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Rush venom immunotherapy program for honeybee sting sensitivity
Rush venom immunotherapy for honeybee sting sensitivity
program
J. W. Yunginger, M.D., * B. R. Paull, M.D., R. T. Jones, B.S., and P. J. Santrach, B.A. Rochester, Minn.
The clinical and immunological effects of honeybee venom (HBV) immunotherapy were studied in 20 HBV-sensitive patients. Patients were treated once monthly in a hospital setting by “rusk” immunotherapy; we aimed for a maximum single dose of 200 pg. One patient withdrew shortly after beginning therapy. Eleven patients required epinephrine during treatment for reversal of anaphylaxis. Patients who tolerated maximum doses were readmitted one month later for deliberate honeybee sting challenges. Stings were tolerated by 11 of 19 patients; 5 of the remaining 8 required epinephrine therapy. Twelve of 16 patients achieved long-term protection as determined by deliberate stings; 4 of 16 achieved partial protection. Three additional patients, who were treatment failures, were switched to weekly immunotherapy. IgE antibodies to HBV and phospholipase A (PL.A) rose shortly after immunotherapy was begun and in many patients remained elevated above baseline values. Serum IgG antibodies to PU rose steadily during therapy; there was no absolute level of IgG antibody which was protective for all patients. Although satisfactory long-term clinical results were achieved for most patients, the frequency of systemic reactions during the rush immunization caused us to abandon this form of treatment in favor of a once-weekly outpatient injection program.
Systemic allergic reactions to Hymenoptera stings are mediated by IgE antibodies to venom and venom components.’ Although venom immunotherapy programs have been reported previously by some allergists,’ conventional allergy practice has been to treat Hymenoptera sting- sensitive patients with injections of whole insect body extracts. Previous case reports have documented the clinical and immunological consequences of venom immunotherapy in two honeybee-sensitive patients. 3, 4 A recent controlled clinical From the Departments of Pediatrics and Internal Medicine (Allergy), and the Allergic Diseases ResearchLaboratory, Mayo Clinic. Supportedin part by a grant (Al- 11483)and contract (A l-42546) from the National Institute of Allergy and Infectious Diseases;a contract from the Food and Drug Administration (223-77-1202); a Clinical ResearchCenter grant from the National Institutes of Health (RR-585); and by Mayo Foundation. Presentedin part at the American Congressof Allergy and Immunology, New York, N. Y., March 28, 1977, and at the meeting of the American Pediatric Society and Society for Pediatric Research, San Francisco, Calif., April 28, 1977. Received for publication March 6, 1978. Accepted for publication Nov. 13, 1978. Reprint requests to: John W. Yunginger, M.D., Allergic Diseases Research Laboratory, Mayo Clinic, 200 First St. SW, Rochester, MN 55901. *Recipient of an Allergic DiseasesAcademic Award (Al-00107). Vol. 63, No. 5, pp. 340-347
study which compared venom, whole body, and placebo immunotherapy has demonstrated the clear superiority of the venom treatment, as judged by deliberate sting challenges following therapy.5 The present study was conducted in 1975 and 1976 to determine the clinical and immunological changes occurring during an inpatient “rush” honeybee venom immunotherapy program in honeybee sting- sensitive beekeepers or beekeeper family members; these persons represent a “high-risk” category with respect to receiving future stings. MATERIALS Patients
AND METHODS
Twenty honeybee sting-sensitive patients (12 females, 8 males) were identified during visits to the annual conventions of the Minnesota Beekeepers Association or were referred via other beekeepers or their physicians. The clinical characteristics of the study group are shown in Table I. Patients ranged in age from 4 to 66 yr (X = 32.6 yr). All patients had clinical histories of systemic anaphylactic reactions following honeybee stings, with symptoms ranging from generalized urticaria to profound shock. Three patients were beekeepers: 16 were members of beekeepers’ families (9 wives, 7 children); and 1 lived on a farm containing bee yards. In all cases the insect responsible for the sting reaction could reliably be identified as a honeybee. Eleven of the
0091-6749/79/050340+08$00.80/0
63 1979 The C. V. Mosby
Co.
VOLUME NUMBER
TABLE
Rush venom immunotherapy
63 5
I. Clinical
characteristics
of the study group Whole
Patient No.1 sex/ age (W
body extract
immunotherapy
Stung while Skin test Occupation*
BW BC BC BW BW F BW BW BC BC BC BC B B BW BW BW BC BW B
I /F/52 2/F/17 3/M/9 4/F/40 5/F/56 6/M/l I 7/F/50 8/F/54 9/M/4 10/M/5 1l/M/1.7 12/M/8 13/M/43 14/M/66 15/F/60 16/F/32 17/F/36 18/F/18 19/F/38 20/F/36
341
(pglml)t
10 0.01 0.01 1 10 0.01 0.1 10 0.01 0.1 0.01 I 10 0.01 0.1 0.01 0.01 1 0.1 10
Clinical reaction*
AE, U A, AC, AE, S A, U A. U A, R, U A A. AE AC, AE, U A, AE, U A, AE, U AE, U AE, U A, AE, U AE, U A, AE, S R, S, U A A, AE, U A, S, U AC, U
Previous
Rx
Anaphylaxis
+ +
+ +
+
+
+ + +
+
+ +
on Rx No reaction
Not stung while on Rx
+
+ +
+ + +
+
*B, Beekeeper; BC, beekeeper’s child; BW, beekeeper’s wife; F, lived on farm containing bee yards. t Lowest concentrationof HBV capable of producing a 2+ (6 x 6 mm wheal) or greater intrademral skin test response. $A, Asthma: AC, abdominal cramps; AE, angioedema; R, rhinitis; S, shock; U, urticaria.
patients had received mixed Hymenoptera whole body immunotherapy previously or were receiving such injections at the time of rheir initial evaluation. In 7 instances the patient had continued to experience generalized reactions to bee stings while receiving whole body therapy.
Honeybee
venom
Honeybee venom (HBV) was collected by electrical stimulation of intact colonies in the Rochester, Minn., vicinity.a Dried venom was scraped from glass collection plates, taken up in 0.1 M NH,HCOa (pH 8.4), and centrifuged to remove par:iculate material. The venom was then dialyzed using 3500 mw cutoff casing against 3 changes of 0.1 M NH,HCOa for 24 hr at 4” C, and the protein content of the dialyzed venom was measured by the biuret reaction using human serum albumin (HSA) as the standard.’ The dialyzed venom was sterilized by sequential filtration through Millipore filters and lyophilized in individual ampules. Venom was reconstituted using Sorenson’s phosphate buffer containing 0.5% HSA. Venom solutions were sterile when cultured in thioglycolate broth and were pyrogen-free as determined by injection into rabbits. The phospholipase A (PLA) content of the dialyzed HBV was estimated by the egg yolk-agarose plate assay of Habermann and Hardt.” The hyaluronidase content was determined by radial diffusion in hyaluronic acid-containing agarose plates (Worthington Biochemical Corporation,
II. Enzymatic honeybee venom
TABLE
Enzyme
content
Activity
Phospholipase A Hyaluronidase Acid phosphatase
of dialyzed
per mg dialyzed
venom
337 u* 1,040 NF units 0.77 Sigma unitst
*One unit will hydrolyze 1.Opmole of L-o-lecithin to lysolecithin and fatty acid per minute at pH 8.5 at 37” C. tone unit will liberate 1.0 pmole of p-nitrophenol per hour at pH 4.8 at 37” C.
