Recombinant allergens in the US

Recombinant allergens may increase the safety and efficacy of allergen immunodiagnostics and immunotherapy, and may prove to be excellent tools for the standardization of existing, natural allergens. However, there are several biological and regulatory issues that need to be addressed as these products are moved from bench to bedside. This article discusses some of these issues.     

Proteomics of allergens

The present state of proteomics research is generally outlined and the character of allergenic compounds briefly elucidated. The principles of experimental approaches to isolation, purification, identification and characterization of allergens and to monitoring of their biological activity are described, with emphasis on the most modern methods. Selected examples are given for illustration and important results are summarized in tables.     

Laboratory animal allergens

Allergic sensitivity to laboratory animals can pose a significant occupational hazard to anyone with regular animal contact. Reactions to mice and rats are most common although all furred animals produce allergens that can lead to sensitization and disease. Most of the relevant allergens of laboratory animals have been defined and characterized, which has revealed that these allergens are typically small, acidic glycoproteins and that many of them are members of a superfamily of extracellular proteins called lipocalins. In addition to understanding their molecular characteristics, the identification of these allergens has also made it possible to measure their distribution in laboratory environments and to relate exposure levels to sensitization and symptoms. These studies have shown that the major laboratory animal allergens are carried on small particles that are both capable of remaining airborne for extended periods and penetrating into the lower airways of exposed workers. These advances in the understanding of these important occupational allergens will allow for the development of better methods of diagnosis and avoidance for affected workers and others who may be at risk for future difficulties.     

Recombinant food allergens

Allergenic (glyco)proteins are the elicitors of food allergies and can cause acute severe hypersensitivity reactions. Recombinant food allergens are available in standardised quantity and constant quality. Therefore, they offer new perspectives to overcome current difficulties in the diagnosis, treatment and investigation of food allergies. This review summarises the expression strategies and characteristics of more than 40 recombinant food allergens that have been produced until today. Their IgE-binding properties are compared to those of their natural counterparts, in addition their application as diagnostic tools, the generation of hypoallergenic recombinant isoforms and mutants for therapeutic purposes, the determination of epitopes and cross-reactive structures are described.     

Lipocalins as allergens

The term allergy refers to clinical conditions caused by an inappropriate immune response to innocuous proteins in genetically predisposed persons. Allergens of animal origin are responsible for a significant proportion of allergies. In recent years, it has become evident that practically all respiratory animal allergens characterized at the molecular level belong to the lipocalin family of proteins. The current list comprises the major allergens of horse, cow, dog, mouse, rat and cockroach as well as beta-lactoglobulin of cow's milk. While the molecular structure of all these allergens is known, far less information is available regarding their immunological characteristics. Knowing the way the immune system recognizes these allergens and reacts to them might, however, be the key for discovering the common denominator of the allergenicity of lipocalins. The human body contains numerous endogenous lipocalins, and the immune system has to adapt to their presence. We have proposed that under these conditions the immune response against the lipocalin allergens which are structurally related to endogenous lipocalins might be the pathway to allergy in genetically predisposed persons. The same might well apply also to other allergens with homologous endogenous counterparts.     

Production of recombinant allergens in plants

A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed.     

Polymorphism in myristoylpalmitoylphosphatidylcholine

This study focuses on the mixed-chain lipid myristoylpalmitoylphosphatidylcholine (MPPC) near full hydration. The lipid, synthesized according to the procedure of (Mason et al., 1981a, has a low degree of acyl chain migration. When MPPC is temperature-jumped (T-jumped) from the L alpha phase (T = 38 degrees C) to T = 20 degrees C or below, a subgel phase forms; this formation takes less than 1 h at a temperature below T = 12 degrees C. The subgel remains stable up to T = 29 degrees C. When MPPC is T-jumped from the L alpha phase to T = 24 degrees C or above, a ripple phase forms with coexisting ripple wavelengths of 240 A and 130 A. In contrast, when MPPC is melted from the subgel phase, the ripple phase is characterized by bilayers having a single ripple wavelength of 130 A. In agreement with earlier studies (Stumpel et al., 1983; Serrallach et al., 1984. Structure and thermotropic properties of mixed-chain phosphatidylcholine bilayer membranes. Biochemistry 23:713-720.), no stable gel phase was observed. Instead, an ill-defined low-angle X-ray pattern is initially observed, which gradually transforms into the subgel phase below 20 degrees C, or into the ripple phase above 24 degrees C. In the wide-angle X-ray diffraction, a single peak is observed, similar to the ripple phase wide-angle pattern, that either persists above 24 degrees C or transforms into a multi-peaked subgel wide-angle pattern below 20 degrees C. The absence of a gel phase can be understood phenomenologically as the relative dominance of the subgel phase in mixed-chain PCs compared to same-chain PCs. The subgel structure and molecular interactions responsible for this comparative behavior are interesting open issues.     

