ha hecho una modificación química para reducir la alergenicidad a la vez que se conservaba la inmunogenicidad, y por lo tanto, permitir una inmunoterapia más segura y eficiente. Métodos: Se produjeron alergoides experimentales a partir de extracto de alérgeno intacto para abedul, hierba y ácaros del polvo doméstico (HDM) y se evaluaron los efectos de la modificación química realizada. Las preparaciones se compararon con alergoides comerciales y se analizaron mediante SDS-PAGE/inmunotransferencia, ensayos de inhibición de IgE e inmunoelectroforesis cruzada (CIE). Las vacunas de Dermatophagoides pteronyssinus (Der p) también se probaron para determinar la actividad de la proteasa y la capacidad de inmunización en un modelo de ratón. Resultados: La composición de los epítopos de unión a IgE en los alergoides difería de las vacunas de alérgenos intactas. Los alergoides de hierba y abedul produjeron manchas de agregados de proteínas en el SDS-PAGE, mientras que las preparaciones de alérgenos intactos mostraron distintas bandas de proteínas como se esperaba. Las vacunas alergoides Der p, sin embargo, mostraron una banda de proteína distinta de la correspondiente al alérgeno principal Der p 1 en los análisis SDS-PAGE y CIE. Las vacunas alergoides comerciales Der p mostraron actividad de cisteína proteasa relacionada con Der p 1.Conclusión: Los alergoides y las preparaciones de alérgenos intactos difieren con respecto a la composición de los epítopos de unión a IgE; sin embargo, el entrecruzamiento químico no afecta a todas las moléculas de alérgenos de un modo similar. Der p 1, por ejemplo, permanece prácticamente sin modificar. Además, las vacunas alergoides de HDM produjeron respuestas inmunitarias reducidas y tardías cuando se usaron para la inmunización de ratones (AU)

Comparison of Intact Allergen Extracts and Allergoids For Subcutaneous Immunotherapy: The Effect of Chemical Modification Differs Both Between Species and Between Individual Allergen Molecules.

BACKGROUND: Allergen products for subcutaneous immunotherapy (SCIT) contain intact allergen extracts or chemically modified allergoids. Chemical modification was introduced to reduce allergenicity while retaining immunogenicity and thereby enable safer and more efficient allergy immunotherapy. METHODS: Experimental allergoids were produced from intact allergen extract for birch, grass, and house dust mite (HDM) to evaluate the effects of chemical modification. Preparations were compared with commercial allergoids and analyzed using SDS-PAGE/immunoblotting, IgE-inhibition assays, and crossed immunoelectrophoresis (CIE). Dermatophagoides pteronyssinus (Der p) vaccines were also tested for protease activity and immunizing capacity in a mouse model. RESULTS: The composition of IgE-binding epitopes in allergoids differed from that of intact allergen vaccines. Birch and grass allergoids produced smears of protein aggregates on SDS-PAGE, whereas intact allergen preparations showed distinct protein bands as expected. Der p allergoid vaccines, however, showed a distinct protein band corresponding to major allergen Der p 1 in both SDS-PAGE and CIE analysis, and commercial Der p allergoid vaccines showed Der p 1-related cysteine protease activity. CONCLUSION: Allergoids and intact allergen preparations differ with respect to the composition of IgE-binding epitopes. However, chemical cross-linking does not affect every allergen molecule to the same degree. Der p 1, for example, remains largely unmodified. Furthermore, the investigational HDM allergoid vaccines showed reduced and delayed immune responses when used for immunization of mice.

Environmental Surveillance for Noroviruses in Selected South African Wastewaters 2015-2016: Emergence of the Novel GII.17.