Freehold, N. J.). The acid phosphatase activity was measured by a calorimetric technique using a p-nitrophenol substrate (Sigma Chemical Company, St. Louis, MO.). The results are summarized in Table II. The relative enrichment produced by dialysis was assumed to be 2- to 4-fold for both hyaluronidase and PLA based on previously published findings with native honeybee venoms from a variety of s0urces.a
Immunologic
tests
HBV* was fractionated over a 5 x 100 cm calibrated Sephadex G-75 column equilibrated and eluted with 0.1 M *Sigma Chemical Company, St. Louis, MO.; Grade 1.
342
Yunginger
et al.
J. ALLERGY
I
10’
10.000
FIG. 1. Representative dose-response curves produced with the standard IgE serum pool in the HBV (O-O) and PLA (o-o) RAST. The correlation coefficient for each of these least squares regression lines was +0.99.
f \ 3
B
: LA
x
.
.
.
x 0
o
x
0
x
0 loo-
I
.
0
d 4”
ammonium formate (pH 4.5). Fractions eluting at approximately 15,000 to 20,000 daltons containing PLA were pooled, concentrated, and rechromatographed over the same column. Fractions were pooled, concentrated, dialyzed against 0.1 M ammonium formate (pH 4.75), and applied to a 2.5 X 18 cm column of CM-Sephadex equilibrated with the same buffer. The PLA eluted as a sharp, single peak after application of a linear gradient with a limiting buffer of 1.5 M ammonium formate (pH 4.75). Peak tubes were pooled, concentrated, dialyzed against 0.2 M NH,HCO, (pH 8.3), and lyophilized. This purified PLA (36 mg) was reacted with cyanogen bromide-activated microcrystalline cellulose (3 gm) to prepare a PLA radioallergosorbent test (RAST) reagent.‘O Crude HBV (500 mg) from the same source was also coupled to microcrystalline cellulose (2 gm). Serum IgE antibodies to HBV and PLA were measured by the direct RAST procedure as described previously.l” In each assay we included a standard IgE antibody pool composed of sera from 7 HBV-sensitive patients who were not receiving immunotherapy. This pool was assigned an arbitrary IgE antibody content of 1,000 U/ml; a dose-response curve using this pool was linear from 0.2 to 20 U (Fig. 1). IgE antibody levels in test serums were expressed in units by interpolation from this curve. The coefficient of variation for the assay ranged from 9.0% (PLA, 7 assays) to 11.6% (HBV, 10 assays). Serum from a nonallergic control patient contained 5 to 9 U IgE antibody per milliliter to HBV and 10 to 15 U IgE antibody per milliliter to PLA when tested repetitively in these RAST assays. Pretreatment IgE antibody levels to HBV or PLA in sting-sensitive patients were at least two times that of this negative control serum. Serum IgG antibody to PLA was measured by radioimmunoprecipitation. PLA was isolated from HBV by a single Sephadex G-75 gel filtration step, and 1 mg PLA was radiolabeled with 1 mCi 13’1 by the chloramine-T procedure.” Unreacted radioiodine was removed by gel filtration through Sephadex G-25 equilibrated with 1% bovine serum albumin (BSA) in 0.1 M phosphate buffer (pH 7.5). Void volume fractions were pooled and dialyzed for 3 days at 4”
-
1.000
CLIN. IMMUNOL. MAY 1979
x o
”
0
.
10 -
9
01 0
I 20 IgG
/ 40 Ab
PLA.
60
yg/ml
FIG. 2. Correlation between IgE antibodies to HBV and PLA, and IgG antibodies to PLA at the time of initial sting challenge following HBV immunotherapy. Patients challenged showed no reaction (0); mild reactions which did not require epinephrine (01; or moderate to severe reactions which required epinephrine therapy (“1. There was no correlation between the clinical response to stings and any of the three immunologic parameters.
C against 0.15 M NaCl. Phosphotungstic acid precipitated greater than 98% total radioactive counts in this preparation, which was then diluted in additional 1% BSA. A standard IgG antibody pool was prepared using equal quantities of serum from five nonallergic, frequently stung beekeepers. This pool contained 141 pg IgG antibody to PLA per milliliter, as calibrated against a standard serum in an immunoprecipitation experiment at Johns Hopkins University through the courtesy of Drs. L. M. Lichtenstein and A. K. Sobotka. This pool was diluted 1: 20 using 1% BSA; further twofold dilutions were made using a 1:20 dilution of a normal non-beekeeper control serum as the diluent. Radiolabeled PLA ( 1.5 pg) was added to 100 ~1 patient serum (1: 20) or to 100 ~1 of the standard IgG antibody pool (7 points from 11 to 705 ng) and diluted to 0.3 ml with 1% BSA. Tubes were incubated for 1 hr at 37” C, then overnight at 4” C, after which 0.25 ml burro anti-IgG (Fc) was added. After a second incubation for 1 hr at 37” C and overnight at 4” C, the resulting precipitates were washed three times with 0.1 M phosphate buffer, and the radioactivity associated with the washed precipitate was counted in a gamma spectrometer. The background binding produced by a nonimmune control serum was subtracted from all tubes. As performed, the lower limit of sensitivity of the assay was 0.4 pg/ml. The coefficient of variation for the assay was 5.7% for 28 assays.
VOLUME NUMBER
TABLE
Rush venom
63 5
ill. Clinical
results
of initial
HBV immunotherapy Initial
Patient
Original sting reaction*
I
AE, U
2
A. AC, AE, S
3 4 5
A, U A, U A, R, U
6 7 8 9 10 II I2 I4 IS
A A, AE AC, AE, U A, AE, U A, AE, U AE, U AE, U A, AE, U AE, U A, AE, S
I6
R, S, U
I7 I8 I9
A A, AE, U A, S, U AC, U
13
20 *A, Asthma; t Epinephrirle
No. courses 1 3 3 6 1
Cumulative dose (pg)
2
I I
657 1,124
2 3 9 2 2 4 2 2
3,858 2,062 5,657 1,371 1,148 2,888 2,940
I
2
AC, Abdominal cramps; AE, angioedema; therapy required.
P. pruritus
Patients were skin-tested by the puncture and intradermal techniques with dialyzed HBV at concentrations ranging from 0.0 I to 100 pg/ml. Irritant-positive intradermal tests were noted at venom concentrations of 100 pg/ml in 2 of 4 nonallergic patients and at 1,000 pg/ml in 4 of 4 nonallergic patients had significant
positive
intradermal skin tests at HBV concentrations of 10 pg/ml or less. Skin 1:estingwas not repeated following venom immunotherapy.
Treatment
protocol
by the patient. We aimed to admin-
ister a maximum single dose of 200 /*g HBV, which is equivalent to approximately four honeybee stings3 When this dose was achieved, or whenever a systemic reaction requiring epinephrine treatment was produced consistently by a given dose of venom, the patient was discharged and readmitted one month later either for further venom injections or for deliberate honeybee sting challenges. Between 1
Reaction*
3 3 3
A, AC
P
A, Ut Withdrew from study 4 NR 2 AE, Ut 3 NR 3 A 2 NR 2 NR 2 AE, S, Ut 2 4 4 I 3 2 3 3 4
ut
A, AE, Ut AE, S, Ut
A, AC, AE, Ut A, R, Ut A, R, U
AC, P urticaria;
sting challenge
No. stings
P A, AC, AE, R, U: A. R, U: A, AE, Ut NR NR AC, U: A, AE, R, S, Uf AE, Ut NR U U U
R, rhinitis;
ut
U NR A
NR NR AE, R, Ui U NR
S, shock; U, urticaria;
NR, no reaction,
and 4 honeybee sting challenges (depending on clinical response to each sting) were administered in one day, the greater number being administered to patients who anticipated having daily contact with bees in the future. Maintenance of immunity
was achieved
either by periodic
injec-
tions of 200 pg HBV or by deliberate bee stings. Baseline laboratory tests included complete blood count with leukocyte differential, urinalysis, and chest roentgenogram. Later in the course of the study, we also obtained a blood chemistry group and plasma corticoid determinations on all patients. Blood and urine tests and chest x-rays were repeated at periodic
Informed consent was obtained from all participants. Patients were admitted for treatment to a clinical research center, and an intravenous cannula was placed. Increasing increment:. of HBV were administered subcutaneously at 15 to 60-min intervals throughout the day for 2 to 3 days. Initial doses ranged from 0.05 ng to I pg and were usually increased by 5- to lo-pg increments depending on the clinical reactio:a experienced
Reaction*
without
343
challenge initial
1,975
Skin tests
patients. All sting-sensitive
sting
HBV immunotherapy
4,080 2,132 2,018 69 360 1,090 3,985 1,924 2,243 2,732
4 3
and initial
immunotherapy
intervals
throughout
the study period.
