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Hymenoptera venoms used to produce allergen extracts
Ann Allergy Asthma Immunol 118 (2017) 649e654
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How Allergen Extracts Are MadedFrom Source Materials to Allergen Extracts
Hymenoptera venoms used to produce allergen extracts Greg Plunkett, PhD *; Robert S. Jacobson, MS y; David B.K. Golden, MD z * ALK-Abello y z
Inc, Round Rock, Texas Greer Laboratories, Lenoir, North Carolina Johns Hopkins University, Baltimore, Maryland
A R T I C L E
I N F O
Article history: Received for publication April 2, 2016. Received in revised form May 20, 2016. Accepted for publication May 31, 2016.
A B S T R A C T
Objective: To review the methods and materials used for collection, purification, commercial production, and clinical application of Hymenoptera venoms. Data Sources: Most of the sources for this review are the experience and expertise of the authors. Published reports and review articles on Hymenoptera venom collection and production were identified through database searches (PubMed). Study Selections: Studies describing the methods for Hymenoptera venom collection and production were selected for review. Results: Meticulous methods for identification and collection of the insects are required. Collection and purification of the venoms from the insects are based on validated methods and result in a commercial extract that is standardized for the major allergenic proteins required for accurate diagnosis and safe and effective treatment of patients allergic to insect sting. The steps required for mixing, purifying, testing, and standardizing the products are described. Conclusion: Hymenoptera venom extracts were developed using many new methods for the collection, purification, and commercial production of the unique materials required for this product. Clinical applications for diagnosis and treatment are affected by the integrity and stability of the allergens after processing and purification. Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Important Hymenoptera Insects Bees, Wasps, Yellow Jackets, and Hornets The flying insects of the order Hymenoptera (bees and wasps) that are responsible for anaphylaxis attributable to venom hypersensitivity are largely similar between the United States and Europe. Honeybees (Apis mellifera), native to Europe, were introduced into the United States nearly 400 years ago, becoming established throughout the country, and are valued on both continents for pollinating crops, honey, and wax.1 Stinging risk occurs throughout the year from their perennial colonies but minimally during colder months. Some strains are more defensive than others. The introduced Africanized or “killer” bees, which migrated from Brazil and were intentionally brought from Africa, have reached the Southern and Southwestern United States, where their extremely
Reprints: David B.K. Golden, MD, Johns Hopkins University, 7939 Honeygo Blvd, Room 219, Baltimore, MD 21236; E-mail: [email protected]. Disclosures: Dr Golden reported receiving honoraria and serving on the speaker’s bureau for Genentech and Greer Laboratories, working as a consultant for Greer Laboratories, and receiving a research grant and working on a clinical trial for Genentech.
defensive behavior poses a serious risk. Honeybees are unique among important stinging species because their barbed stings readily detach and remain in the skin, although yellow jackets are sometimes unable to remove their stings.2 The genera Vespa, Dolichovespula, and Vespula (hornets and yellow jackets, with Vespula often simply called wasps or true wasps in Europe) are important in Europe and the United States, although a member of the first genusdVespa crabro or European hornetdwas introduced into the United States around 1840. Although V crabro is widespread in Europe, 2 other hornets (one native, another introduced) have limited distributions. All 3 genera make paper nests composed of several combs usually covered by an envelope, with colonies typically lasting only one season, and will defend their nests aggressively if disturbed. Vespula and Dolichovespula are native and widespread in the United States and Europe, with the former genus building large colonies, often in the ground or in structures, and the latter usually building exposed nests on tree branches or under eaves. Several species of yellow jackets are scavengers with large colonies; a prime example is Vespula germanica, native to Europe (which usually includes Paravespula) and established in much of the United States. Scavenging species are attracted to human food, making
http://dx.doi.org/10.1016/j.anai.2016.05.027 1081-1206/Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
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unintentional contact or ingestion of the insect a risk. European species cross-react well with most American species but less so with the American Vespula squamosa.3 Dolichovespula includes several species whose American members, particularly the large and widespread baldfaced species (Dolichovespula maculata) with its large gray nests, are sometimes called hornets. The closely related aerial yellow jacket or yellow hornet (Dolichovespula arenaria) is a smaller member of this genus. They both have sibling species in Europe (where they are sometimes called tree wasps) with which they can be expected to crossreact.4 Paper wasps (Polistes species) are rather slender insects that build single exposed combs (ie, no covering envelope). They are found through most of the contiguous United States and much of Europe, particularly warmer regions. Mass stinging is unlikely because of their small colonies. American and European species are partially cross-reactive, although one of the European species (Polistes dominula) has become established in the United States.5,6 Fire Ants and Other Ants Five species of fire ants (Solenopsis)d3 native and 2 importeddare in the United States; no species are found in Europe. Two imported species (Solenopsis richteri, the black species, and especially Solenopsis invicta, the red species) are introductions from South America that present important stinging hazards throughout much of the southeastern United States and occasionally westward, particularly when their soil mounds are disturbed.7 Additional medically important ants in the United States include harvester ants (Pogonomyrmex) distributed mostly in the Western United States, with one species reaching the Southeast and Asian needle ants (Brachyponera chinensis) currently restricted to the Southeast. Extracts are available only for imported fire ants; their venoms are not cross-reactive with the other genera.8,9 Collection of Stinging Insects Obtaining stinging insects for venom production presents several challenges. A collector must obtain sufficient leads from property owners or managers who can report active colonies, and such reports must be screened to avoid wasting time with unwanted species of insects (eg, honeybees) and to be sure the nest is accessible or at least the insects can be trapped. Before collecting, the collector must be certain no pesticides or chemicals have been or are used (although carbon dioxide is permitted). Insects must be kept alive until frozen for storage. Beekeepers and pest control operators often receive gratuitous calls, whereas others must advertise free insect removal. There are many ways of collecting, and collectors have their own trade secrets. Techniques range from collecting entire nests (which are frozen intact and dissected later while maintained frozen), trapping insects, and using a special vacuum cleaner designed to prevent killing and desiccating the insects; in the last 2 cases, prompt freezing is critical. Species are kept segregated, so the collector must bag the insects from each nest separately. A chesttype freezer is used for storage to avoid loss of cold when opened. Because only female insects sting, collectors often time their collecting for maximum female populations. Normally, this is late summer or autumn for vespines, but this may be past the peak for Polistes. Collected insects must remain frozen until just before dissection. On receipt at the manufacturer, insects are examined. This entails verification that the insects are still frozen (usually by the presence of dry ice in the shipping container), confirmation of the species, certification that no pesticides or other chemicals were used, confirmation there are no odors atypical of the species (ie, attributable to chemicals or spoilage), random sampling to assess
female percentage, and subtraction of weight due to debris and water ice. Finally, female insects are randomly sampled, thawed, and dissected to ensure the venom sacs are light yellow or whitish in color (depending on the species) as evidence that insects were frozen alive. The usable weight of female insects is calculated for both payment and raw material inventory purposes. If criteria are not met, insects are rejected. Honeybee venom (HBV) is collected by electrical stimulation, as discussed below. Because fire ant extracts are whole body, their colonies are scooped into containers, and then water is used to float the ants from the soil, which is a natural behavior for fire ants. The floating ants are scooped and stored frozen.