Norovirus (NoV) GII.4 is the predominant genotype associated with gastroenteritis pandemics and new strains emerge every 2-3 years. Between 2008 and 2011, environmental studies in South Africa (SA) reported NoVs in 63% of the sewage-polluted river water samples. The aim of this study was to assess whether wastewater samples could be used for routine surveillance of NoVs, including GII.4 variants. From April 2015 to March 2016, raw sewage and effluent water samples were collected monthly from five wastewater treatment plants in SA. A total of 108 samples were screened for NoV GI and GII using real-time RT-qPCR. Overall 72.2% (78/108) of samples tested positive for NoVs with 4.6% (5/108) GI, 31.5% (34/108) GII and 36.1% (39/108) GI + GII strains being detected. Norovirus concentrations ranged from 1.02 × 102 to 3.41 × 106 genome copies/litre for GI and 5.00 × 103 to 1.31 × 106 genome copies/litre for GII. Sixteen NoV genotypes (GI.2, GI.3, GI.4, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GII.9, GII.10, GII.14, GII.16, GII.17, GII.20, and GII.21) were identified. Norovirus GII.2 and GII.17 co-dominated and the majority of GII.17 strains clustered with the novel Kawasaki 2014 variant. Sewage surveillance facilitated detection of Kawasaki 2014 in SA, which to date has not been detected with surveillance in children with gastroenteritis <5 years of age. Combined surveillance in the clinical setting and environment appears to be a valuable strategy to monitor emergence of NoV strains in countries that lack NoV outbreak surveillance.

Strong and frequent T-cell responses to the minor allergen Phl p 12 in Spanish patients IgE-sensitized to Profilins.

BACKGROUND: Profilins are dominant pan-allergens known to cause cross-sensitization, leading to clinical symptoms such as pollen-food syndrome. This study aimed to determine the T-cell response to Phl p 12 in profilin-sensitized patients, by measuring the prevalence, strength and cross-reactivity to clinically relevant profilins. METHODS: The release of Phl p allergens from pollen was determined by mass spectrometry and immunochemistry. T-cell responses, epitope mapping and cross-reactivity to profilins (Phl p 12, Ole e 2, Bet v 2 and Mal d 4) were measured in vitro using PBMCs from 26 Spanish grass-allergic donors IgE-sensitized to profilin. Cross-reactivity was addressed in vivo using 2 different mouse strains (BALB/c and C3H). RESULTS: Phl p 12 and Phl p 1 are released from pollen simultaneously and in similar amounts. Both T-cell response frequency (17/26 donors) and strength were comparable between Phl p 12 and Phl p 1. T-cell cross-reactivity to other profilins correlated with overall sequence homology, and 2 immunodominant epitope regions of Phl p 12 were identified. Data from mice immunized with Phl p 12 showed that cross-reactivity to Bet v 2 was mediated by conserved epitopes and further influenced by additional genetic factors, likely to be MHC II. CONCLUSION: The strength, prevalence and cross-reactivity of T-cell responses towards Phl p 12 are comparable to the major allergen Phl p 1, which supports the hypothesis that T cells to Phl p 12 can play an important role in development of allergic symptoms, such as those associated with pollen-food syndrome.

The structure of the mite allergen Blo t 1 explains the limited antibody cross-reactivity to Der p 1.

The Blomia tropicalis (Blo t) mite species is considered a storage mite in temperate climate zones and an important source of indoor allergens causing allergic asthma and rhinitis in tropical and subtropical regions. Here, we report the crystal structure of one of the allergens from Blo t, recombinant proBlo t 1 (rproBlo t 1), determined at 2.1 Å resolution. Overall, the fold of rproBlo t 1 is characteristic for the pro-form of cysteine proteases from the C1A class. Structural comparison of experimentally mapped Der f 1/Der p1 IgG epitopes to the same surface patch on Blo t 1, as well as of sequence identity of surface-exposed residues, suggests limited cross-reactivity between these allergens and Blo t 1. This is in agreement with ELISA inhibition results showing that, although cross-reactive human IgE epitopes exist, there are unique IgE epitopes for both Blo t 1 and Der p 1.

Allergen structures and epitopes.