RESULTS Clinical results The clinical results of HBV immunotherapy are summarized in Tables III and IV. One patient withdrew early in the study because of anxiety over receiving injections. This patient had bronchial asthma, which repeatedly flared one to two days prior to admission;
wheezing
made
evaluation
of response
to
HBV injections impossible to assess. The remaining 19 patients required from one to nine monthly treatment sessions to reach the 200-wg dose. All patients developed warm, erythematous, swollen injection sites by the evening of the first day of therapy. These
344 Yunginger et al.
J. ALLERGY
CLIN. IMMUNOL. MAY 1979
TABLE IV. Clinical course after initial immunotherapy Subsequent sting challenges Patient,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
No. stings
Multiple 2 1 3 4 Multiple 3 1 2 1 3 1 1 Multiple 1 Multiple 1 2 2 Multiple 3 2 Multiple Multiple
Reaction*
Additional No. courses
Cumulative
dose (fig)
NR NR
1
825 -
1
100
-
-
-
NR -
1
Reaction *
k,ACR,Ut
-
-
-
-
NR NR
2 -
1
NR UT
-
-
-
1,900 1,650 -
NR
4
LJ? -
-
2,050
ut
NR U NR -
-
AE,Ut
At NR PSJ NR R AACSt NR NR
No. stings
-
A,R,UI NR NR NR U NR
NR UT NR
Reaction+
A,AC,AE,R’F
A,AC,Rt
A,Ut A,AE,U?
Subsequent sting challenges
HBV immunotherapy
-
1
I
-
-
$
2.800
A,Ut
-
-
-
I
A,Rt
-
-
*A, Asthma; AC, abdominal cramps; AE, angioedema;P, pruritis without urticaria; R, rhinitis; S, shock; U, urticaria; NR, no reaction. t Epinephrine therapy required. $ Weekly outpatient therapy for several months.
local reactions subsided within 24 to 36 hr following the last dose of HBV. Venom doses greater than 50 pg were accompanied by transient deep pain at the injection site. Systemic anaphylactic symptoms were common during treatment; 11 of 20 patients required subcutaneous administration of epinephrine for reversal of these symptoms at some point during their treatment. While most reactions were mild or moderate, consisting mainly of generalized urticaria and/or mild wheezing, 4 patients (Nos. 8, 14, 15, 16) experienced more severe episodes involving shock, severe bronchospasm, and/or cyanosis which required intravenous epinephrine therapy for reversal. No episodes of laryngeal edema were encountered. Cumulative HBV doses prior to sting challenge in 19 patients ranged between 360 and 5,657 @g (j? = 2,329 + 1,345 /&. Initial sting challenges following therapy were tolerated with minimal or no symptoms by 11 of 19
patients. Of the remaining 8 patients (Nos. 2, 3, 6, 8, 11, 12, 15, 18), 5 required epinephrine therapy (Table III). These 8 patients were given either additional courses of HBV therapy (Nos. 2, 11, 12, 15, 18) or additional deliberate stings (Nos. 3, 6, 8) (Table IV). Five of these patients were subsequently able to tolerate stings without difficulty. The remaining 3 patients (Nos. 2, 8, 18) continued to experience systemic reactions following sting challenges; all 3 were switched to once-weekly venom injections as outpatients. Two patients (Nos. 7 and 9) who experienced no reaction to their initial sting challenges later reacted to subsequent stings; both were given additional courses of HBV injections and were then stung again. Both achieved either partial (No. 7) or complete (No. 9) clinical protection. With regard to long-term maintenance therapy, 3 of 16 patients (Nos. 6, 9, 14) who successfully completed venom immunotherapy are receiving mainte-
VOLUME NUMBER
Rush venom immunotherapy
63 5
Long-term
follow-up
Maintenance dose/interval
Reaction*
I sting/ l-2 wk Variable/ l-4 wk
Oct. AE,P Freq. A,AC,Rt
I sting/4 wk
NR
Dropped from study 2 stings/4 wk 200 pug/4 wk
NR NR
1 sting/l-2 wk Variable/l-2 wk 1 sting/,? wk
Oct. A,Ut Freq. A,Ut NR
1 sting/G6 wk 1 sting/imeg. 1 sting/2-4 wk 1 sting/,4 wk 200 pg/ 12 wk 1 sting/ l-2 wk 1 sting/irreg.
NR NR occ. u NR NR Oct. At NR
1 sting/irreg. Variable/ l-4 wk
NR Freq. At
1 sting/irreg.
NR
Discontinued beekeeping; not stung
nance stings or venom injections every 4 to 12 wk at the Mayo Clinic; all are doing well. The remaining 13 patients have elected to self-administer maintenance stings at home. In some cases the compliance with recommendations regarding the timing of these booster stings has been less than ideal, either due to a lack of personal initiative on the part of the patient or to lack of access to bees during a bitterly cold winter in the upper midwestem United States. As a result, 4 of the 13 patients (Nos. 1, 7, 12, 15) have experienced some mild to moderate systemic reactions to repeat stings, always when the interval between stings has exceeded 3 to 8 wk. The remaining 9 patients have continued to do well. Immunologic
results
Table V summarizes IgE and IgG antibody levels prior to therapy, at the time of sting challenge, and at 12 to 24 months after beginning treatment. In two patients (Nos. 1 and 5), baseline IgE antibody levels to PLA were similar to those seen in nonallergic con-
345
trol sera, but both patients had elevated IgE antibody levels to HBV. Patient 14 had a minimal but definite elevation in baseline IgE antibody to HBV. IgE antibodies to both HBV and PLA rose in most patients after initiation of venom immunotherapy, each rising by a mean of 330% after the first treatment session. In approximately half of the patients, these IgE antibodies remained elevated above baseline values during a 12- to 29-month period of follow-up. The ultimate success or failure of HBV treatment could not be correlated with pretreatment IgE antibody levels to either HBV or PLA. Serum IgG antibodies rose steadily throughout the treatment period in all patients. In 7 patients the maximum IgG antibody level exceeded 40 pglml; this level is comparable to that seen in many commercial beekeepers who are stung daily or weekly without ill effects.” However, there was no absolute level of IgG antibody which was protective for all patients. The relationship between IgE and IgG antibody levels at the time of initial sting challenge is shown in Fig. 2. The clinical response to sting challenge could not be correlated with either IgE or IgG antibody levels. Moreover, there were 8 individual patients in whom we could demonstrate no consistent relationship between the level of 1gG antibody to PLA and the clinical response to subsequent deliberate sting challenges Other laboratory
results
There were no consistent abnormalities in hematological or urine tests or in chest roentgenograms after HBV therapy was initiated. In some instances baseline laboratory studies showed mild abnormalities, such as a slightly elevated serum creatinine and + 1 proteinuria in Patient 15, who had a previous nephrectomy for renal calculus disease. These values remained stable throughout her 9-mo period of HBV immunotherapy. DISCUSSION There are relatively few studies on the use of Hymenoptera venoms for the immunotherapy of stingsensitive persons. Because of previous reports on the successful use of “rush” venom immunization programs,2 and because many of our patients lived at some distance from Rochester, we elected to initiate our venom treatments by using a monthly inpatient program. Our use of an admittedly selected group of study patients was advantageous in many respects. The patients were knowledgeable about honeybees and their habits; they were also highly motivated to receive therapy, because in most cases they would have continued exposure to bees. Finally,