Venom Processing Hymenoptera Venom Sources: History Testing and treatment for allergy to flying insect stings began nearly 100 years ago when in 1925 Braun10 treated a HBV allergic patient with an extract from bee posterior bodies. From 1930 to 1954, studies reported that treatment with HBV and whole-body extracts was equally effective.11 In the 1960s, Benton and colleagues12 from Penn State University designed an improved apparatus for collecting pure HBV using electrostimulation.12 Collection of vespid venom was described by Loveless and Fackler13 in 1956. As these purified venom sources improved and venom became more readily available, clinical trials during the 1970s and 1980s established that whole-body extracts were not effective for testing or treatment.14e16 Venoms were found to be better than whole-body extracts, resulting in commercial venom products becoming available from Hollister-Stier (now Jubilant Hollister Stier) and Pharmacia Labs (now ALK-Abello Inc) in 1979 to 1980 (Table 1).17e19 Table 1 History of Venom Extracts Used for Immunotherapy Year
Event
1925
Braun successfully tested and treated honeybee allergic patient with extract made from posterior 1/800 of HB bodies (WBE) Benson and Semenov reported on a honeybee allergic beekeeper who had positive skin test results to HBV, HB sting apparatuses, and HB bodies w/o sting apparatuses Markovic and Molnar described electrical stimulation for the collection of honeybee Loveless and Fackler reported on successful diagnostic and therapeutic use of venom sac extracts Benton and colleagues reported an improved method for electrical collection of honeybee venom Bermon and Brown reported positive whole-body extract skin test results in 37% of inmates without positive histories (n ¼ 200) and in 40% of inmates who had never been stung (n ¼ 15) Schwartz reported no significant difference between skin test reactivity to whole-body extract in sting allergic patients and control patients Torsney reported 8 insect sting allergy fatalities in patients treated with whole-body extract Small samples of Hymenoptera venoms to Johns Hopkins University Ramp up of venom source materials from Penn State University (Dr Benton, honeybee venom) and Rockefeller University (Dr King, vespids) Sobotka et al reported that Hymenoptera venoms induced histamine release in basophil leukocytes from insect allergic patients Hopkins asked to treat a beekeeper’s 4-year-old son, whole-body extract failure, whose sister had previously died of a honeybee sting Hunt et al reported on the diagnostic specificity of venom skin tests in 30 patients Favorable results from the Johns Hopkins clinical trials Hunt reported blinded placebo-controlled trial with 60 patients US Food and Drug Administration grants licenses to Hollister-Stier, Pharmacia Labs (ALK purchased in 1987)
1930
1954 1956 1963 1965
1965
1973 1973 1970s
1974 1974
1976 1976 1978 1979e1980
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How Are Hymenoptera Venoms Obtained? Honeybee venom The method for collection of HBV using electrostimulation was perfected by Benton and colleaques12 from Penn State University, and Dr Benton, along with Miles Guralnick, formed Vespa Labs in Spring Mills, PA, to supply insect venoms for immunotherapy. Electrostimulation involves exposing a bee to a small electric current from a wire grid connected to a carlike battery, causing the bee to automatically sting through a plastic wrap, leaving a small bead of venom (Fig 1). The battery and grid apparatus is placed near a commercial hive. As the bees explore the device and land on the grid, the stinging impulse also releases attack pheromones and soon is covered in hundreds of bees. The plastic wrap covering the grid does not impede the barbed stinger because it retracts, so the bees are not harmed. Each sting through the plastic deposits approximately 50 mL of pure venom so after a short time several grams of pure venom can be collected. The purified HBV source material is dried on the plastic wrap then scraped off. More recently, the plastic wrap has been replaced by a latex-free membrane that is safer for the bees. Vespid and wasp venom In 1956, Loveless and Fackler13 reported the successful use of venom sac extracts for immunotherapy. Because electrostimulation of hives was not practical for these insects, venom sac isolation was the preferred path for collecting venom. The scientists at Vespa Labs along with scientists at Hollister-Stier Labs, Spokane, WA, developed for commercial purposes the technique described by King et al,20 who described removing venom sacs and characterizing the components in the venoms (Fig 2). The venoms collected by these methods in the middle 1970s were used for immunotherapy clinical trials at Johns Hopkins University and Mayo Clinic that were the basis of US Food and Drug Administration (FDA) approval of the commercial venom products.17,21,22 Today there is an extensive network of private collectors and beekeepers that supplies the purified HBV and insects for further processing at Vespa Labs and Jubilant Hollister Stier. This network is still the sole supplier of venom source material used for FDAapproved immunotherapy. Vespa Labs is now part of the global allergy extract company ALK (Hørsholm, Denmark), and ALK Inc (Round Rock, Texas) distributes Hymenoptera venom products in the United States. The vespid and paper wasp insect collectors are responsible for finding nests for at least 6 species of yellow jacket and 4 to 5 species of Polistes wasps. The insects are rendered harmless using carbon dioxide and then delivered to the sacpulling teams frozen.
Figure 1. Collection of honey bee venom by electrostimulation. Reproduced with permission of Vespa Labs (ALK-Abello Inc).
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Imported fire ant source material Venom from Solenopsis richteri and invicta fire ants is not available for immunotherapy. Manufacturers supply weight/volume whole-body extracts that are nonstandardized (no standard of potency). Insects are collected from nests and then freeze-dried before extraction. Analysis of these extracts has revealed sufficient venom proteins for use in immunotherapy diagnosis and treatment.23e25 Batch to batch variability and stability are not known for fire ant extracts in the United States. Manufacture of Insect Extracts FDA approval of venom products The FDA approved stinging insect venom products in 1979 to 1980, subsequently removing the corresponding whole-body extracts from the market. Six venom product types are approved in the United States. Honeybee, domestic (Apis mellifera), yellow jacket (Vespula species), yellow hornet (Dolichovespula arenaria), white-faced hornet (Dolichovespula maculata), paper wasps (Polistes species), and a mixture of yellow jacket, yellow hornet, and white-faced hornet (mixed vespids). There are various configurations, of single use or multidose vials, and they are formulated to be 100 mg/mL when reconstituted (300 mg/mL for mixed vespids). In Europe, there are some alternate venom product types, such as dialyzed venom to remove low-molecular-weight nonallergenic material and vaccines of venom adsorbed to alum. Below is a discussion of US manufactured products only. Manufacturing Steps Species mixtures Yellow jacket and paper wasp are formulated with a mixture of species within the genus and include the most common species that cause allergic reactions. Yellow jacket venom contains Vespula species of Vespula vulgaris, V germanica, Vespula maculifrons, Vespula pensylvanica, Vespula flavopilosa, and Vespula squamosa. The ALK venom package inserts specify that these species are present in their yellow jacket venom extract. These are the most commonly used species in the Jubilant Hollister Stier venoms, although their package insert allows for other Vespula species. From the ALK package insert, the Polistes paper wasp products are mixtures of Polistes annularis, Polistes apachus, Polistes exclamans, Polistes fuscatus, and Polistes metricus. Jubilant Hollister Stier paper wasp products have contained mixtures of P metricus, P exclamans, and P fuscatus, recently removing P annularis. There is extensive (but not absolute) cross-reactivity among the venoms of the Vespula genus and also among those of the Polistes genus. The species chosen for the commercial mixtures are the ones that most commonly sting people. Venom of V squamosa was added to the yellow jacket mixture early (1980) because it possesses some unique allergenic activity and is less cross-reactive with other Vespula species. European venom extracts can also contain mixtures of Vespula and Polistes species. Some contain the same mixtures as used in the United States, whereas others have fewer species, such as V germanica and V vulgaris. There has also been the addition in some markets of P dominula, also called the Mediterranean paper wasp, as a single species extract. This species, even though its presence is increasing in the United States, has not been introduced in FDAapproved venoms. Preparation of intermediate venom products HBV intermediate product is pure dried venom collected from electrostimulation. The vespid and wasp venoms are extracts of venom sacs that are individually pulled from insects collected and frozen alive. Each sac is pulled from the insect and placed in an extraction fluid containing a small amount of an acetic acid/ b-alanine buffer. Hundreds of sacs are extracted, and the mixture is
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Figure 2. Collection of Vespula and Polistes venom. Reproduced with permission of Vespa Labs (ALK-Abello Inc).
