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Polymerized whole ragweed: An improved method of immunotherapy
Polymerized whole ragweed: An improved method of immunotherapy Edita Bacal, M.D., C. R. Zeiss, M.D., lrena Suszko, Roy Patterson, M.D., F.A.C.P. Chicago, Ill.
B.S., Doris
Levitz,
B.S., and
A single-blind study compared the effectiveness qf glutaraldehyde-treated polymerized ragweed with nonpolymerized monomeric ragweed. These studies are an extension of those previously reported for polymerized AgE using a readily available ragweed preparation containing all ragweed antigens. Nineteen ragweed-sensitive patients were randomized into 2 groups: 10 received the polymerized form and 9 received the monomeric form. Four parameters were followed: serum-specijc IgE against antigen E, total blocking antibody against antigen E, local and systemic reactions to injection therapy, and symptom score indices. Pretreatment levels of antigen E-specijic IgE and blocking antibody activity were similar in both groups. Ajier a total of 15,000 protein nitrogen units (PNU) had been given, blocking antibody activity in the monomer group rose from a mean of 173 ng AgE bound per ml to A mean of 2,813. The rise in blocking antibody activity in the polymer group WAS from a mean of 181 ng AgE bound per ml to 1,574. At 15,000 PNU, blocking antibody activity levels were not statistically difSerent in the 2 groups. After I year of treatmenf. no consistent decrease in postseasonal spec$ic IgE rise could be shown in either group. Forty times less erythema and 15 times less induration were found with polymerized ragweed. There were 7 systemic reactions with the monomer and none with the polymer. Both groups experienced symptomatic improvement with treatment.
may be clinically significant and which were not included in the AgE preparationspreviously used. Polymerization of the whole ragweed extract and a comparisonof its immune reactivity in rabbits to nonpolymerized monomer ragweed have been described previously.3 The polymerized preparation consistsof a partially purified, glutaraldehyde-treated whole ragweed extract containing a mixture of ragweed protein polymers with a molecular weight range of 200,000 to 20,000,OOOdaltons. Polymerized whole ragweed had 100 to 1,OOO-foldlessallergenicity than the monomer as shown by end point cutaneoustiters (skin reactivity) in humans.3Despite this decreased allergenicity, animal studiesshowedthat the polymer induced a greater immune responseagainst AgE than was induced by the monomer. This differed from results of studies with polymerized AgE in which a similar responsewas found using the polymeric and monomeric forms. 4 In the current study, polymerized whole ragweed was compared to monomer ragweed in 19 ragweedsensitive patients. The results suggestthat polymerized whole ragweed has increasedsafety becauseof decreased allergenicity. The polymer has retained immunogenicity and clinical efficacy. In this report, allergenicity refers to the ability to elicit an IgE-me-
Immunotherapy has been recognized to be an effective form of treatment of ragweed hay fever.’ A recent clinical and immunologic study utilizing a modified method of immunotherapy with polymerized ragweed antigen E (AgE) showed the polymer to be an improvement over therapy with antigen E.2 The polymer was shown to have equal clinical effectiveness compared to the monomer. In addition, the polymer caused fewer immediate-type reactions and required fewer injections to achieve the sametherapeutic response.The purposeof the current study is to investigate the clinical usefulness of polymerized whole ragweed in immunotherapy. Production of large amountsof polymerized whole ragweed is economically and technically more feasible than that for polymerized AgE. In addition, the whole ragweed mixture contains allergensother than antigen E which From the Section of Allergy-Immunology, Department of Medicine, Northwestern University Medical School. Supported by United States Public Health Service Allergy Diseases Centers Grant AI 11403 and the Ernest S. Bazley Grant. Received for publication Jan. 13, 1978. Accepted far publication July 24, 1978. Reprint requests to: Dr. C. R. Zeiss, Department of Medicine, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, Ill. 6061 I. 0091-6749~78/110289+06$00.60/0
@ 1978
The
C. V. Mosby
Co.
Vol.
62, No.
5, pp. 289-294
290
Bacal
J. ALLERGY
et al.