346 Yunginger et al.
TABLE
V. Serum
venom
treatment
J. ALLERGY
IgE (U/ml) and IgG (pglml)
Baseline
antibody
W Patient
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
antibody
HBV
PLA
PLA
361 1,977 796 2,903 224 2,932 66 2,267 1,025 150 58 73 990 13 2,916 198 289 4,600 1,295 56
10 1,228 1,642 2,160
0 7.2 0 0 0 7.9 0 0 0 0 0 0 0 0 18.2 0.5 0 5.0 9.4 0
510 149 660 89 220 28 74 116 41 1,030 262 350 5,655 2,005 42
levels to HBV and PLA before and after
initial IS
sting challenge
IgE HBV
Long-term W
PLA
PLA
44 79 16.7 2,234 1,592 30.3 2,780 3,285 31.1 Withdrew from study 224 7 0 2,932 510 7.9 796 698 18.4 24,665 4,560 21.0 1,267 156 10.9 906 1,052 53.2 1,532 554 32.1 474 236 34.0 1,326 70 0 162 279 5.6 1,615 2,420 56.9 745 594 44.3 624 2,542 9.8 4,475 7,220 16.3 2,750 3,200 24.1 40 58 3.3
the insect responsible for the sting reaction could be identified with certainty, thus circumventing possible diagnostic dilemmas. Early in the study we became impressed with the wide variety of individual patient responses to HBV injections. Moreover, when the HBV doses were pushed beyond the patients’ tolerance, the signs and symptoms of anaphylaxis tended to be stereotyped for a given individual. These symptoms almost always duplicated those experienced by the patients at the time of their original sting reactions. Most anaphylactic episodes occurred during the treatment of the first several patients. With further experience with the venom and modifications of the dosage schedule, fewer reactions were encountered as time went by. There was also a definite tendency for reactions to occur in late afternoon, probably as a cumulative effect of several injections during the day. Although almost all treated patients could ultimately receive 200 pg by injection, and the majority of patients are doing well on a long-term basis, we were somewhat surprised at the number of patients who had difficulty in maintaining their immunity. The program for maintenance of immunity was individualized and, in most cases, empirical. Most of our patients take 1 or 2 deliberate stings one day each
CLIN. IMMUNOL. MAY 1979
follow-up
W HBV
IS PLA
PLA
84 2,897 524
135 742 902
18.3 32.8 25.8
109 328 453 1,507 462 262 659 205 148 44 617 159 81 2,060 864 30
64 384 425 723 132 397 469 122 52 54 977 316 175 2,981 705 50
21.5 25.2 26.5 15.7 15.1 16.3 35.3 26.7 4.5 1.4 52.0 22.2 0 19.8 10.8 0
month; a few receive stings as infrequently as once every twelve weeks. However, there are a few patients who require stings once every week or two in order to maintain their clinical immunity. In at least 3 of our patients, attempts to prolong the interval between stings have resulted in a recurrence of mild systemic symptoms. This is contrary to the report of Loveless,13 who recommends booster stings at annual intervals to maintain immunity in wasp-sensitive patients. None of the various immunological parameters utilized in this study was able to successfully predict the clinical response to sting challenges in all the patients. Although PLA was the first well-characterized allergen described in HBV,r4 reports of other allergenic components in HBV are also emerging.‘“-l8 Two of our HBV-sensitive patients were initially unreactive to PLA by RAST, a finding previously noted by Light and co-workers.rs The fact that dialyzed venom was used for immunotherapy rather than whole venom may have influenced the clinical results. The dialysis procedure probably removed a portion of the melittin as well as apamin and other polypeptide components, although these materials were not assayed. While melittin has been noted to be allergenic in selected patients,15, 17, 2o apamin has not been found to be
VOLUME NUMBER
63 5
allergenic.‘!’ In the future it will probably be necessary to me.asureserum IgE antibodies to a variety of HBV components in order to ascertain the spectrum of sensitivity for each individual patient. It would also be logical to monitor serum IgG antibody levels to those venom components to which a patient exhibits elevated pretreatment IgE antibodies. We emphasize that the treatment regimen employed in this study was aggressive and that anaphylactic reactions during therapy were frequent. All patients were under the direct observation of the physicians in charge of the study during the period when venom was being administered. At present, we have abandoned the inpatient rush immunotherapy program; we have substituted a conventional outpatient, once-weekly injection program and are achieving a greater percentage of clinical successes with markedly lessened morbidity. When venoms are released for marketing by the Food and Drug Administration, there would appear to be little or no advantage in using a rush immunotherapy program as opposed to conventional once-weekly injections. We are indebted to Mr. Dennis Lind for obtaining the honeybeevenom used in this project; to Dr. G. J. Gleich for reviewing the manuscript; to Dr. S. F. Phillips, Director of the Clinical ResearchCenter: and to Ms. Lee Fast, R.N. and her nursing staff, without whose support these studies would not have been possible. REFERENCES 1. Sobotka AK, Valentine MD, Benton AW, Lichtenstein LM: Allergy to insect stings. 1. Diagnosis of IgE-mediated Hymenoptera sensitivity by venom-induced histamine release, .I ALLER’SY CLIN IMMUNOL 53: 170, 1974. 2. Loveless MH, Fackler WR: Wasp venom allergy and immunity, Ann Allergy 14~347, 1956. 3. Lichtenstein LM, Valentine MD, Sobotka AK: A case for venom treatment in anaphylactic sensitivity to Hymenoptera sting. N Engl J Med 290: 1223, 1974. 4. Busse WW. Reed CE, Lichtenstein LM, Reisman RE: Immunotherapy in beesting anaphylaxis. Use of honeybee venom, JAMA 231: 1154, 1975. 5. Hunt KJ, Valentine MD, Sobotka AK, Amodio FJ, Lichten-
Rush venom
immunotherapy
347
stein LM: A controlled trial of immunotherapy in insect hy-
persensitivity,N Engl J Med 299:157, 1978. 6. Benton AW, Morse RA, Stewart JD: Venom collection from honey bees, Science 142228, 1963. 7. Kabat EA. Mayer MM: In Experimental immunochemistry, ed. 2, Springfield, III., 1967, Charles C Thomas, Publisher, ch. 27, p. 559. 8. Habermann E, Hardt KL: A sensitive and specific plate test for the quantitation of phospholipase, Anal Biochem 50~163, 1972. 9. Franklin R, Baer H: Comparison of honeybee venoms and their
components from various sources. J ALLERGY CLIN IMMUNOL 1975. 10. Gleich GJ, Yunginger JW: Standardization of allergens, in Rose NR, Friedman H, editors: Manual of clinical immunology, Washington D. C., 1976, American Society for Microbiology, p. 575. 1I. Greenwood FC, Hunter WM, Glover JS: The preparation of ‘“‘I-labelled human growth hormone of high specific radioactivity. Biochem J 89: 114, 1963. 12. Yunginger JW, Jones RT, Leiferman KM, Paul1 BR, Welsh PW, Gleich GJ: Immunological and biochemical studies in beekeepers and their family members, J ALLERGY CLIN IM55:285,
MUNOL
61:93,
1978.