blended, filtered, and freeze-dried into bulk vials. Yellow jacket species and wasp species are mixed into the final bulk vials. All the venom bulk vials are stable for at least 30 months according to one manufacturer. Intermediate testing Initial estimates of venom potency are made by measuring the total protein content (even though this is not a direct measure of allergenic potency). The bulk intermediate vials are tested for total protein by the FDA-approved Lowry method (Laboratory of Immunobiology of the FDA, Methods of the Allergenics Products Testing Laboratory, Docket No. 94N0012 October 1993). During the development of commercial products in the late 1970s, 2 enzymes were discovered to be major allergen proteins, phospholipase and hyaluronidase. Enzyme activity identified the presence of these 2 proteins and therefore became part of the potency testing. Even though the proteins are not cross-reactive between honeybees and vespids, the same enzyme assays were performed for all venom bulk vials. Each batch of venom bulk vials has minimum enzyme level requirements. Final commercial venom products The final venom products are reconstituted to 100 mg/mL based on the Lowry total protein content of the bulk vials. For mixed vespid venom, yellow jacket, yellow hornet, and white-faced hornet intermediate vials are combined and formulated to 300 mg/mL. The reconstitution solution contains mannitol as a freeze-drying agent and helps stabilize the venom protein. ALK adds human serum albumin along with the mannitol as an additional freezedrying stabilizing agent and is the primary difference in the venom products in the United States. The vials are commercially available as freeze-dried single or multidose vials at 100 mg/mL (300 mg/mL for mixed vespid venom) when reconstituted. Standardization Venom source material is analyzed for the allergen enzyme activity, and native polyacrylamide electrophoresis is performed. Each venom has a unique pattern. One of the major allergens in vespid venom is antigen 5 or vespid 5. The enzyme activity is not known, and no immunoassay was developed, so this important allergen is not measured in US extracts. It is a prominent protein band in electrophoresis. Final commercial vials are tested for total protein, phospholipase, and hyaluronidase and must meet certain limits as specified in the manufacturers’ FDA licenses. IgE binding is not determined for FDA-licensed products; however, potency by IgE binding is determined in European venoms in addition to the enzyme assays. Sterility and safety testing is also performed before release for sale. Expiration dating of the freeze-dried powder is determined by stability studies performed by each manufacturer. Expiration studies of the reconstituted vials using human serum
albumin with phenol diluent have also been performed with recommendations stated in the package insert. Composition of venoms Since the introduction of venom products for immunotherapy in the late 1970s, much as been learned about their makeup. Lowmolecular-weight components are present, many of which are active pharmacologic agents composed of vasoactive amines and peptides. These components are not removed from the venom products and most likely are responsible for limiting intradermal skin test diagnosis to concentration not exceeding 1 mg/mL. Many additional allergen proteins have been described in addition to the phospholipase and hyaluronidase enzyme allergens. In the vespids, group 5, such as Ves v 5, has been found to be an important allergen, but the enzymatic function is not known. Purified Ves v 5 was used as an electrophoresis marker to demonstrate the presence of this protein in US venom products. Implications for Clinical Practice Hymenoptera insect extracts are necessary for accurate diagnosis and preventive treatment of insect sting allergy. The process from source materials to allergenic extracts has considerable effect on the clinical applications. The selection and collection of insect species are critical to all subsequent characterization of the commercial product for diagnosis and treatment. The purity and allergenic activity of the collected materials must be optimal and consistent. There are numerous species of Vespula and Polistes, which vary in different regions of the United States. For any nonentomologist, the accurate identification of these species is difficult and unreliable.26 A trained and skilled network of collectors and vigilance in review of the materials that arrive at the laboratory are required to ensure the allergenic specificity, activity, and purity of the products. There are only 2 allergen laboratories in the United States that supply Hymenoptera extracts for clinical use, and their products have been described above. The clinician must always be aware that there are species that are not represented in the commercial products and may be antigenically distinct from the more common species. This could lead to negative diagnostic test results in a patient who is allergic to a specific species of vespid.27 If an extract varies in the type or quantity of certain species, then the ability to detect the allergy on testing or to protect with venom immunotherapy could be reduced. The preparation of the extract from the raw materials is critical in determining the reliability of testing and treatment. At each of the steps described above for intermediate products and standardization, any change or deficiency could greatly affect the clinical outcome. At risk is the integrity and stability of each allergenic protein in each of the venoms being prepared. Loss of activity could
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result from susceptibility to reagents used in the processing, mechanical degradation, enzymatic degradation, molecular instability, and binding characteristics. For example, the activity of the major vespid allergen Ves v 5 is not optimal for diagnosis, and the detection of yellow jacket allergy can be improved by spiking the extract with this protein.28 The procedures for standardization can also have an effect on clinical practice. As described above, the Hymenoptera venoms are standardized by the FDA based on measurement of phospholipase (HBV) or hyaluronidase (vespid venoms), although there are specifications for both enzymes in all Hymenoptera venom extracts. Without quantification of the major vespid allergen, Ves v 5, it is difficult to be certain of the equivalence of products from different batches or different laboratories. Notably, despite numerous occasions that required changing a patient’s treatment to a different product, there has been no report of any adverse outcomes. Diagnostic applications of Hymenoptera extracts are for in vivo and in vitro testing methods. Skin tests use the commercial extract at concentrations found to provide optimal sensitivity and specificity (up to 1.0 mg/mL for intradermal testing). Higher concentrations give improved sensitivity but can also cause false-positive results because of local irritative effects.29 Greater diagnostic accuracy is achieved with a concentration of 10 mg/mL if the venom is dialyzed to reduce these irritative effects.30 Dialyzed venoms are not commercially available in the United States but are common in Europe.31 Commercial reagents for skin prick testing are not available because skin prick testing with venoms is much less sensitive than intradermal testing and is often omitted in favor of intradermal tests starting with very low concentrations (0.001 mg/mL). When skin prick tests are performed as a preliminary test, the concentrations used range from 1 to 100 mg/mL.32,33 Diagnostic testing for fire ant allergy uses the commercial whole-body extracts. Although venoms might be superior, they are not commercially available. Laboratory methods that use Hymenoptera extracts are primarily enzyme-linked immunoassays for allergen specific IgE antibodies. In these assays, the accuracy may be affected not only by the activity of the many allergens in each venom but also by their binding characteristics for the solid phase used in the assay.34 Nevertheless, neither skin tests nor in vitro tests can detect all cases of insect sting allergy. Another in vitro method that shows promise is the basophil activation test, which uses the same