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Background: Hyaluronidase (Hya) is one of several allergens in honeybee venom. Its cDNA sequence was recently described., Objective: We sought to express recombinant Hya in prokaryotic and eukaryotic systems and to compare it with natural (n)Hya for biologic activity., Methods: In Escherichia coli Hya was produced as inclusion body 6 x His-fusion protein. In baculovirus-infected insect cells expression was obtained by cotransfection of linearized Bac-N-Blue DNA and pMelBac transfer vector into Spodoptera frugiperda cells., Results: Enzymatic activity of Hya from the baculovirus system was equal to nHya, and that of the enzyme expressed in E. coli was only 20% to 30% of nHya. In vitro IgE binding was similar in nHya and the enzyme from baculovirus but markedly lower in Hya expressed in E. coli., Conclusions: Biologic activity of Hya expressed in baculovirus-infected insect cells was comparable with that of the natural enzyme, indicating a native-like conformation of the recombinant protein. In contrast, the enzyme expressed in E. coli as an inclusion-body protein and reconstituted in vitro reached only 20% to 30% of the activity of nHya.

Previous work has demonstrated a close association between certain histocompatibility antigens and the gene that controls the IgE response to certain ragweed allergens. For example, there is a 90% association between IgE production to the short ragweed allergen, Amb a V, and an HLA class II allele. To assess whether these HLA linkages are specific for ragweed, we have investigated the association between HLA antigens and the capacity of individuals to mount a specific IgE response to melittin in patients with bee-venom allergy. Twenty-two subjects with bee-venom sensitivity, 22 healthy beekeepers without bee-venom allergy, and a normal population of 149 unselected individuals were studied. With serologic tissue typing and restriction fragment length polymorphism analysis, we have demonstrated a significant decrease in the HLA-DR4 and DQw3 alleles in subjects who are allergic to melittin compared to the control populations. There was also a negative association between the presence of HLA-DR4 and DQw3 alleles with the capacity of the individuals to mount an IgE response to phospholipase A2 (PLA2). The bee-venom sensitive subjects had a slightly lower titer of anti-PLA2 IgG when these subjects were compared to the bee-venom insensitive beekeepers. These results support the view that either HLA-DR or HLA-DQ has a protective role in controlling the IgE immune response. Lack of an IgE response to melittin or PLA2 is unlikely to be due to a failure to recognize allergen.

During immunotherapy with wasp venom, levels of venom-specific IgE antibodies increase and then fall, whereas the concentration of IgG antibodies rises and then remains at a high level. Successful treatment is therefore associated with both increased concentrations of serum IgG and decreased serum IgE antibodies to venom. In this study we have investigated the possible role of auto-antibodies in inducing the decrease in serum IgE antibody. Levels of auto-anti-IgE were measured by a radioimmunoassay. Anti-IgE auto-antibodies were not generated during immunotherapy and there was no significant difference in the levels of anti-IgE auto-antibodies between patients whose venom-specific IgE antibody levels fell more than fivefold after immunotherapy and those patients in whom IgE antibody levels did not change significantly. We conclude that anti-IgE auto-antibodies do not play a part in IgE suppression induced by immunotherapy.

The radioallergosorbent test (RAST) has been widely used in studying allergy to hymenoptera stings, but with variable results. We report here a modification of the RAST method which in a total of 157 bee keepers and family members, gave a close correlation between a positive RAST, and a history of generalized (100%) or localized (95%) allergic reactions. There was no positive RAST among forty-nine non-atopic control subjects but a striking finding was that 58% of non-allergic bee keepers had significant, but usually low, levels of IgE antibody to bee venom. IgG antibodies to bee venom have also been measured by a paper disc RAST which is quick and convenient. Levels were highest in non-allergic bee keepers. In the presence of high levels of IgG antibody, IgE antibody levels may be underestimated.