13. Loveless MH: Triple stings by captive wasps to appraise and to booster immunity in venom allergy. Ann Allergy 38:299. 1977. (Abst.) 14. Sobotka AK, Franklin RM, Adkinson NF Jr, Valentine MD, Baer H. Lichtenstein LM: Allergy to insect stings. II. Phospholipase A: The major allergen in honeybee venom, J ALLERGYCLIN IMMUNOL 51~29, 1976. 15. Hoffman DR. Shipman WH: Allergens in bee venom. I. Separation and identification of the major allergens, J ALLERGY CLIN IMMUNOL S&551. 1976. 16. Hoffman DR. Shipman WH, Babin D: Allergens in bee venom. II. Two new high molecular weight allergenic specificities, J ALLERGY CLIN IMMUNOL 59: 147, 1977. 17. Paul1 BR, Yunginger JW, Gleich GJ: Mel&in: An allergen of honeybee venom, J ALLERGY CLM IMMUNOL 59334, 1977. 18. Karpas AB, Baer H, Hooton ML, Evans R: A high molecular weight allergenic fraction of honeybee venom, 3 ALLERGY CLIN IMMUNOL 60: 155, 1977. 19. Light WC, Reisman RE, Ilea VS, Wypych JI, Okazaki T, Arbesman CE: Studies of the antigenicity and allergenicity of phospholipase A, of bee venom, J ALLERGY CLIN IMMUNOL 58:322, 1976. 20. King TP, Sobotka AK, Kochoumian L, Lichtenstein LM: Al-
lergens of honeybee venom, Arch Biochem Biophys 172:661, 1976.
program
J. W. Yunginger, M.D., * B. R. Paull, M.D., R. T. Jones, B.S., and P. J. Santrach, B.A. Rochester, Minn.
The clinical and immunological effects of honeybee venom (HBV) immunotherapy were studied in 20 HBV-sensitive patients. Patients were treated once monthly in a hospital setting by “rusk” immunotherapy; we aimed for a maximum single dose of 200 pg. One patient withdrew shortly after beginning therapy. Eleven patients required epinephrine during treatment for reversal of anaphylaxis. Patients who tolerated maximum doses were readmitted one month later for deliberate honeybee sting challenges. Stings were tolerated by 11 of 19 patients; 5 of the remaining 8 required epinephrine therapy. Twelve of 16 patients achieved long-term protection as determined by deliberate stings; 4 of 16 achieved partial protection. Three additional patients, who were treatment failures, were switched to weekly immunotherapy. IgE antibodies to HBV and phospholipase A (PL.A) rose shortly after immunotherapy was begun and in many patients remained elevated above baseline values. Serum IgG antibodies to PU rose steadily during therapy; there was no absolute level of IgG antibody which was protective for all patients. Although satisfactory long-term clinical results were achieved for most patients, the frequency of systemic reactions during the rush immunization caused us to abandon this form of treatment in favor of a once-weekly outpatient injection program.
Systemic allergic reactions to Hymenoptera stings are mediated by IgE antibodies to venom and venom components.’ Although venom immunotherapy programs have been reported previously by some allergists,’ conventional allergy practice has been to treat Hymenoptera sting- sensitive patients with injections of whole insect body extracts. Previous case reports have documented the clinical and immunological consequences of venom immunotherapy in two honeybee-sensitive patients. 3, 4 A recent controlled clinical From the Departments of Pediatrics and Internal Medicine (Allergy), and the Allergic Diseases ResearchLaboratory, Mayo Clinic. Supportedin part by a grant (Al- 11483)and contract (A l-42546) from the National Institute of Allergy and Infectious Diseases;a contract from the Food and Drug Administration (223-77-1202); a Clinical ResearchCenter grant from the National Institutes of Health (RR-585); and by Mayo Foundation. Presentedin part at the American Congressof Allergy and Immunology, New York, N. Y., March 28, 1977, and at the meeting of the American Pediatric Society and Society for Pediatric Research, San Francisco, Calif., April 28, 1977. Received for publication March 6, 1978. Accepted for publication Nov. 13, 1978. Reprint requests to: John W. Yunginger, M.D., Allergic Diseases Research Laboratory, Mayo Clinic, 200 First St. SW, Rochester, MN 55901. *Recipient of an Allergic DiseasesAcademic Award (Al-00107). Vol. 63, No. 5, pp. 340-347
study which compared venom, whole body, and placebo immunotherapy has demonstrated the clear superiority of the venom treatment, as judged by deliberate sting challenges following therapy.5 The present study was conducted in 1975 and 1976 to determine the clinical and immunological changes occurring during an inpatient “rush” honeybee venom immunotherapy program in honeybee sting- sensitive beekeepers or beekeeper family members; these persons represent a “high-risk” category with respect to receiving future stings. MATERIALS Patients
AND METHODS
Twenty honeybee sting-sensitive patients (12 females, 8 males) were identified during visits to the annual conventions of the Minnesota Beekeepers Association or were referred via other beekeepers or their physicians. The clinical characteristics of the study group are shown in Table I. Patients ranged in age from 4 to 66 yr (X = 32.6 yr). All patients had clinical histories of systemic anaphylactic reactions following honeybee stings, with symptoms ranging from generalized urticaria to profound shock. Three patients were beekeepers: 16 were members of beekeepers’ families (9 wives, 7 children); and 1 lived on a farm containing bee yards. In all cases the insect responsible for the sting reaction could reliably be identified as a honeybee. Eleven of the
0091-6749/79/050340+08$00.80/0
63 1979 The C. V. Mosby
Co.
VOLUME NUMBER
TABLE
Rush venom immunotherapy
63 5
I. Clinical
characteristics
of the study group Whole
Patient No.1 sex/ age (W
body extract
immunotherapy
Stung while Skin test Occupation*
BW BC BC BW BW F BW BW BC BC BC BC B B BW BW BW BC BW B
I /F/52 2/F/17 3/M/9 4/F/40 5/F/56 6/M/l I 7/F/50 8/F/54 9/M/4 10/M/5 1l/M/1.7 12/M/8 13/M/43 14/M/66 15/F/60 16/F/32 17/F/36 18/F/18 19/F/38 20/F/36
341
(pglml)t
10 0.01 0.01 1 10 0.01 0.1 10 0.01 0.1 0.01 I 10 0.01 0.1 0.01 0.01 1 0.1 10
Clinical reaction*
AE, U A, AC, AE, S A, U A. U A, R, U A A. AE AC, AE, U A, AE, U A, AE, U AE, U AE, U A, AE, U AE, U A, AE, S R, S, U A A, AE, U A, S, U AC, U
Previous
Rx
Anaphylaxis
+ +
+ +
+
+
+ + +
+
+ +
on Rx No reaction
Not stung while on Rx
+
+ +
+ + +
+
*B, Beekeeper; BC, beekeeper’s child; BW, beekeeper’s wife; F, lived on farm containing bee yards. t Lowest concentrationof HBV capable of producing a 2+ (6 x 6 mm wheal) or greater intrademral skin test response. $A, Asthma: AC, abdominal cramps; AE, angioedema; R, rhinitis; S, shock; U, urticaria.
patients had received mixed Hymenoptera whole body immunotherapy previously or were receiving such injections at the time of rheir initial evaluation. In 7 instances the patient had continued to experience generalized reactions to bee stings while receiving whole body therapy.