TABLE 1. Comparison
of local and systemic Total
Antigen
Monomer (9 pt .) Polymer (10 pt.)
Total
PNU
254,500 438,000
reactions
occurring
with polymerized
CLIN. IMMUNOL. NOVEMBER 1978
and monomeric
ragweed
AgE
(cc91
Erythema*
Induration*
360 9
60 4
916 1,577
Generalized
reaction
7 0
*Mean cm of induration and erythema for entire immunotherapy treatment program. PNU: protein nitrogen unit; AgE: antigen E, pt.: patient.
TABLE II. Patients’
blocking
antibody
response
Pretreatment
Monomer group Polymer group
173* (l-600) 181* (9-700)
to immunotherapy 100 PNU RWP I
(6wg
420 (l-1,000) 325 (10-700)
1,000 PNU (60 fig RWP 1
584 (l-1,600) 346 (lo-1,600)
15,ooO PNU ( 900 pg RWP )
2,813 (l-5,400) 1,574 (88-5,000)
*Mean ng of AgE bound per ml serum; range in brackets.
diated response and immunogenicity refers to the ability to induce an IgG-mediated responsein animals or humans. MATERIALS AND METHODS Selection of subjects Nineteen atopic patients with a history of seasonal rhinitis compatible with ragweed pollinosis and marked skin reactivity to short ragweed (Ambrosia eliator) (1 : 20 Hollister-Stier Laboratories, Spokane, Wash.) were selected for study. Eleven patients had received prior immunotherapy which had been discontinued for at least three years. Of those who had previous immunotherapy, there were 6 in the monomer group and 5 in the polymer group. Patients with asthma or other significant medical illnesses were excluded. In addition, all patients underwent an initial screening hemogram and urinalysis which were normal in all cases. The patient population consisted of I3 men and 6 women ranging in age from 22 to 53 years. The subjects were randomized into 2 groups, one to receive polymerized ragweed and one to receive monomer ragweed. There were 10 patients in the polymer group and 9 in the monomer group.
Methods of therapy and clinical evaluation Treatment was conducted in a single-blind fashion with either the monomer or the polymer. The polymerized antigen was made as previously described by polymerization of monomer antigen (obtained by the extraction of giant and short ragweed pollen) with glutaraldehyde treatment.3 The stock monomer and polymer ragweed preparations contained 0.3 mg ragweed protein (P)/ml. The monomer preparation contained 18 pg AgE/ml. These preparations approximated a 1: 100 preparation of crude ragweed extract based on the starting preparation of ragweed and the pro-
tein content. In order to record doses as positive numbers, I ml of the polymer and monomer preparation was considered to contain 5,000 protein nitrogen units (PNU). Each preparation was filtered through a 0.20-p bacterial filter (Nalge Filter Unit, Nalge Sybron Corp., Rochester, N. Y.) and cultured for bacteria and fungi. Fresh polymer and monomer extracts were prepared every 6 months because previous studies showed the polymer to be stable for at least 6 months.” Both were diluted in a solution of phosphate-buffered 0.15 M NaCI, pH 7.35, containing 0.5% phenol and stored at 4” C. The schedule of immunization was comparable to that used at the Northwestern University Allergy Service for routine pollen immunotherapy. One injection per week was given until a maintenance dose of 0.5 ml of 1: 100 (0. I.5 mg ragweed P) was achieved. Maintenance dosage should be achieved in 27 wk. Once a maintenance dose was reached, the frequency of injections was reduced to once every four weeks. Therapy was started in November, 1976. The patients completed symptom score sheets for the 6-wk ragweed season (August I4 to September 30) of 1976 prior to onset of therapy and for the same period during the ragweed season in 1977 after approximately 10 months of treatment. The daily symptom scores were derived from weighted total scores recorded by the patient for symptoms experienced and the amount of medication needed to control his symptoms. A daily record was kept by the patient of the following symptoms: sneezing, nasal congestion, nasal discharge, red pruritic eyes, cough, and wheezing. The severity was graded on a scale of 0 to 3, the former having no symptoms and the latter severe symptoms. In addition, the amounts of antihistamines, bronchodilators, and nasal spray were recorded and given a standard score. Local reactions at injection sites were determined by measuring the diameters of erythema and wheals by a nurse who did not know which of the two types of immunotherapy
VOLUME NUMBER
Polymerized
62 5
ragweed
immunotherapy
291
MONOMER
POLYMER PRE TREATMENT
whole
POST TREATMENT
PRE TREATMENT
POST TREATMENT
10000
*
1ooc
E & c ” P z
z a
100
W o! a
1c
FIG. 1. Antibody activity against ragweed AgE. Pretreatment sera are compared with posttreatment sera in the polymer and monomer groups after 15,000 PNU had been given. Note log scale. Values for two patients in the monomer group (---) are not reported at 15,000 because one patient was able to reach only 6,000 PNU after 1 year of treatment. The other patient had no response at 15,000 PNU and the response at 45,000 is shown.