venom extracts to elicit basophil activation (not necessarily IgE dependent).35e37 One of the dilemmas of diagnostic specificity for Hymenoptera venoms is the cross-reactivity between genera and species. Radioallergosorbent assay inhibition tests have been used to determine whether patients with dual yellow jacket and Polistes wasp venom sensitivity can be treated with just 1 of the venoms.38 The use of recombinant venom allergens for in vitro measurement of IgE can identify the primary allergy in patients with dual positive test results for honeybee and yellow jacket venoms.39e41 However, for reliable accuracy, these tests would have to include multiple recombinant allergens for each venom. Therapeutic application of Hymenoptera products prevents severe allergic reactions to stings. Early clinical trials revealed the safety and efficacy of venom immunotherapy.17 Clinical experience in thousands (if not millions) of patients during the past 40 years has confirmed that venom immunotherapy prevents systemic reactions in 80% to 98% of affected patients.29 The safety of venom immunotherapy has been comparable to other standard types of allergen immunotherapy (eg, grass or cat). The choice of which venoms to use for treatment can be facilitated with in vitro tests to clarify the cross-reactivity of the venoms, as described above. Immunotherapy for fire ant sting allergy is performed using wholebody extracts, which contain sufficient venom allergens to provide significant clinical protection from anaphylaxis.23
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Conclusions The development of specific materials to be used for diagnosis and treatment of Hymenoptera venom allergy posed unique challenges in the collection and processing of the allergens. Insects of known species must be obtained by knowledgeable collectors and must be handled from the outset in a way that preserves the integrity and activity of the venom. Identification of the relevant species requires expertise, and locating and collecting them require understanding of their nesting and foraging habits. In the case of honeybee, the method of venom collection is unique and requires live insects. Fire ant extracts are prepared from whole insect bodies without separation of the venom. The crude venoms must be assessed qualitatively and quantitatively for purity and allergenic activity. The intermediate venom product must be purified and stabilized to eliminate most small molecules that can increase adverse effects and to avoid enzymatic degradation of the allergens. The choice of specific species and their relative quantities for the yellow jacket and Polistes products must be consistent. Finally, venoms must be standardized by quantitation of specific allergenic proteins. The use of commercial Hymenoptera venoms for diagnosis and treatment of insect sting allergy is both improved and hampered by the methods of production. Hymenoptera venoms were the first allergenic extracts to be standardized by the FDA. On the other hand, the vespid venoms are not routinely standardized for the major allergen Ves v 5. Recent studies found that some allergens may be underrepresented in the commercial venoms, which might reduce the diagnostic accuracy and therapeutic efficacy of the venoms.28,42 In the future, the use of recombinant venom allergens to supplement the commercial venoms might address this problem (and some of those below as well). Another potential improvement in the commercial venom products would be more efficient purification (eg, dialysis) to reduce the irritative components that limit the concentration used for skin tests and contribute to local reactions to immunotherapy.30,31 Purified venoms are available in Europe but not in the United States. Another possible improvement would be the availability of additional species, either individually or within a species mix (eg, Vespula, Polistes). There are patients with specific sensitivity to bumble bees, Mediterranean wasps (P dominula), or other unique species of wasp or yellow jacket that are not currently represented in the commercial products.27 It is believed that fire ant venom would be superior to the whole-body extract for testing and immunotherapy. It would useful if fire ant venom could be made available in a commercial product. References [1] Turpin T. Honeybees Not Native to North America. On Six Legs; Purdue Extension. West Lafayette, IN. https://http://www.agriculture.purdue.edu/agcomm/ newscolumns/archives/OSL/1999/November/111199OSL.html. Accessed November 28, 2015. [2] Greene A, Breisch NL, Golden DB, Kelly D, Douglass LW. Sting embedment and avulsion in yellowjackets (Hymenoptera:Vespidae): a functional equivalent to autotomy. Am Entomol. 2012;58:50e57. [3] Hoffman DR. Allergens in Hymenoptera venom, XXV: the amino acid sequence of antigen 5 molecules: the structural basis of antigenic crossreactivity. J Allergy Clin Immunol. 1993;92:707e716. [4] Ross KG, Matthews RW. The Social Biology of Wasps. Ithaca, NY: Cornell University Press; 1991. [5] Hoffman DR, Jacobson R, Blanca M. Allergy to venom of Polistes dominulus, a paper wasp introduced from Europe [abstract]. J Allergy Clin Immunol. 1990; 85:211. [6] Severino MG, Campi P, Macchia D, et al. European Polistes venom allergy. Allergy. 2006;61:860e863. [7] Kemp SF, deShazo RD, Moffitt JE, et al. Expanding habitat of the imported fire ant: a public health concern. J Allergy Clin Immunol. 2000;105:683e691. [8] Hoffman DR. Allergens in Hymenoptera venom, XXIV: the amino acids sequences of imported fire ant venom allergens Sol i II, Sol i III, and Sol i IV. J Allergy Clin Immunol. 1993;91:71e78.
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[9] Kim S-S, Park H-S, Kim H-Y, Lee S-K, Nahm D-H. Anaphylaxis caused by the new ant, Pachycondyla chinensis: demonstration of specific IgE and IgEbinding components. J Allergy Clin Immunol. 2001;107:1095e1099. [10] Braun LIB. Notes on desensitization of a patient hypersensitive to bee stings. S Afr Med Rec. 1925;23:408e409. [11] Benson R, Semenov H. Allergy in its relation to bee sting. J Allergy Clin Immunol. 1930;1:105e111. [12] Benton AW, Morse RA, Stewart JD. Venom collection from honeybees. Science. 1963;142:228e230. [13] Loveless MH, Fackler WR. Wasp venom allergy and immunity. Ann Allergy. 1956;14:347e366. [14] Schwartz HJ. Skin sensitivity in insect allergy. JAMA. 1965;194:703e705. [15] Torsney PJ. Treatment failure: insect desensitization: case reports of fatalities. J Allergy Clin Immunol. 1973;52:303e306. [16] Bermon HS, Brown H. Studies on Hymenoptera, I: skin reactions of normal persons to honeybee (Apis mellifera) extract. J Allergy. 1965;36:315e320. [17] Hunt KJ, Valentine MD, Sobotka AK, Benton AW, Amodio FJ, Lichtenstein LM. A controlled trial of immunotherapy in insect hypersensitivity. N Engl J Med. 1978;299:157e161. [18] Lichtenstein LM. Insect allergy: the state of the art. J Allergy Clin Immunol. 1979;64:5e12. [19] Muller U, Thurnheer U, Patrizzi R, Spiess J, Hoigne R. Immunotherapy in bee sting hypersensitivity: bee venom versus wholebody extract. Allergy. 1979; 34:369e378. [20] King T, Joslyn A, Kochoumian L. Antigenic cross-reactivity of venom proteins from hornets, wasps and yellow jackets. J Allergy Clin Immunol. 1985;75: 621e628. [21] Hunt KJ, Valentine MD, Sobotka AK, Lichtenstein LM. Diagnosis of allergy to stinging insects by skin testing with Hymenoptera venoms. Ann Intern Med. 1976;85:56e59. [22] Yunginger JW, Paull BR, Jones RT, Santrach PJ. Rush venom immunotherapy program for honeybee sting sensitivity. J Allergy Clin Immunol. 1979;63: 340e347. [23] Freeman TM, Hyghlander R, Ortiz A, Martin ME. Imported fire ant immunotherapy: effectiveness of whole body extracts. J Allergy Clin Immunol. 1992; 90:210e215. [24] Hoffman DR, Jacobson RS, Schmidt M, Smith AM. Allergens in Hymenoptera venoms. XXIII. Venom content of imported fire ant whole body extracts. Ann Allergy. 1991;66:29e31. [25] Strom GB, Boswell MD, Jacobs RL. In vivo and in vitro comparison of fire ant venom and fire ant whole body extract. J Allergy Clin Immunol. 1983;72: 46e53. [26] Baker TW, Forester JP, Johnson ML, Stolfi A, Stahl MC. The HIT study: Hymenoptera identification test e how accurate are people at identifying flying insects? Ann Allergy Asthma Immunol. 2014;113:267e270.