Honeybee
venom
Honeybee venom (HBV) was collected by electrical stimulation of intact colonies in the Rochester, Minn., vicinity.a Dried venom was scraped from glass collection plates, taken up in 0.1 M NH,HCOa (pH 8.4), and centrifuged to remove par:iculate material. The venom was then dialyzed using 3500 mw cutoff casing against 3 changes of 0.1 M NH,HCOa for 24 hr at 4” C, and the protein content of the dialyzed venom was measured by the biuret reaction using human serum albumin (HSA) as the standard.’ The dialyzed venom was sterilized by sequential filtration through Millipore filters and lyophilized in individual ampules. Venom was reconstituted using Sorenson’s phosphate buffer containing 0.5% HSA. Venom solutions were sterile when cultured in thioglycolate broth and were pyrogen-free as determined by injection into rabbits. The phospholipase A (PLA) content of the dialyzed HBV was estimated by the egg yolk-agarose plate assay of Habermann and Hardt.” The hyaluronidase content was determined by radial diffusion in hyaluronic acid-containing agarose plates (Worthington Biochemical Corporation,
II. Enzymatic honeybee venom
TABLE
Enzyme
content
Activity
Phospholipase A Hyaluronidase Acid phosphatase
of dialyzed
per mg dialyzed
venom
337 u* 1,040 NF units 0.77 Sigma unitst
*One unit will hydrolyze 1.Opmole of L-o-lecithin to lysolecithin and fatty acid per minute at pH 8.5 at 37” C. tone unit will liberate 1.0 pmole of p-nitrophenol per hour at pH 4.8 at 37” C.
Freehold, N. J.). The acid phosphatase activity was measured by a calorimetric technique using a p-nitrophenol substrate (Sigma Chemical Company, St. Louis, MO.). The results are summarized in Table II. The relative enrichment produced by dialysis was assumed to be 2- to 4-fold for both hyaluronidase and PLA based on previously published findings with native honeybee venoms from a variety of s0urces.a
Immunologic
tests
HBV* was fractionated over a 5 x 100 cm calibrated Sephadex G-75 column equilibrated and eluted with 0.1 M *Sigma Chemical Company, St. Louis, MO.; Grade 1.
342
Yunginger
et al.
J. ALLERGY
I
10’
10.000
FIG. 1. Representative dose-response curves produced with the standard IgE serum pool in the HBV (O-O) and PLA (o-o) RAST. The correlation coefficient for each of these least squares regression lines was +0.99.
f \ 3
B
: LA
x
.
.
.
x 0
o
x
0
x
0 loo-
I
.
0
d 4”
ammonium formate (pH 4.5). Fractions eluting at approximately 15,000 to 20,000 daltons containing PLA were pooled, concentrated, and rechromatographed over the same column. Fractions were pooled, concentrated, dialyzed against 0.1 M ammonium formate (pH 4.75), and applied to a 2.5 X 18 cm column of CM-Sephadex equilibrated with the same buffer. The PLA eluted as a sharp, single peak after application of a linear gradient with a limiting buffer of 1.5 M ammonium formate (pH 4.75). Peak tubes were pooled, concentrated, dialyzed against 0.2 M NH,HCO, (pH 8.3), and lyophilized. This purified PLA (36 mg) was reacted with cyanogen bromide-activated microcrystalline cellulose (3 gm) to prepare a PLA radioallergosorbent test (RAST) reagent.‘O Crude HBV (500 mg) from the same source was also coupled to microcrystalline cellulose (2 gm). Serum IgE antibodies to HBV and PLA were measured by the direct RAST procedure as described previously.l” In each assay we included a standard IgE antibody pool composed of sera from 7 HBV-sensitive patients who were not receiving immunotherapy. This pool was assigned an arbitrary IgE antibody content of 1,000 U/ml; a dose-response curve using this pool was linear from 0.2 to 20 U (Fig. 1). IgE antibody levels in test serums were expressed in units by interpolation from this curve. The coefficient of variation for the assay ranged from 9.0% (PLA, 7 assays) to 11.6% (HBV, 10 assays). Serum from a nonallergic control patient contained 5 to 9 U IgE antibody per milliliter to HBV and 10 to 15 U IgE antibody per milliliter to PLA when tested repetitively in these RAST assays. Pretreatment IgE antibody levels to HBV or PLA in sting-sensitive patients were at least two times that of this negative control serum. Serum IgG antibody to PLA was measured by radioimmunoprecipitation. PLA was isolated from HBV by a single Sephadex G-75 gel filtration step, and 1 mg PLA was radiolabeled with 1 mCi 13’1 by the chloramine-T procedure.” Unreacted radioiodine was removed by gel filtration through Sephadex G-25 equilibrated with 1% bovine serum albumin (BSA) in 0.1 M phosphate buffer (pH 7.5). Void volume fractions were pooled and dialyzed for 3 days at 4”
-
1.000
CLIN. IMMUNOL. MAY 1979
x o
”
0
.
10 -
9
01 0
I 20 IgG
/ 40 Ab
PLA.
60
yg/ml
FIG. 2. Correlation between IgE antibodies to HBV and PLA, and IgG antibodies to PLA at the time of initial sting challenge following HBV immunotherapy. Patients challenged showed no reaction (0); mild reactions which did not require epinephrine (01; or moderate to severe reactions which required epinephrine therapy (“1. There was no correlation between the clinical response to stings and any of the three immunologic parameters.
C against 0.15 M NaCl. Phosphotungstic acid precipitated greater than 98% total radioactive counts in this preparation, which was then diluted in additional 1% BSA. A standard IgG antibody pool was prepared using equal quantities of serum from five nonallergic, frequently stung beekeepers. This pool contained 141 pg IgG antibody to PLA per milliliter, as calibrated against a standard serum in an immunoprecipitation experiment at Johns Hopkins University through the courtesy of Drs. L. M. Lichtenstein and A. K. Sobotka. This pool was diluted 1: 20 using 1% BSA; further twofold dilutions were made using a 1:20 dilution of a normal non-beekeeper control serum as the diluent. Radiolabeled PLA ( 1.5 pg) was added to 100 ~1 patient serum (1: 20) or to 100 ~1 of the standard IgG antibody pool (7 points from 11 to 705 ng) and diluted to 0.3 ml with 1% BSA. Tubes were incubated for 1 hr at 37” C, then overnight at 4” C, after which 0.25 ml burro anti-IgG (Fc) was added. After a second incubation for 1 hr at 37” C and overnight at 4” C, the resulting precipitates were washed three times with 0.1 M phosphate buffer, and the radioactivity associated with the washed precipitate was counted in a gamma spectrometer. The background binding produced by a nonimmune control serum was subtracted from all tubes. As performed, the lower limit of sensitivity of the assay was 0.4 pg/ml. The coefficient of variation for the assay was 5.7% for 28 assays.
VOLUME NUMBER
TABLE
Rush venom
63 5
ill. Clinical
results
of initial
HBV immunotherapy Initial
Patient
Original sting reaction*
I
AE, U
2
A. AC, AE, S
3 4 5
A, U A, U A, R, U
6 7 8 9 10 II I2 I4 IS
A A, AE AC, AE, U A, AE, U A, AE, U AE, U AE, U A, AE, U AE, U A, AE, S
I6
R, S, U
I7 I8 I9
A A, AE, U A, S, U AC, U
13
20 *A, Asthma; t Epinephrirle
No. courses 1 3 3 6 1
Cumulative dose (pg)
2
I I
657 1,124
2 3 9 2 2 4 2 2
3,858 2,062 5,657 1,371 1,148 2,888 2,940
I
2
AC, Abdominal cramps; AE, angioedema; therapy required.
P. pruritus
Patients were skin-tested by the puncture and intradermal techniques with dialyzed HBV at concentrations ranging from 0.0 I to 100 pg/ml. Irritant-positive intradermal tests were noted at venom concentrations of 100 pg/ml in 2 of 4 nonallergic patients and at 1,000 pg/ml in 4 of 4 nonallergic patients had significant
positive
intradermal skin tests at HBV concentrations of 10 pg/ml or less. Skin 1:estingwas not repeated following venom immunotherapy.