The response of 15 strongly bee-venom-allergic patients to highly purified venom allergens was compared using skin prick test titration, peripheral blood leukocyte (PBL) histamine release and radioallergosorbent test with three highly purified bee venom allergens: phospholipase (PLA2), hyaluronidase (HYAL) and acid phosphatase (ACID P). Sensitivity to the three allergens ranked in the same order for all three tests and in each case PLA2 was found to the most potent allergen. In the presence of hyperimmune beekeeper plasma, maximum histamine release was reduced significantly for all three allergens (p less than 0.001). Furthermore, hyperimmune beekeeper plasma increased the amount of allergen required for a comparable release of histamine (mean shift in dilution curve PLA2 917-fold; HYAL, 492-fold; ACID P, 61-fold). The release of histamine from whole blood was also compared with PBL + 10% normal human serum (NHS). For all three allergens maximum release was much lower from whole blood compared with washed cells + 10% NHS (p less than 0.001). These data confirm PLA2 as the major bee venom allergen by all three tests. Hyperimmune beekeeper plasma reduces maximum histamine release and increases its threshold. Histamine release in response to ACID P appears harder to block with hyperimmune beekeeper plasma than that provoked by PLA2 or HYAL (p less than 0.01). Whole blood releases less histamine and requires more allergen than washed cells, indicating that sensitivity of PBL in vivo is unlikely to be as great as washed PBL in vitro.

Hyaluronidase has been purified from the venom of the honey bee, Apis mellifera. The purification proved remarkably difficult, requiring a large number of chromatographic steps culminating in the removal of traces of phospholipase A2 with an affinity purified rabbit anti-phospholipase A2 immunosorbent column. The purified enzyme showed a 1143-fold increase in specific activity and was homogeneous. Electrophoresis in polyacrylamide gels (12%) containing sodium dodecyl sulphate (pH 8.9) or urea (pH 2.8) and electrofocusing in polyacrylamide (5%) gave a single band. The final product contained less than 0.1% phospholipase A2 and less than 1.5% acid phosphatase and gave a single line of precipitation against rabbit anti-hyaluronidase but was not precipitated by rabbit anti-phospholipase A2. Previous reports of instability were not confirmed, and we found the enzyme to be highly stable over a wide range of temperature and pH, and to denaturing agents. Purified hyaluronidase was found to be 'sticky' when highly pure and at low concentration, and adhered strongly to Sephadex G-75. The relative molecular mass was estimated at 35 000-37 000 by gel filtration, and at 41 000 by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. A value of 50 000 was obtained by ultracentrifugation assuming a partial specific volume of 0.73 cm3/g. Hyaluronidase was found to be a minor allergen in bee venom allergic patients.

Antibodies to individual bee venom antigens were studied in detail in nine bee sting-allergic patients who received venom immunotherapy without side effects, in two patients who failed to reach maintenance, and in two whose sensitivity returned. The study was confined to patients who had IgE antibodies to at least one of four purified bee venom antigens at the start of treatment. IgE and IgG antibodies to phospholipase A2 (PLA2), hyaluronidase (HYAL), and acid phosphatase (ACID P) and IgE antibodies to melittin (MEL) were measured, and changes in the antibody levels were followed during bee venom immunotherapy. Two contrasting patterns of antibody response were seen in the nine successfully treated patients. In five patients there was a rise in serum IgG antibodies to the same antigens as the IgE antibodies. In two patients' serum IgE antibody to HYAL or ACID P fell without a marked IgG antibody response to these antigens, although high levels of IgG antibody to PLA2 were present in both. Although the first pattern is consistent with a "blocking" role for IgG antibody, clearly the second is not. Not all patients can be conveniently divided into these two categories, and two patients did not show any significant change in either IgG or IgE antibody but were nevertheless able to tolerate the maintenance dose of 100 micrograms of venom. Two patients who failed to reach the maintenance dose of 100 micrograms because of their allergic reactions to the injections of venom were distinguished by (1) very high serum IgE antibody and (2) a low ratio of IgG/IgE antibody. Passive immunization with IgG antibody from a hyperimmune beekeeper was, however, protective in these patients, although it did not raise their overall serum IgG antibody level very much. We are unable to explain either the failure of conventional therapy or the beneficial effect of passive immunization in these two patients. Two bee sting--allergic beekeepers lost their sensitivity to stings, but later, when their sera contained IgE antibody to another bee venom antigen, they reacted to stings and inhalation of beehive dander. These data suggest that either falling IgE antibody or IgG- "blocking" antibody could be responsible for providing clinical protection to bee venom--allergic subjects. Renewed clinical sensitivity was observed when the IgE response was modulated, with patients making IgE antibody first to one antigen and then to another.