[27] Tracy J, Olsen J, Carlson J. A “difficult” insect allergy patient: reliable history of a sting, but all testing negative. Curr Opin Allergy Clin Immunol. 2015;15:358e363. [28] Vos B, Kohler J, Muller S, Stretz E, Rueff F, Jacob T. Spiking venom with rVes v 5 improves sensitivity of IgE detection in patients with allergy to Vespula venom. J Allergy Clin Immunol. 2013;131:1225e1227. [29] Golden DBK. Advances in diagnosis and management of insect sting allergy. Ann Allergy Asthma Immunol. 2013;111:84e89. [30] Golden DBK, Kelly D, Hamilton RG, Wang NY, Kagey-Sobotka A. Dialyzed venom skin tests for identifying yellow jacket-allergic patients not detected using standard venom. Ann Allergy Asthma Immunol. 2009;102:47e50. [31] Bilo MB, Severino M, Cilia M, et al. The VISYT trial: venom immunotherapy safety and tolerability with purified vs nonpurified extracts. Ann Allergy Asthma Immunol. 2009;103:57e61. [32] Bilo BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JNG. EAACI. Diagnosis of Hymenoptera venom allergy. Allergy. 2005;60:1339e1349. [33] Zeleznick LD, Hunt KJ, Sobotka AK, Valentine MD, Tippett LO, Lichtenstein LM. Diagnosis of Hymenoptera hypersensitivity by skin testing with Hymenoptera venoms. J Allergy Clin Immunol. 1977;59:2e9. [34] Hamilton RG. Clinical laboratory assessment of immediate-type hypersensitivity. J Allergy Clin Immunol. 2010;125:S284eS296. [35] Eberlein-Konig B, Rakoski J, Behrendt H, Ring J. Use of CD63 expression as marker of in vitro basophil activation in identifying the culprit in insect venom allergy. J Investig Allergol Clin Immunol. 2004;14:10e16. [36] Ebo DG, Bridts CH, Hagendorens MM, deClerck LS, Stevens WJ. The basophil activation test in the diagnosis and follow-up of Hymenoptera venom allergy: an alternative point of view. J Investig Allergol Clin Immunol. 2008;18:482e495. [37] Erdmann SM, Sachs B, Kwiecien R, Moll-Slodowy S, Sauer I, Merk HF. The basophil activation test in wasp venom allergy: sensitivity, specificity and monitoring venom immunotherapy. Allergy. 2004;59:1102e1109. [38] Hamilton RH, Wisenauer JA, Golden DBK, Valentine MD Jr. Selection of Hymenoptera venoms for immunotherapy based on patients’ IgE antibody cross-reactivity. J Allergy Clin Immunol. 1993;92:651e659. [39] Eberlein B, Krischan L, Darsow U, Ollert M. Double positivity to bee and wasp venom: improved diagnostic procedure by recombinant allergen-based IgE testing and basophil activation test including data about cross-reactive carbohydrate determinants. J Allergy Clin Immunol. 2012;130:155e161. [40] Mitterman I, Zidarn M, Silar M, Markovic-Housley Z, Aberer W. Recombinant allergen based IgE testing to distinguish bee and wasp allergy. J Allergy Clin Immunol. 2010;125:1300e1307. [41] Muller UR, Schmid-Grendelmeier P, Hausmann O, Helbling A. IgE to recombinant allergens Api m 1, Ves v 1, and Ves v 5 distinguish double sesnitization from crossreaction in venom allergy. Allergy. 2012;67:1069e1073. [42] Kohler J, Blank S, Muller S, et al. Component resolution reveals additional major allergens in patients with honeybee venom allergy. J Allergy Clin Immunol. 2014;133:1383e1389.
Contents lists available at ScienceDirect
How Allergen Extracts Are MadedFrom Source Materials to Allergen Extracts
Hymenoptera venoms used to produce allergen extracts Greg Plunkett, PhD *; Robert S. Jacobson, MS y; David B.K. Golden, MD z * ALK-Abello y z
Inc, Round Rock, Texas Greer Laboratories, Lenoir, North Carolina Johns Hopkins University, Baltimore, Maryland
A R T I C L E
I N F O
Article history: Received for publication April 2, 2016. Received in revised form May 20, 2016. Accepted for publication May 31, 2016.
A B S T R A C T
Objective: To review the methods and materials used for collection, purification, commercial production, and clinical application of Hymenoptera venoms. Data Sources: Most of the sources for this review are the experience and expertise of the authors. Published reports and review articles on Hymenoptera venom collection and production were identified through database searches (PubMed). Study Selections: Studies describing the methods for Hymenoptera venom collection and production were selected for review. Results: Meticulous methods for identification and collection of the insects are required. Collection and purification of the venoms from the insects are based on validated methods and result in a commercial extract that is standardized for the major allergenic proteins required for accurate diagnosis and safe and effective treatment of patients allergic to insect sting. The steps required for mixing, purifying, testing, and standardizing the products are described. Conclusion: Hymenoptera venom extracts were developed using many new methods for the collection, purification, and commercial production of the unique materials required for this product. Clinical applications for diagnosis and treatment are affected by the integrity and stability of the allergens after processing and purification. Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Important Hymenoptera Insects Bees, Wasps, Yellow Jackets, and Hornets The flying insects of the order Hymenoptera (bees and wasps) that are responsible for anaphylaxis attributable to venom hypersensitivity are largely similar between the United States and Europe. Honeybees (Apis mellifera), native to Europe, were introduced into the United States nearly 400 years ago, becoming established throughout the country, and are valued on both continents for pollinating crops, honey, and wax.1 Stinging risk occurs throughout the year from their perennial colonies but minimally during colder months. Some strains are more defensive than others. The introduced Africanized or “killer” bees, which migrated from Brazil and were intentionally brought from Africa, have reached the Southern and Southwestern United States, where their extremely
Reprints: David B.K. Golden, MD, Johns Hopkins University, 7939 Honeygo Blvd, Room 219, Baltimore, MD 21236; E-mail: [email protected]. Disclosures: Dr Golden reported receiving honoraria and serving on the speaker’s bureau for Genentech and Greer Laboratories, working as a consultant for Greer Laboratories, and receiving a research grant and working on a clinical trial for Genentech.