Human type 1 hypersensitivity diseases such as allergic rhinoconjunctivitis are characterized by allergen-specific IgE antibodies produced in allergic individuals after allergen exposure. IgE antibodies bound to receptors on the surface of effector cells trigger an allergic response by interacting with three-dimensional (conformational) epitopes on the allergen surface. Crystal structures are available for complexes of antibody specifically bound to five allergens, from birch pollen, bee venom, cockroach, cow's milk and timothy grass pollen. The details of the antibody-allergen interaction extending all the way to atomic resolution are available from such complexes. In vitro investigations using recombinant monoclonal antibodies and human basophils show that binding affinity is a key to triggering the allergic response. Continued molecular characterization of antibody-allergen interactions is paving the way for the use of recombinant allergens in allergen-specific diagnosis and immunotherapy.

Major venom allergen of yellow jackets, Ves v 5: structural characterization of a pathogenesis-related protein superfamily.

Ves v 5 is one of three major allergens found in yellow-jacket venom: phospholipase A(1) (Ves v 1), hyaluronidase (Ves v 2), and antigen 5 (Ves v 5). Ves v 5 is related by high amino acid sequence identity to pathogenesis-related proteins including proteins from mammals, reptiles, insects, fungi, and plants. The crystal structure of Ves v 5 has been solved and refined to a resolution of 1.9 A. The majority of residues conserved between the pathogenesis-related proteins can be rationalized in terms of hydrogen bonding patterns and hydrophobic interactions defining an alpha-beta-alpha sandwich core structure. A small number of consensus residues are solvent exposed (including two adjacent histidines) and located in an elongated cavity that forms a putative active site. The site has no structural resemblance to previously characterized enzymes. Homologous antigen 5's from a large number of different yellow jackets, hornets, and paper wasps are known and patients show varying extents of cross-reactivity to the related antigen 5's. The structure of Ves v 5 allows a detailed analysis of the epitopes that may participate in antigenic cross-reactivity, findings that are useful for the development of a vaccine for treatment of insect allergy.

Thermally induced changes of lipoate acetyltransferase inner core isolated from the Bacillus stearothermophilus pyruvate dehydrogenase complex.

Incubation at 70 degrees C converted the Bacillus stearothermophilus lipoate acetyltransferase inner core into an unidentified active molecular form, X, yielding an inactive aggregate. The core and X showed similar thermostabilities, but they were different in the recovery of enzyme activity after incubation with 1.2-2.0 M guanidine hydrochloride and its subsequent removal; the core was hardly recovered, but X was well recovered.

Thermally induced disintegration of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase.

Upon heat treatment of the pyruvate dehydrogenase complex from Bacillus stearothermophilus, the most thermostable component is a dihydrolipoamide dehydrogenase (E3c). To understand this stability, the thermal disintegration of E3 dissociated from the complex (E3d) was examined, comparing with that of E3c. Judging from residual activity and inactivation rate, E3d was less thermostable than E3c; E3d and E3c lost half of their original activities upon incubations for 30 min at 79 degrees C and 90 degrees C, respectively. Heat treatment of E3d raised the fluorescence intensities of Trp residue, intrinsic FAD, and extrinsic 8-anilinonaphthalene-1-sulfonate. E3d lost FAD, and inactive E3d polypeptides were aggregated. The sulfonate bound to the aggregate became notably fluorescent. The thermal disintegration of E3d was speculated to be a consecutive reaction that was different from the concurrent disintegration reaction of the complex. Some interactions with other component polypeptides was suggested to improve the thermostability of E3c.

Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by X-ray crystallography.

The three-dimensional structure of recombinant horseradish peroxidase in complex with BHA (benzhydroxamic acid) is the first structure of a peroxidase-substrate complex demonstrating the existence of an aromatic binding pocket. The crystal structure of the peroxidase-substrate complex has been determined to 2.0 A resolution with a crystallographic R-factor of 0.176 (R-free = 0. 192). A well-defined electron density for BHA is observed in the peroxidase active site, with a hydrophobic pocket surrounding the aromatic ring of the substrate. The hydrophobic pocket is provided by residues H42, F68, G69, A140, P141, and F179 and heme C18, C18-methyl, and C20, with the shortest distance (3.7 A) found between heme C18-methyl and BHA C63. Very little structural rearrangement is seen in the heme crevice in response to substrate binding. F68 moves to form a lid on the hydrophobic pocket, and the distal water molecule moves 0.6 A toward the heme iron. The bound BHA molecule forms an extensive hydrogen bonding network with H42, R38, P139, and the distal water molecule 2.6 A above the heme iron. This remarkably good match in hydrogen bond requirements between the catalytic residues of HRPC and BHA makes the extended interaction between BHA and the distal heme crevice of HRPC possible. Indeed, the ability of BHA to bind to peroxidases, which lack a peripheral hydrophobic pocket, suggests that BHA is a general counterpart for the conserved hydrogen bond donors and acceptors of the distal catalytic site. The closest aromatic residue to BHA is F179, which we predict provides an important hydrophobic interaction with more typical peroxidase substrates.