Treatment
protocol
by the patient. We aimed to admin-
ister a maximum single dose of 200 /*g HBV, which is equivalent to approximately four honeybee stings3 When this dose was achieved, or whenever a systemic reaction requiring epinephrine treatment was produced consistently by a given dose of venom, the patient was discharged and readmitted one month later either for further venom injections or for deliberate honeybee sting challenges. Between 1
Reaction*
3 3 3
A, AC
P
A, Ut Withdrew from study 4 NR 2 AE, Ut 3 NR 3 A 2 NR 2 NR 2 AE, S, Ut 2 4 4 I 3 2 3 3 4
ut
A, AE, Ut AE, S, Ut
A, AC, AE, Ut A, R, Ut A, R, U
AC, P urticaria;
sting challenge
No. stings
P A, AC, AE, R, U: A. R, U: A, AE, Ut NR NR AC, U: A, AE, R, S, Uf AE, Ut NR U U U
R, rhinitis;
ut
U NR A
NR NR AE, R, Ui U NR
S, shock; U, urticaria;
NR, no reaction,
and 4 honeybee sting challenges (depending on clinical response to each sting) were administered in one day, the greater number being administered to patients who anticipated having daily contact with bees in the future. Maintenance of immunity
was achieved
either by periodic
injec-
tions of 200 pg HBV or by deliberate bee stings. Baseline laboratory tests included complete blood count with leukocyte differential, urinalysis, and chest roentgenogram. Later in the course of the study, we also obtained a blood chemistry group and plasma corticoid determinations on all patients. Blood and urine tests and chest x-rays were repeated at periodic
Informed consent was obtained from all participants. Patients were admitted for treatment to a clinical research center, and an intravenous cannula was placed. Increasing increment:. of HBV were administered subcutaneously at 15 to 60-min intervals throughout the day for 2 to 3 days. Initial doses ranged from 0.05 ng to I pg and were usually increased by 5- to lo-pg increments depending on the clinical reactio:a experienced
Reaction*
without
343
challenge initial
1,975
Skin tests
patients. All sting-sensitive
sting
HBV immunotherapy
4,080 2,132 2,018 69 360 1,090 3,985 1,924 2,243 2,732
4 3
and initial
immunotherapy
intervals
throughout
the study period.
RESULTS Clinical results The clinical results of HBV immunotherapy are summarized in Tables III and IV. One patient withdrew early in the study because of anxiety over receiving injections. This patient had bronchial asthma, which repeatedly flared one to two days prior to admission;
wheezing
made
evaluation
of response
to
HBV injections impossible to assess. The remaining 19 patients required from one to nine monthly treatment sessions to reach the 200-wg dose. All patients developed warm, erythematous, swollen injection sites by the evening of the first day of therapy. These
344 Yunginger et al.
J. ALLERGY
CLIN. IMMUNOL. MAY 1979
TABLE IV. Clinical course after initial immunotherapy Subsequent sting challenges Patient,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
No. stings
Multiple 2 1 3 4 Multiple 3 1 2 1 3 1 1 Multiple 1 Multiple 1 2 2 Multiple 3 2 Multiple Multiple
Reaction*
Additional No. courses
Cumulative
dose (fig)
NR NR
1
825 -
1
100
-
-
-
NR -
1
Reaction *
k,ACR,Ut
-
-
-
-
NR NR
2 -
1
NR UT
-
-
-
1,900 1,650 -
NR
4
LJ? -
-
2,050
ut
NR U NR -
-
AE,Ut
At NR PSJ NR R AACSt NR NR
No. stings
-
A,R,UI NR NR NR U NR
NR UT NR
Reaction+
A,AC,AE,R’F
A,AC,Rt
A,Ut A,AE,U?
Subsequent sting challenges
HBV immunotherapy
-
1
I
-
-
$
2.800
A,Ut
-
-
-
I
A,Rt
-
-
*A, Asthma; AC, abdominal cramps; AE, angioedema;P, pruritis without urticaria; R, rhinitis; S, shock; U, urticaria; NR, no reaction. t Epinephrine therapy required. $ Weekly outpatient therapy for several months.
local reactions subsided within 24 to 36 hr following the last dose of HBV. Venom doses greater than 50 pg were accompanied by transient deep pain at the injection site. Systemic anaphylactic symptoms were common during treatment; 11 of 20 patients required subcutaneous administration of epinephrine for reversal of these symptoms at some point during their treatment. While most reactions were mild or moderate, consisting mainly of generalized urticaria and/or mild wheezing, 4 patients (Nos. 8, 14, 15, 16) experienced more severe episodes involving shock, severe bronchospasm, and/or cyanosis which required intravenous epinephrine therapy for reversal. No episodes of laryngeal edema were encountered. Cumulative HBV doses prior to sting challenge in 19 patients ranged between 360 and 5,657 @g (j? = 2,329 + 1,345 /&. Initial sting challenges following therapy were tolerated with minimal or no symptoms by 11 of 19
patients. Of the remaining 8 patients (Nos. 2, 3, 6, 8, 11, 12, 15, 18), 5 required epinephrine therapy (Table III). These 8 patients were given either additional courses of HBV therapy (Nos. 2, 11, 12, 15, 18) or additional deliberate stings (Nos. 3, 6, 8) (Table IV). Five of these patients were subsequently able to tolerate stings without difficulty. The remaining 3 patients (Nos. 2, 8, 18) continued to experience systemic reactions following sting challenges; all 3 were switched to once-weekly venom injections as outpatients. Two patients (Nos. 7 and 9) who experienced no reaction to their initial sting challenges later reacted to subsequent stings; both were given additional courses of HBV injections and were then stung again. Both achieved either partial (No. 7) or complete (No. 9) clinical protection. With regard to long-term maintenance therapy, 3 of 16 patients (Nos. 6, 9, 14) who successfully completed venom immunotherapy are receiving mainte-
VOLUME NUMBER
Rush venom immunotherapy
63 5
Long-term
follow-up
Maintenance dose/interval
Reaction*
I sting/ l-2 wk Variable/ l-4 wk
Oct. AE,P Freq. A,AC,Rt
I sting/4 wk
NR
Dropped from study 2 stings/4 wk 200 pug/4 wk
NR NR
1 sting/l-2 wk Variable/l-2 wk 1 sting/,? wk
Oct. A,Ut Freq. A,Ut NR
1 sting/G6 wk 1 sting/imeg. 1 sting/2-4 wk 1 sting/,4 wk 200 pg/ 12 wk 1 sting/ l-2 wk 1 sting/irreg.
NR NR occ. u NR NR Oct. At NR
1 sting/irreg. Variable/ l-4 wk
NR Freq. At
1 sting/irreg.