defensive behavior poses a serious risk. Honeybees are unique among important stinging species because their barbed stings readily detach and remain in the skin, although yellow jackets are sometimes unable to remove their stings.2 The genera Vespa, Dolichovespula, and Vespula (hornets and yellow jackets, with Vespula often simply called wasps or true wasps in Europe) are important in Europe and the United States, although a member of the first genusdVespa crabro or European hornetdwas introduced into the United States around 1840. Although V crabro is widespread in Europe, 2 other hornets (one native, another introduced) have limited distributions. All 3 genera make paper nests composed of several combs usually covered by an envelope, with colonies typically lasting only one season, and will defend their nests aggressively if disturbed. Vespula and Dolichovespula are native and widespread in the United States and Europe, with the former genus building large colonies, often in the ground or in structures, and the latter usually building exposed nests on tree branches or under eaves. Several species of yellow jackets are scavengers with large colonies; a prime example is Vespula germanica, native to Europe (which usually includes Paravespula) and established in much of the United States. Scavenging species are attracted to human food, making
http://dx.doi.org/10.1016/j.anai.2016.05.027 1081-1206/Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
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unintentional contact or ingestion of the insect a risk. European species cross-react well with most American species but less so with the American Vespula squamosa.3 Dolichovespula includes several species whose American members, particularly the large and widespread baldfaced species (Dolichovespula maculata) with its large gray nests, are sometimes called hornets. The closely related aerial yellow jacket or yellow hornet (Dolichovespula arenaria) is a smaller member of this genus. They both have sibling species in Europe (where they are sometimes called tree wasps) with which they can be expected to crossreact.4 Paper wasps (Polistes species) are rather slender insects that build single exposed combs (ie, no covering envelope). They are found through most of the contiguous United States and much of Europe, particularly warmer regions. Mass stinging is unlikely because of their small colonies. American and European species are partially cross-reactive, although one of the European species (Polistes dominula) has become established in the United States.5,6 Fire Ants and Other Ants Five species of fire ants (Solenopsis)d3 native and 2 importeddare in the United States; no species are found in Europe. Two imported species (Solenopsis richteri, the black species, and especially Solenopsis invicta, the red species) are introductions from South America that present important stinging hazards throughout much of the southeastern United States and occasionally westward, particularly when their soil mounds are disturbed.7 Additional medically important ants in the United States include harvester ants (Pogonomyrmex) distributed mostly in the Western United States, with one species reaching the Southeast and Asian needle ants (Brachyponera chinensis) currently restricted to the Southeast. Extracts are available only for imported fire ants; their venoms are not cross-reactive with the other genera.8,9 Collection of Stinging Insects Obtaining stinging insects for venom production presents several challenges. A collector must obtain sufficient leads from property owners or managers who can report active colonies, and such reports must be screened to avoid wasting time with unwanted species of insects (eg, honeybees) and to be sure the nest is accessible or at least the insects can be trapped. Before collecting, the collector must be certain no pesticides or chemicals have been or are used (although carbon dioxide is permitted). Insects must be kept alive until frozen for storage. Beekeepers and pest control operators often receive gratuitous calls, whereas others must advertise free insect removal. There are many ways of collecting, and collectors have their own trade secrets. Techniques range from collecting entire nests (which are frozen intact and dissected later while maintained frozen), trapping insects, and using a special vacuum cleaner designed to prevent killing and desiccating the insects; in the last 2 cases, prompt freezing is critical. Species are kept segregated, so the collector must bag the insects from each nest separately. A chesttype freezer is used for storage to avoid loss of cold when opened. Because only female insects sting, collectors often time their collecting for maximum female populations. Normally, this is late summer or autumn for vespines, but this may be past the peak for Polistes. Collected insects must remain frozen until just before dissection. On receipt at the manufacturer, insects are examined. This entails verification that the insects are still frozen (usually by the presence of dry ice in the shipping container), confirmation of the species, certification that no pesticides or other chemicals were used, confirmation there are no odors atypical of the species (ie, attributable to chemicals or spoilage), random sampling to assess
female percentage, and subtraction of weight due to debris and water ice. Finally, female insects are randomly sampled, thawed, and dissected to ensure the venom sacs are light yellow or whitish in color (depending on the species) as evidence that insects were frozen alive. The usable weight of female insects is calculated for both payment and raw material inventory purposes. If criteria are not met, insects are rejected. Honeybee venom (HBV) is collected by electrical stimulation, as discussed below. Because fire ant extracts are whole body, their colonies are scooped into containers, and then water is used to float the ants from the soil, which is a natural behavior for fire ants. The floating ants are scooped and stored frozen.
Venom Processing Hymenoptera Venom Sources: History Testing and treatment for allergy to flying insect stings began nearly 100 years ago when in 1925 Braun10 treated a HBV allergic patient with an extract from bee posterior bodies. From 1930 to 1954, studies reported that treatment with HBV and whole-body extracts was equally effective.11 In the 1960s, Benton and colleagues12 from Penn State University designed an improved apparatus for collecting pure HBV using electrostimulation.12 Collection of vespid venom was described by Loveless and Fackler13 in 1956. As these purified venom sources improved and venom became more readily available, clinical trials during the 1970s and 1980s established that whole-body extracts were not effective for testing or treatment.14e16 Venoms were found to be better than whole-body extracts, resulting in commercial venom products becoming available from Hollister-Stier (now Jubilant Hollister Stier) and Pharmacia Labs (now ALK-Abello Inc) in 1979 to 1980 (Table 1).17e19 Table 1 History of Venom Extracts Used for Immunotherapy Year
Event
1925
Braun successfully tested and treated honeybee allergic patient with extract made from posterior 1/800 of HB bodies (WBE) Benson and Semenov reported on a honeybee allergic beekeeper who had positive skin test results to HBV, HB sting apparatuses, and HB bodies w/o sting apparatuses Markovic and Molnar described electrical stimulation for the collection of honeybee Loveless and Fackler reported on successful diagnostic and therapeutic use of venom sac extracts Benton and colleagues reported an improved method for electrical collection of honeybee venom Bermon and Brown reported positive whole-body extract skin test results in 37% of inmates without positive histories (n ¼ 200) and in 40% of inmates who had never been stung (n ¼ 15) Schwartz reported no significant difference between skin test reactivity to whole-body extract in sting allergic patients and control patients Torsney reported 8 insect sting allergy fatalities in patients treated with whole-body extract Small samples of Hymenoptera venoms to Johns Hopkins University Ramp up of venom source materials from Penn State University (Dr Benton, honeybee venom) and Rockefeller University (Dr King, vespids) Sobotka et al reported that Hymenoptera venoms induced histamine release in basophil leukocytes from insect allergic patients Hopkins asked to treat a beekeeper’s 4-year-old son, whole-body extract failure, whose sister had previously died of a honeybee sting Hunt et al reported on the diagnostic specificity of venom skin tests in 30 patients Favorable results from the Johns Hopkins clinical trials Hunt reported blinded placebo-controlled trial with 60 patients US Food and Drug Administration grants licenses to Hollister-Stier, Pharmacia Labs (ALK purchased in 1987)
1930
1954 1956 1963 1965
1965
1973 1973 1970s
1974 1974
1976 1976 1978 1979e1980
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How Are Hymenoptera Venoms Obtained? Honeybee venom The method for collection of HBV using electrostimulation was perfected by Benton and colleaques12 from Penn State University, and Dr Benton, along with Miles Guralnick, formed Vespa Labs in Spring Mills, PA, to supply insect venoms for immunotherapy. Electrostimulation involves exposing a bee to a small electric current from a wire grid connected to a carlike battery, causing the bee to automatically sting through a plastic wrap, leaving a small bead of venom (Fig 1). The battery and grid apparatus is placed near a commercial hive. As the bees explore the device and land on the grid, the stinging impulse also releases attack pheromones and soon is covered in hundreds of bees. The plastic wrap covering the grid does not impede the barbed stinger because it retracts, so the bees are not harmed. Each sting through the plastic deposits approximately 50 mL of pure venom so after a short time several grams of pure venom can be collected. The purified HBV source material is dried on the plastic wrap then scraped off. More recently, the plastic wrap has been replaced by a latex-free membrane that is safer for the bees. Vespid and wasp venom In 1956, Loveless and Fackler13 reported the successful use of venom sac extracts for immunotherapy. Because electrostimulation of hives was not practical for these insects, venom sac isolation was the preferred path for collecting venom. The scientists at Vespa Labs along with scientists at Hollister-Stier Labs, Spokane, WA, developed for commercial purposes the technique described by King et al,20 who described removing venom sacs and characterizing the components in the venoms (Fig 2). The venoms collected by these methods in the middle 1970s were used for immunotherapy clinical trials at Johns Hopkins University and Mayo Clinic that were the basis of US Food and Drug Administration (FDA) approval of the commercial venom products.17,21,22 Today there is an extensive network of private collectors and beekeepers that supplies the purified HBV and insects for further processing at Vespa Labs and Jubilant Hollister Stier. This network is still the sole supplier of venom source material used for FDAapproved immunotherapy. Vespa Labs is now part of the global allergy extract company ALK (Hørsholm, Denmark), and ALK Inc (Round Rock, Texas) distributes Hymenoptera venom products in the United States. The vespid and paper wasp insect collectors are responsible for finding nests for at least 6 species of yellow jacket and 4 to 5 species of Polistes wasps. The insects are rendered harmless using carbon dioxide and then delivered to the sacpulling teams frozen.