Guanidine hydrochloride-induced changes of the E2 inner core of the Bacillus stearothermophilus pyruvate dehydrogenase complex.

The limited proteolysis of the Bacillus stearothermophilus pyruvate dehydrogenase complex by V8 protease yields its core structure solely composed of lipoate acetyltransferase (E2) fragments. The changes in the core with guanidine hydrochloride (GdnHCl) were biphasic: below 0.8 M (first) and above 1.0 M (second) GdnHCl. The changes in the first phase were slight but significant: decreases in ellipticity and light scattering, and an increase in E2 activity. Insignificant changes in the molecular shape and size of the core were detected on fluorescence spectroscopy, ultracentrifugation, gel filtration, and electron microscopy. On the other hand, the changes in the second phase were drastic; the core was disassembled and denatured.

Potassium Iodide-Induced Changes in Pyruvate Dehydrogenase Complex from Bacillus stearothermophilus.

Treatment with KI and its subsequent removal induced disassembly of Bacillus stearothermophilus pyruvate dehydrogenase complex (PDC) and association of disassembly products, respectively. The disassembly yielded neither completely dissociated components nor aggregate, but did yield a few molecular forms smaller than PDC. Depending on the KI concentration, these changes were of three phases: K-1, below 0.6 M; K-2, 1.0-1.5 M; K-3, above 1.8 M. PDC was disassembled in K-1 to C1 comprising pyruvate decarboxylase and lipoate acetyltransferase mainly and C2 comprising the decarboxylase and dihydrolipoamide dehydrogenase. In K-1, the removal of KI resulted in an apparent reconstitution of PDC. The mixing of C1 with an excess of C2 yielded an assembly larger than PDC and restored enzyme activities, but specific activities were different from those of PDC. In K-2 and K-3 phases, complexes smaller than PDC were yielded from disassembly products, and activities except for that of the acetyltransferase were not restored.

Inhibition of prophenoloxidase-activating enzyme from Bombyx mori by endogenous chymotrypsin inhibitors.

Inhibitory activities for the activation of prophenoloxidase with its activating enzyme were detected in the gel-filtration fractions of hemolymph from Bombyx mori. The fractions with the highest activity contained chymotrypsin inhibitors; CI-13a, 13b, and 13c. They inhibited the activation of prophenoloxidase. The activating enzyme affected a synthetic substrate for trypsin, Boc-Gln-Ala-Arg-MCA: Km = 0.62 mM. CI-13c partially suppressed the peptidase activity.

Serum diatrizoate level during intraoperative cholangiography in patients without choledochal obstructions.

To assess the degree of regurgitation of contrast media during intraoperative cholangiography, serum diatrizoate levels were measured in 15 patients without demonstrable bile duct obstructions. The maximum pressure achieved during the injection was measured in seven cases and ranged from 13 to 39 cm H2O (average 21.7 +/- 9.3 cm H2O). Only two of the 15 patients studied had detected serum diatrizoate (in one patient 7-14 micrograms I/ml and the other patient 119-200 micrograms I/ml) after completion of the injection. The data suggest that regurgitation of contrast media into the blood during intraoperative cholangiography does not depend solely on injection pressure. Moreover the data suggest that in order to prevent adverse reactions to accidental intravenous contrast administration nonionic contrast media should be used in most, if not all, radiographic studies as some contrast media reaches the bloodstream.