We have measured in allergic and non-allergic beekeepers IgE and IgG antibodies to the bee venom allergens, phospholipase A2 (PLA2) and hyaluronidase (HYAL), by the radioallergosorbent test (RAST) and a 125I-radiolabelled antigen-binding assay. The absolute amount of IgG antibody in a reference serum was determined by saturation analysis using 125I-radiolabelled PLA2 and HYAL. Using monoclonal anti-IgE coated microtitre plates, the absolute amount of IgE antibody to the same antigen was also determined by saturation analysis. Regardless of the IgE response to the different allergens, IgG antibody concentrations to PLA2 were invariably higher than those to HYAL. In addition, the ratio of IgG to IgE antibody was higher for PLA2 (220:1) than for HYAL (10:1). Higher levels of IgG antibody to both allergens (especially HYAL) were found in those who had had prolonged exposure to bee stings. These data suggest that the level of IgG antibody produced is related to the dose administered, while the amount of IgE antibody may be regulated by other factors.

Acid phosphatase from bee venom was purified by a combination of saturated ammonium sulphate precipitation, gel filtration and ion exchange chromatography. The final product which is a glycoprotein contained less than 0.1% phospholipase A2 or hyaluronidase activity and existed in two molecular weight (96,000 and 45,000) forms. Acid phosphatase is a potent allergen, in bee venom allergic patients, which is capable of releasing histamine from sensitized human basophils and of inducing a wheal and flare reactions in sensitized human skin.

The antibody response to phospholipase A2 (PLA2) was studied by using radiolabelled antigen-binding techniques for both IgG and IgE antibodies. The results are compared with results for antigen P1 from Dermatophagoides pteronyssinus and for rye I from rye grass pollen. The response to PLA2 differs from the response to the inhalant allergens in several ways: almost all exposed bee keepers produce IgG antibodies to PLA2; among the allergic bee keepers there was no quantitative correlation between IgG and IgE antibodies; and the antibodies to PLA2 are not produced locally in the nose. Bee keepers with hay fever did not appear to have an increased incidence of allergic reactions to bee venom. In addition, the bee keepers with hay fever and the same levels of IgG and IgE antibodies to rye I as other individuals with hay fever. However, the bee keepers who had no history of hay fever were found to have high levels of IgG antibodies to rye I. These antibodies did not cross-react with PLA2 or bee venom, and the results suggest that bee stings may act as an adjuvant for IgG antibodies to pollen antigens.

IgE antibodies to purified proteins and peptides from honeybee venom have been measured by the RAST. Trace amounts (less than 0.1%) of the major venom protein phospholipase A2 (PLA2) grossly distorted the measurement of IgE antibody to the other venom proteins, acid phosphatase (Acid P) and hyaluronidase (HYAL), and overemphasized their importance. Reduction of antigen coupled to the cellulose paper discs, which were used in the assay, diluted out the contaminating PLA2 without apparent loss in sensitivity. The reduction of disc-bound antigen increased the competition between IgE and IgG antibodies but did not affect measurement of IgE antibodies in sera taken from 35 untreated patients who had a history of general allergic reactions to bee stings. In 54% of sera from bee venom--allergic patients, the greatest IgE antibody response was to PLA2. In all, IgE antibodies to PLA2 were present in 91% of these sera. IgE antibodies to Acid P, HYAL, or melittin were present in 60%, 51%, and 31% of sera, respectively, and accounted for the highest level of binding in 17%, 17%, and 6% of these. Only 6% of sera were positive for whole venom but negative for the isolated antigens. A low level of IgE antibody was found to peptide 401 in 6% of sera. No IgE antibodies were found to apamin. While confirming the central role played by PLA2 in bee sting allergy, these results show that other venom components are also important in some patients.

IgG and IgE antibody levels have been followed for a period of 2 years in patients receiving immunotherapy with bee and wasp venom. 106 adult patients who had had anaphylactic reactions to wasp stings had initially low IgG antibody levels to wasp venom which rose with therapy (p less than 0.001). IgE antibody levels also showed an initial rise but subsequently fell (p less than 0.001). The pattern was similar to that previously reported in children who had had anaphylactic reactions to bee stings, but who, after a course of immunotherapy, were able to tolerate stings with impunity. 60 adults who had had anaphylactic reactions to bee stings showed a different pattern, with initially high IgG antibody levels which did not rise further. Since two thirds of this group were beekeepers or members of beekeeper's families, the high initial IgG antibody levels could have been a response to the frequent stings to which such individuals are prone. The fact that high levels did not protect against anaphylaxis shows, however, that the classical concept of 'blocking antibody' is in need of revision.