Figure 1. Collection of honey bee venom by electrostimulation. Reproduced with permission of Vespa Labs (ALK-Abello Inc).
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Imported fire ant source material Venom from Solenopsis richteri and invicta fire ants is not available for immunotherapy. Manufacturers supply weight/volume whole-body extracts that are nonstandardized (no standard of potency). Insects are collected from nests and then freeze-dried before extraction. Analysis of these extracts has revealed sufficient venom proteins for use in immunotherapy diagnosis and treatment.23e25 Batch to batch variability and stability are not known for fire ant extracts in the United States. Manufacture of Insect Extracts FDA approval of venom products The FDA approved stinging insect venom products in 1979 to 1980, subsequently removing the corresponding whole-body extracts from the market. Six venom product types are approved in the United States. Honeybee, domestic (Apis mellifera), yellow jacket (Vespula species), yellow hornet (Dolichovespula arenaria), white-faced hornet (Dolichovespula maculata), paper wasps (Polistes species), and a mixture of yellow jacket, yellow hornet, and white-faced hornet (mixed vespids). There are various configurations, of single use or multidose vials, and they are formulated to be 100 mg/mL when reconstituted (300 mg/mL for mixed vespids). In Europe, there are some alternate venom product types, such as dialyzed venom to remove low-molecular-weight nonallergenic material and vaccines of venom adsorbed to alum. Below is a discussion of US manufactured products only. Manufacturing Steps Species mixtures Yellow jacket and paper wasp are formulated with a mixture of species within the genus and include the most common species that cause allergic reactions. Yellow jacket venom contains Vespula species of Vespula vulgaris, V germanica, Vespula maculifrons, Vespula pensylvanica, Vespula flavopilosa, and Vespula squamosa. The ALK venom package inserts specify that these species are present in their yellow jacket venom extract. These are the most commonly used species in the Jubilant Hollister Stier venoms, although their package insert allows for other Vespula species. From the ALK package insert, the Polistes paper wasp products are mixtures of Polistes annularis, Polistes apachus, Polistes exclamans, Polistes fuscatus, and Polistes metricus. Jubilant Hollister Stier paper wasp products have contained mixtures of P metricus, P exclamans, and P fuscatus, recently removing P annularis. There is extensive (but not absolute) cross-reactivity among the venoms of the Vespula genus and also among those of the Polistes genus. The species chosen for the commercial mixtures are the ones that most commonly sting people. Venom of V squamosa was added to the yellow jacket mixture early (1980) because it possesses some unique allergenic activity and is less cross-reactive with other Vespula species. European venom extracts can also contain mixtures of Vespula and Polistes species. Some contain the same mixtures as used in the United States, whereas others have fewer species, such as V germanica and V vulgaris. There has also been the addition in some markets of P dominula, also called the Mediterranean paper wasp, as a single species extract. This species, even though its presence is increasing in the United States, has not been introduced in FDAapproved venoms. Preparation of intermediate venom products HBV intermediate product is pure dried venom collected from electrostimulation. The vespid and wasp venoms are extracts of venom sacs that are individually pulled from insects collected and frozen alive. Each sac is pulled from the insect and placed in an extraction fluid containing a small amount of an acetic acid/ b-alanine buffer. Hundreds of sacs are extracted, and the mixture is
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Figure 2. Collection of Vespula and Polistes venom. Reproduced with permission of Vespa Labs (ALK-Abello Inc).
blended, filtered, and freeze-dried into bulk vials. Yellow jacket species and wasp species are mixed into the final bulk vials. All the venom bulk vials are stable for at least 30 months according to one manufacturer. Intermediate testing Initial estimates of venom potency are made by measuring the total protein content (even though this is not a direct measure of allergenic potency). The bulk intermediate vials are tested for total protein by the FDA-approved Lowry method (Laboratory of Immunobiology of the FDA, Methods of the Allergenics Products Testing Laboratory, Docket No. 94N0012 October 1993). During the development of commercial products in the late 1970s, 2 enzymes were discovered to be major allergen proteins, phospholipase and hyaluronidase. Enzyme activity identified the presence of these 2 proteins and therefore became part of the potency testing. Even though the proteins are not cross-reactive between honeybees and vespids, the same enzyme assays were performed for all venom bulk vials. Each batch of venom bulk vials has minimum enzyme level requirements. Final commercial venom products The final venom products are reconstituted to 100 mg/mL based on the Lowry total protein content of the bulk vials. For mixed vespid venom, yellow jacket, yellow hornet, and white-faced hornet intermediate vials are combined and formulated to 300 mg/mL. The reconstitution solution contains mannitol as a freeze-drying agent and helps stabilize the venom protein. ALK adds human serum albumin along with the mannitol as an additional freezedrying stabilizing agent and is the primary difference in the venom products in the United States. The vials are commercially available as freeze-dried single or multidose vials at 100 mg/mL (300 mg/mL for mixed vespid venom) when reconstituted. Standardization Venom source material is analyzed for the allergen enzyme activity, and native polyacrylamide electrophoresis is performed. Each venom has a unique pattern. One of the major allergens in vespid venom is antigen 5 or vespid 5. The enzyme activity is not known, and no immunoassay was developed, so this important allergen is not measured in US extracts. It is a prominent protein band in electrophoresis. Final commercial vials are tested for total protein, phospholipase, and hyaluronidase and must meet certain limits as specified in the manufacturers’ FDA licenses. IgE binding is not determined for FDA-licensed products; however, potency by IgE binding is determined in European venoms in addition to the enzyme assays. Sterility and safety testing is also performed before release for sale. Expiration dating of the freeze-dried powder is determined by stability studies performed by each manufacturer. Expiration studies of the reconstituted vials using human serum
albumin with phenol diluent have also been performed with recommendations stated in the package insert. Composition of venoms Since the introduction of venom products for immunotherapy in the late 1970s, much as been learned about their makeup. Lowmolecular-weight components are present, many of which are active pharmacologic agents composed of vasoactive amines and peptides. These components are not removed from the venom products and most likely are responsible for limiting intradermal skin test diagnosis to concentration not exceeding 1 mg/mL. Many additional allergen proteins have been described in addition to the phospholipase and hyaluronidase enzyme allergens. In the vespids, group 5, such as Ves v 5, has been found to be an important allergen, but the enzymatic function is not known. Purified Ves v 5 was used as an electrophoresis marker to demonstrate the presence of this protein in US venom products. Implications for Clinical Practice Hymenoptera insect extracts are necessary for accurate diagnosis and preventive treatment of insect sting allergy. The process from source materials to allergenic extracts has considerable effect on the clinical applications. The selection and collection of insect species are critical to all subsequent characterization of the commercial product for diagnosis and treatment. The purity and allergenic activity of the collected materials must be optimal and consistent. There are numerous species of Vespula and Polistes, which vary in different regions of the United States. For any nonentomologist, the accurate identification of these species is difficult and unreliable.26 A trained and skilled network of collectors and vigilance in review of the materials that arrive at the laboratory are required to ensure the allergenic specificity, activity, and purity of the products. There are only 2 allergen laboratories in the United States that supply Hymenoptera extracts for clinical use, and their products have been described above. The clinician must always be aware that there are species that are not represented in the commercial products and may be antigenically distinct from the more common species. This could lead to negative diagnostic test results in a patient who is allergic to a specific species of vespid.27 If an extract varies in the type or quantity of certain species, then the ability to detect the allergy on testing or to protect with venom immunotherapy could be reduced. The preparation of the extract from the raw materials is critical in determining the reliability of testing and treatment. At each of the steps described above for intermediate products and standardization, any change or deficiency could greatly affect the clinical outcome. At risk is the integrity and stability of each allergenic protein in each of the venoms being prepared. Loss of activity could