were administered. Late local reactions occurring after several hours or systemic symptoms were reported and evaluated by the physician.
Immunologic
studies
Blood specimens were drawn from each patient before and after the 1976 ragweed season after 100, I ,000, 15,000 PNU had been given and at the end of the 1977 ragweed season. The serum obtained at the end of both ragweed seasons was collected in late October or early November. The sera were analyzed for antigen E specific IgE and total serum antigen E binding capacity. Measurement of&E specific IgE. IgE specific for antigen E was measured by the polystyrene tube radioimmunoassay technique previously described.‘j Briefly, polystyrene tubes were coated with purified myeloma IgEps. Anti-IgE (Behring Diagnostics, Sommerville, N. J.) was added in excess to form an IgE immunoabsorbent. A I : 10 dilution of the patients’ serum was added to the tubes and incubated in the cold for 48 hr, during which time a known fraction of the IgE in the serum was bound to the tube. After discarding the unreacted serum, the tubes were washed, and ‘? AgE was added in excess. The tubes were incubated for 48 hr in the cold. Unbound AgE was removed by washing
and bound counts were determined. The ng of AgE bound by specific IgE in I ml of serum was determined and the ng of IgE antibody specific for AgE per ml calculated.” Measurement of total antibody against AgE. Total serum AgE binding capacity was measured by a modification of the ammonium sulfate technique of Lidd and Farr.7, x Briefly, 0.1 ml aliquots of sera were incubated with 100 and 1,000 ng of ‘%I AgE. After appropriate incubation, the antibody-bound AgE was separated from the free AgE by precipitation with 40% saturated ammonium sulfate. The ng AgE bound by 1 ml serum in antigen excess was then calculated and the results expressed as ng AgE bound/ml serum.
RESULTS Therapeutic
safety
All patients in both treatment groups were monitored with hemograms and urinalyses. No abnormalities were noted in these laboratory teststhroughout one year of treatment. After approximately one year of treatment, the total amount of polymerized ragweed injected was approximately twice the amount of monomer. Despite the fact that the number of injections in both groups was
292
J. ALLERGY
Bacal et al.