We studied 55 subjects who had had anaphylactic reactions to bee stings within the previous 3 years. 38 out of 54 tested had IgE antibody to honey bee venom (HBV) as measured by radioallergosorbent test (RAST). On skin testing, 30 out of 34 had a positive test to HBV. Of these, 26 had a positive RAST. A positive skin test to HBV at high dilution or else a high anti-HBV RAST score appeared to identify those who, in a 6-month follow-up period, were at risk of developing further anaphylaxis following bee stings or immunotherapy. Of the two tests, RAST appeared to be the less sensitive. Measurements of IgG antibody to phospholipase A were seldom available for the period immediately preceding an anaphylactic episode and proved to be a poor means of predicting the liability to bee sting anaphylaxis in subsequent months.

Intradermal (ID) and prick tests with bee or wasp venom (Pharmalgen) have been performed on 102 subjects with a history of adverse reactions to stings and forty-six control subjects giving no such history. Venom was diluted 100, 10 and 1 microgram/ml for prick testing and 10(-2), 10(-2), 10(-3) and 10(-4) micrograms/ml for ID injections. In forty-six control subjects all were tested with the highest concentration of prick testing solution (100 micrograms/ml), eight (17%) had positive reactions, a similar reaction rate to that reported in control subjects using 10(-1) micrograms/ml ID. In our 102 test subjects skin tests were therefore regarded as positive only if the reaction was elicited by 10 micrograms/ml or less by prick test of 10(-2) micrograms/ml or less ID. In general the results with skin prick tests and ID tests were comparable when the prick solution was 1000 times the concentration of that used for ID testing. ID tests were positive in thirteen with negative skin prick, seven of whom had detectable antibodies when tested by RAST. Conversely four with a positive skin prick test (two of whom were RAST positive) were considered negative on ID testing. As judged either by RAST or skin tests it appeared that sensitivity diminished with the time interval from the last sting (P less than 0.001).

The IgG sub-class antibody response to bee venom in the four sub-classes was investigated in ten patients during and after venom immunotherapy. All patients tolerated a bee sting challenge 1, 2 and 3 years after the start of treatment as well as 1 and 2 years after treatment was stopped. Anti-phospholipase A2 (PLA2) antibodies were of IgG1 and IgG4 sub-class and rose early in treatment, IgG1 anti-PLA2 fell to pre-treatment levels after 3 years in contrast to IgG4 anti-PLA2 levels, which remained high during maintenance therapy and declined relatively little in the 2 years after the termination of treatment. This data shows that IgG4 antibodies are maintained in the absence of monthly maintenance injections and suggests that they may provide long lasting clinical protection from insect stings.

High functional affinity and high titer IgG4 antibodies to phospholipase A2 were produced by allergic patients in response to bee-venom immunotherapy. In contrast, the affinity of IgG1 antibodies decreased after immunotherapy, and both the titer and affinity of IgG1 antiphospholipase A2 remained significantly lower compared to IgG4 1 to 2 years after treatment. Analysis of affinity heterogeneity suggested a loss of IgG1 high-affinity antibody-producing clones during immunotherapy and a preferential expansion of IgG4 clones. High-affinity IgE antibodies were found in untreated allergic patients, and preliminary results suggest that immunotherapy may result in an early marked decrease in the affinity of IgE antibodies.

The immunological response to individual bee-venom allergens was studied in blood samples collected at frequent intervals from four bee-venom allergic patients who had suffered systemic allergic reactions to injections of bee venom during immunotherapy. All had high IgE antibody levels, at the upper end of the range found in bee-sting allergic patients, and all had antibodies to the minor allergens at the time of the reactions. These did not, however, provide a simple explanation for the reactions that occurred. We were able to observe two interesting phenomena--in one patient IgE antibodies to the individual venom antigens appeared to be 'switched off' sequentially. In another, IgE antibodies to hyaluronidase rose substantially after 4 years of therapy. We believe that these results provide evidence to support the view that the regulation of IgE antibodies is controlled by mechanisms that are both isotype- and antigen-specific.