Polymorphism in Pomatoceros

Allelic variation at 13 sites for the intertidal Pomatoceros lamarckii (Quatrefages) and at six sites for the sublittoral P. triqueter (L.) in North-West Europe, when examined at 19 metabolic enzyme loci, showed significant differences between sites in the frequencies of the various enzyme morphs. There was no readily interpretable geographic or environmental trend. The Hardy-Weinberg equilibrium was maintained at almost all loci and all sites. The genetic distance between populations was very small in comparison with the difference between the two species.
Colour morphs of P. triqueter showed a clear latitudinal cline with blue and brown morphs predominating at more northerly latitudes, and grey, orange and red morphs at more southerly latitudes.
Interlocality variations in allele frequency, though statistically significant, were never sufficient to alter the order of frequency of the two or three commonest alleles, so that the frequency of all but rare alleles showed a consensus throughout the ranges studied for each species. Accounting for local genetic patchiness in gene frequencies in species with pelagic larvae raises problems if the alleles are selectively neutral; accounting for wide geographic uniformity raises problems if alleles are selected for or against by environmental factors.     

Isolation of food allergens

The identification of food allergens is a priority in the management of food allergy, because of the need to obtain standardized extracts and pure allergens for diagnosis and therapy. It is thus important to develop methods for purification of allergenic molecules in order to study their biological and immunological characteristics. Protocols for protein extraction from foods and for allergen purification are reviewed in this paper. We report published methods for extraction of allergens from either animal and vegetable foods and detailed purification methodologies including ion-exchange, gel filtration and reversed-phase chromatography of well known allergens.     

The Blomia tropicalis allergens

Blomia tropicalis allergens are the most important mite allergens in tropical regions. Most of them only have 30-40% sequence identity with their Dermatophagoides counterparts and they share low IgE cross reactivity and exhibit different immunobiology. Unlike the pyroglyphid counterparts, Blo t 5 is the major allergen whereas Blo t 1 only has modest allergenicity.     

Quantitation of latex allergens

Minimizing allergen concentration in latex goods to prevent sensitization to natural rubber latex (NRL) and thereby the development of clinical allergy is acknowledged as of mutual interest for rubber manufacturers and regulatory health authorities. However, measuring total protein, the principal currently available method, cannot be deemed a satisfactory regulatory measure to control allergen content. Specific methods based on human IgE-containing reagents, such as radioallergosorbent test (RAST) inhibition, have been available in certain laboratories for demonstrating NRL allergens in rubber products but the methods lack standardization. Currently, one commercial test has become available for measuring individual NRL allergens by capture ELISA-based assays using monoclonal antibodies and purified or recombinant allergens. Such methods are specific, they can be properly standardized, and they are of sufficient sensitivity and reproducibility. Results from medical gloves collected in two national market surveys in Finland in 1995 and 1999, respectively, show that Hev b 6.02 and Hev b 5, the two major allergens for NRL-allergic adults, are the most abundant allergens regularly detectable in high- and moderate-allergen gloves. In addition, Hev b 3 and Hev b 1, the two major allergens for children with spina bifida, are also commonly found. In general, when the sum of the four allergens exceeded 1 microg/g, most NRL-allergic patients showed positive skin prick test reactions against them. Using these new methods assessment of threshold levels that could in due course become guidelines for the rubber industry and regulatory health authorities is becoming possible. Eventually, this progress is expected to lead to a declining incidence of latex allergy.     

Inhalant allergens in Palm Springs,California

Although Palm Springs, California, offers a pleasant desert climate that is often advantageous in treatment of respiratory allergies, physicians should be made aware in referring their patients that definite pollen factors must be taken into consideration in recommending a visit to Palm Springs, and should try to determine the compatibility of their patient's allergic sensitivity pattern and the particular seasonal incidence of pollen concentration in Palm Springs. It is believed that the most important pollens there are those of Bermuda grass, olive tree, mesquite, dicoria, false ragweed, scales, and hymenoclea salsola. A favorable climate alone does not permit a patient to disregard good allergic management.     

Polymorphism in polydnavirus genomes

Polymorphisms were readily detected in polydnavirus DNA extracted from several different species belonging to two different families of parasitic hymenoptera. Heterogeneity was observed as differences in electrophoretic profiles of genome segments, differences in the number of cross-hybridizing genome segments, and restriction fragment length polymorphisms; polymorphism was also detected at the level of an individual genome segment. Some implications drawn from these observations are discussed.     

Polymorphism in Human Casein

MANY human proteins exist in more than one genetically determined variety¹, but no polymorphism has been reported in human milk proteins, although some components have been sufficiently characterized from the biochemical standpoint^2,3 and extensive polymorphism has been detected in cow's milk. We therefore decided to investigate the possible occurrence of polymorphic variants of human casein, since electrophoresis has now given a clear picture of bovine casein genetics, which can be used as a model for study of the situation in man^4,5. Such variants would obviously be more easily used as genetic markers at the population than at the family level. Moreover, data reported by Grosclaude et al.⁶ suggest the existence of a supergene controlling bovine α_s1, β and k-caseins. For this reason, the possibility of a similar genetic control of human polymorphism (if any) was also tested.