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result from susceptibility to reagents used in the processing, mechanical degradation, enzymatic degradation, molecular instability, and binding characteristics. For example, the activity of the major vespid allergen Ves v 5 is not optimal for diagnosis, and the detection of yellow jacket allergy can be improved by spiking the extract with this protein.28 The procedures for standardization can also have an effect on clinical practice. As described above, the Hymenoptera venoms are standardized by the FDA based on measurement of phospholipase (HBV) or hyaluronidase (vespid venoms), although there are specifications for both enzymes in all Hymenoptera venom extracts. Without quantification of the major vespid allergen, Ves v 5, it is difficult to be certain of the equivalence of products from different batches or different laboratories. Notably, despite numerous occasions that required changing a patient’s treatment to a different product, there has been no report of any adverse outcomes. Diagnostic applications of Hymenoptera extracts are for in vivo and in vitro testing methods. Skin tests use the commercial extract at concentrations found to provide optimal sensitivity and specificity (up to 1.0 mg/mL for intradermal testing). Higher concentrations give improved sensitivity but can also cause false-positive results because of local irritative effects.29 Greater diagnostic accuracy is achieved with a concentration of 10 mg/mL if the venom is dialyzed to reduce these irritative effects.30 Dialyzed venoms are not commercially available in the United States but are common in Europe.31 Commercial reagents for skin prick testing are not available because skin prick testing with venoms is much less sensitive than intradermal testing and is often omitted in favor of intradermal tests starting with very low concentrations (0.001 mg/mL). When skin prick tests are performed as a preliminary test, the concentrations used range from 1 to 100 mg/mL.32,33 Diagnostic testing for fire ant allergy uses the commercial whole-body extracts. Although venoms might be superior, they are not commercially available. Laboratory methods that use Hymenoptera extracts are primarily enzyme-linked immunoassays for allergen specific IgE antibodies. In these assays, the accuracy may be affected not only by the activity of the many allergens in each venom but also by their binding characteristics for the solid phase used in the assay.34 Nevertheless, neither skin tests nor in vitro tests can detect all cases of insect sting allergy. Another in vitro method that shows promise is the basophil activation test, which uses the same venom extracts to elicit basophil activation (not necessarily IgE dependent).35e37 One of the dilemmas of diagnostic specificity for Hymenoptera venoms is the cross-reactivity between genera and species. Radioallergosorbent assay inhibition tests have been used to determine whether patients with dual yellow jacket and Polistes wasp venom sensitivity can be treated with just 1 of the venoms.38 The use of recombinant venom allergens for in vitro measurement of IgE can identify the primary allergy in patients with dual positive test results for honeybee and yellow jacket venoms.39e41 However, for reliable accuracy, these tests would have to include multiple recombinant allergens for each venom. Therapeutic application of Hymenoptera products prevents severe allergic reactions to stings. Early clinical trials revealed the safety and efficacy of venom immunotherapy.17 Clinical experience in thousands (if not millions) of patients during the past 40 years has confirmed that venom immunotherapy prevents systemic reactions in 80% to 98% of affected patients.29 The safety of venom immunotherapy has been comparable to other standard types of allergen immunotherapy (eg, grass or cat). The choice of which venoms to use for treatment can be facilitated with in vitro tests to clarify the cross-reactivity of the venoms, as described above. Immunotherapy for fire ant sting allergy is performed using wholebody extracts, which contain sufficient venom allergens to provide significant clinical protection from anaphylaxis.23
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Conclusions The development of specific materials to be used for diagnosis and treatment of Hymenoptera venom allergy posed unique challenges in the collection and processing of the allergens. Insects of known species must be obtained by knowledgeable collectors and must be handled from the outset in a way that preserves the integrity and activity of the venom. Identification of the relevant species requires expertise, and locating and collecting them require understanding of their nesting and foraging habits. In the case of honeybee, the method of venom collection is unique and requires live insects. Fire ant extracts are prepared from whole insect bodies without separation of the venom. The crude venoms must be assessed qualitatively and quantitatively for purity and allergenic activity. The intermediate venom product must be purified and stabilized to eliminate most small molecules that can increase adverse effects and to avoid enzymatic degradation of the allergens. The choice of specific species and their relative quantities for the yellow jacket and Polistes products must be consistent. Finally, venoms must be standardized by quantitation of specific allergenic proteins. The use of commercial Hymenoptera venoms for diagnosis and treatment of insect sting allergy is both improved and hampered by the methods of production. Hymenoptera venoms were the first allergenic extracts to be standardized by the FDA. On the other hand, the vespid venoms are not routinely standardized for the major allergen Ves v 5. Recent studies found that some allergens may be underrepresented in the commercial venoms, which might reduce the diagnostic accuracy and therapeutic efficacy of the venoms.28,42 In the future, the use of recombinant venom allergens to supplement the commercial venoms might address this problem (and some of those below as well). Another potential improvement in the commercial venom products would be more efficient purification (eg, dialysis) to reduce the irritative components that limit the concentration used for skin tests and contribute to local reactions to immunotherapy.30,31 Purified venoms are available in Europe but not in the United States. Another possible improvement would be the availability of additional species, either individually or within a species mix (eg, Vespula, Polistes). There are patients with specific sensitivity to bumble bees, Mediterranean wasps (P dominula), or other unique species of wasp or yellow jacket that are not currently represented in the commercial products.27 It is believed that fire ant venom would be superior to the whole-body extract for testing and immunotherapy. It would useful if fire ant venom could be made available in a commercial product. References [1] Turpin T. Honeybees Not Native to North America. On Six Legs; Purdue Extension. West Lafayette, IN. https://http://www.agriculture.purdue.edu/agcomm/ newscolumns/archives/OSL/1999/November/111199OSL.html. Accessed November 28, 2015. [2] Greene A, Breisch NL, Golden DB, Kelly D, Douglass LW. Sting embedment and avulsion in yellowjackets (Hymenoptera:Vespidae): a functional equivalent to autotomy. Am Entomol. 2012;58:50e57. [3] Hoffman DR. Allergens in Hymenoptera venom, XXV: the amino acid sequence of antigen 5 molecules: the structural basis of antigenic crossreactivity. J Allergy Clin Immunol. 1993;92:707e716. [4] Ross KG, Matthews RW. The Social Biology of Wasps. Ithaca, NY: Cornell University Press; 1991. [5] Hoffman DR, Jacobson R, Blanca M. Allergy to venom of Polistes dominulus, a paper wasp introduced from Europe [abstract]. J Allergy Clin Immunol. 1990; 85:211. [6] Severino MG, Campi P, Macchia D, et al. European Polistes venom allergy. Allergy. 2006;61:860e863. [7] Kemp SF, deShazo RD, Moffitt JE, et al. Expanding habitat of the imported fire ant: a public health concern. J Allergy Clin Immunol. 2000;105:683e691. [8] Hoffman DR. Allergens in Hymenoptera venom, XXIV: the amino acids sequences of imported fire ant venom allergens Sol i II, Sol i III, and Sol i IV. J Allergy Clin Immunol. 1993;91:71e78.
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