NR
Discontinued beekeeping; not stung
nance stings or venom injections every 4 to 12 wk at the Mayo Clinic; all are doing well. The remaining 13 patients have elected to self-administer maintenance stings at home. In some cases the compliance with recommendations regarding the timing of these booster stings has been less than ideal, either due to a lack of personal initiative on the part of the patient or to lack of access to bees during a bitterly cold winter in the upper midwestem United States. As a result, 4 of the 13 patients (Nos. 1, 7, 12, 15) have experienced some mild to moderate systemic reactions to repeat stings, always when the interval between stings has exceeded 3 to 8 wk. The remaining 9 patients have continued to do well. Immunologic
results
Table V summarizes IgE and IgG antibody levels prior to therapy, at the time of sting challenge, and at 12 to 24 months after beginning treatment. In two patients (Nos. 1 and 5), baseline IgE antibody levels to PLA were similar to those seen in nonallergic con-
345
trol sera, but both patients had elevated IgE antibody levels to HBV. Patient 14 had a minimal but definite elevation in baseline IgE antibody to HBV. IgE antibodies to both HBV and PLA rose in most patients after initiation of venom immunotherapy, each rising by a mean of 330% after the first treatment session. In approximately half of the patients, these IgE antibodies remained elevated above baseline values during a 12- to 29-month period of follow-up. The ultimate success or failure of HBV treatment could not be correlated with pretreatment IgE antibody levels to either HBV or PLA. Serum IgG antibodies rose steadily throughout the treatment period in all patients. In 7 patients the maximum IgG antibody level exceeded 40 pglml; this level is comparable to that seen in many commercial beekeepers who are stung daily or weekly without ill effects.” However, there was no absolute level of IgG antibody which was protective for all patients. The relationship between IgE and IgG antibody levels at the time of initial sting challenge is shown in Fig. 2. The clinical response to sting challenge could not be correlated with either IgE or IgG antibody levels. Moreover, there were 8 individual patients in whom we could demonstrate no consistent relationship between the level of 1gG antibody to PLA and the clinical response to subsequent deliberate sting challenges Other laboratory
results
There were no consistent abnormalities in hematological or urine tests or in chest roentgenograms after HBV therapy was initiated. In some instances baseline laboratory studies showed mild abnormalities, such as a slightly elevated serum creatinine and + 1 proteinuria in Patient 15, who had a previous nephrectomy for renal calculus disease. These values remained stable throughout her 9-mo period of HBV immunotherapy. DISCUSSION There are relatively few studies on the use of Hymenoptera venoms for the immunotherapy of stingsensitive persons. Because of previous reports on the successful use of “rush” venom immunization programs,2 and because many of our patients lived at some distance from Rochester, we elected to initiate our venom treatments by using a monthly inpatient program. Our use of an admittedly selected group of study patients was advantageous in many respects. The patients were knowledgeable about honeybees and their habits; they were also highly motivated to receive therapy, because in most cases they would have continued exposure to bees. Finally,
346 Yunginger et al.
TABLE
V. Serum
venom
treatment
J. ALLERGY
IgE (U/ml) and IgG (pglml)
Baseline
antibody
W Patient
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
antibody
HBV
PLA
PLA
361 1,977 796 2,903 224 2,932 66 2,267 1,025 150 58 73 990 13 2,916 198 289 4,600 1,295 56
10 1,228 1,642 2,160
0 7.2 0 0 0 7.9 0 0 0 0 0 0 0 0 18.2 0.5 0 5.0 9.4 0
510 149 660 89 220 28 74 116 41 1,030 262 350 5,655 2,005 42
levels to HBV and PLA before and after
initial IS
sting challenge
IgE HBV
Long-term W
PLA
PLA
44 79 16.7 2,234 1,592 30.3 2,780 3,285 31.1 Withdrew from study 224 7 0 2,932 510 7.9 796 698 18.4 24,665 4,560 21.0 1,267 156 10.9 906 1,052 53.2 1,532 554 32.1 474 236 34.0 1,326 70 0 162 279 5.6 1,615 2,420 56.9 745 594 44.3 624 2,542 9.8 4,475 7,220 16.3 2,750 3,200 24.1 40 58 3.3
the insect responsible for the sting reaction could be identified with certainty, thus circumventing possible diagnostic dilemmas. Early in the study we became impressed with the wide variety of individual patient responses to HBV injections. Moreover, when the HBV doses were pushed beyond the patients’ tolerance, the signs and symptoms of anaphylaxis tended to be stereotyped for a given individual. These symptoms almost always duplicated those experienced by the patients at the time of their original sting reactions. Most anaphylactic episodes occurred during the treatment of the first several patients. With further experience with the venom and modifications of the dosage schedule, fewer reactions were encountered as time went by. There was also a definite tendency for reactions to occur in late afternoon, probably as a cumulative effect of several injections during the day. Although almost all treated patients could ultimately receive 200 pg by injection, and the majority of patients are doing well on a long-term basis, we were somewhat surprised at the number of patients who had difficulty in maintaining their immunity. The program for maintenance of immunity was individualized and, in most cases, empirical. Most of our patients take 1 or 2 deliberate stings one day each
CLIN. IMMUNOL. MAY 1979
follow-up
W HBV
IS PLA
PLA
84 2,897 524
135 742 902
18.3 32.8 25.8
109 328 453 1,507 462 262 659 205 148 44 617 159 81 2,060 864 30
64 384 425 723 132 397 469 122 52 54 977 316 175 2,981 705 50
21.5 25.2 26.5 15.7 15.1 16.3 35.3 26.7 4.5 1.4 52.0 22.2 0 19.8 10.8 0
month; a few receive stings as infrequently as once every twelve weeks. However, there are a few patients who require stings once every week or two in order to maintain their clinical immunity. In at least 3 of our patients, attempts to prolong the interval between stings have resulted in a recurrence of mild systemic symptoms. This is contrary to the report of Loveless,13 who recommends booster stings at annual intervals to maintain immunity in wasp-sensitive patients. None of the various immunological parameters utilized in this study was able to successfully predict the clinical response to sting challenges in all the patients. Although PLA was the first well-characterized allergen described in HBV,r4 reports of other allergenic components in HBV are also emerging.‘“-l8 Two of our HBV-sensitive patients were initially unreactive to PLA by RAST, a finding previously noted by Light and co-workers.rs The fact that dialyzed venom was used for immunotherapy rather than whole venom may have influenced the clinical results. The dialysis procedure probably removed a portion of the melittin as well as apamin and other polypeptide components, although these materials were not assayed. While melittin has been noted to be allergenic in selected patients,15, 17, 2o apamin has not been found to be
VOLUME NUMBER
63 5
allergenic.‘!’ In the future it will probably be necessary to me.asureserum IgE antibodies to a variety of HBV components in order to ascertain the spectrum of sensitivity for each individual patient. It would also be logical to monitor serum IgG antibody levels to those venom components to which a patient exhibits elevated pretreatment IgE antibodies. We emphasize that the treatment regimen employed in this study was aggressive and that anaphylactic reactions during therapy were frequent. All patients were under the direct observation of the physicians in charge of the study during the period when venom was being administered. At present, we have abandoned the inpatient rush immunotherapy program; we have substituted a conventional outpatient, once-weekly injection program and are achieving a greater percentage of clinical successes with markedly lessened morbidity. When venoms are released for marketing by the Food and Drug Administration, there would appear to be little or no advantage in using a rush immunotherapy program as opposed to conventional once-weekly injections. We are indebted to Mr. Dennis Lind for obtaining the honeybeevenom used in this project; to Dr. G. J. Gleich for reviewing the manuscript; to Dr. S. F. Phillips, Director of the Clinical ResearchCenter: and to Ms. Lee Fast, R.N. and her nursing staff, without whose support these studies would not have been possible. REFERENCES 1. Sobotka AK, Valentine MD, Benton AW, Lichtenstein LM: Allergy to insect stings. 1. Diagnosis of IgE-mediated Hymenoptera sensitivity by venom-induced histamine release, .I ALLER’SY CLIN IMMUNOL 53: 170, 1974. 2. Loveless MH, Fackler WR: Wasp venom allergy and immunity, Ann Allergy 14~347, 1956. 3. Lichtenstein LM, Valentine MD, Sobotka AK: A case for venom treatment in anaphylactic sensitivity to Hymenoptera sting. N Engl J Med 290: 1223, 1974. 4. Busse WW. Reed CE, Lichtenstein LM, Reisman RE: Immunotherapy in beesting anaphylaxis. Use of honeybee venom, JAMA 231: 1154, 1975. 5. Hunt KJ, Valentine MD, Sobotka AK, Amodio FJ, Lichten-
Rush venom
immunotherapy
347
stein LM: A controlled trial of immunotherapy in insect hy-
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