TABLE III. Percent
change in postseasonal and after treatment
specific
CLIN. IMMUNOL. NOVEMBER 1978
total of 15,000 PNU had been given, blocking antibody activity in the monomergroup rose from a mean of 173 ng AgE bound per ml to a mean of 2,8 13. Patient Pretreatment* Patient Posttreetmentt Excluded from these averages was one patient who was unable to reach 15,000 PNU. In the polymerized Monomer + 439% + 87% ragweed group, the rise was from a mean of 181 ng 2 + 189% + 83% AgE bound per ml to 1,574. Blocking antibody activ3 +lOO% + 100% ity at all levels of treatment was not statistically dif4 + 81% - 24% ferent for the 2 groups(analysiswith Student’st test). 5 + 49% + 59% As can be seen in Fig. 1, the pattern of rise in 6 + 9% + 44% blocking antibody activity for individuals in both 7 0% 0% groups was similar. Each individual’s rise in antibody 8 28% + 35% activity (value at 15,000 PNU divided by pretreat9 35% + 42% ment value) was calculated, the individual rises addPolymer 1 +l,loo% + 84% ed, and divided by the number of individuals in each 2 + 111% 2 0% group reaching 15,000 PNU. The average individual 3 + 264% 3 + 87% rise in the monomer group was l&fold (O-56) and in 4 + 220% 4 + 32% the polymer group 18-fold (3-100). It is evident that 5 + 700% 5 0% both groups had low-, mid-, and high-responding in6 + 62% 6 + 81% dividuals. 7 + 41% 7 + 58% Before initiation of therapy, 66% of the monomer 8 + 20% 8 + 173% patients and 80% of the polymer patients showed a 9 36% 9 - 11% rise in postseasonalspecific IgE. In evaluating the 10 58% 10 - 20% decreasedor blunted postseasonalIgE rise after im*Percent increase in AgE-specific IgE after1976ragweed season munotherapy describedby previous investigators,g, lo withnoimmunotherapy. certain patterns could be noted (Table III). In the $-Percent increase in AgE-specific IgE after 1977ragweed season monomergroup, 3 patients out of 9 showeda blunted with ragweed immunotherapy. rise. In the polymer group, 5 out of 10 showed a blunting of the rise in IgE. nearly the same,the polymer group reachedthe maintenancedosagein the preplanned27 injections, while Clinical efficacy the monomer group, becauseof significant local or The majority of patients in both treatment groups systemic reactions, had to advance more slowly and reported subjective clinical improvement during the received less total antigen per injection. All patients ragweed season following treatment. Assessment in the polymer group received 15,000 PNU in 6 basedon analysis of their weighted symptom scores months. In the monomer group it took an average of revealed that 8 out of 9, or 88% of patients in the 8.1 months (6 to 11 months) to receive 15,000 PNU. monomer group, had a decreasein symptom scores Although a greater total amount of antigen was given after treatment. Seven out of 10 patientsor 70% of the to the polymer group, there was 40 times as much polymer group had a decrease in symptoms. The erythema and 15 times as much induration in the groups were not statistically different by this analysis monomer group as compared to the polymer group of their symptom scores(chi square). (Table I). Seven systemic reactions occurred in the Ragweedcounts were similar for seasonspreceding monomer group while none occurred in the polymer and following treatment. Fig. 2 compares symptom group. The systemic reactions consisted of genscores for 1976 and 1977 ragweed seasonfor both eralized urticaria, pruritus with throat tightness, or monomer and polymer groups. In both treatment nasalsymptomswith or without asthma.These sympgroups, the mean symptom score after treatment was toms were reversed by subcutaneousepinephrine and oral antihistaminesand did not progressto any serious less than pretreatment. The statistical significance varies on individual days (statistical analysis by Stusequelae. dent’s t test). Averages of symptom scoreswere taken Immunologic results for 2-day periods and the number of days demonstrating statistical significance was totaled. The monomer Blocking antibody activity against AgE rose in all group had approximately two weeks of statistically subjects treated (Table II and Fig. 1). Pretreatment significant decrease in symptoms scores while the levels of blocking antibody were not statistically difpolymer group had approximately one week. Of note ferent in the monomer and polymer groups. After a IgE before
VOLUME NUMBER
Polymerized
62 5
POLYMER
whole
ragweed
immunotherapy
293
-1976 ---*
1077 +
Pd.05
50,
40 30
20 10 it 0
0
:: MONOMER
I 0 i
l
l
50
l
@ 40
30 20 10 0 I
18
I
2’2
J
2’6’dOl
I
AUGUST
I
b
’
‘e’,‘3’?7’2: SEPTEMBER
‘?I5’29
I
FIG. 2. Mean symptom score indices for polymer and monomer groups for the 1976 (-) and 1977 (---) ragweed seasons. Symptom scores are averaged for two days at a time. Means and standard deviation for selected days are shown by vertical lines. Levels of significance less than 0.05 for selected days are shown by an asterisk.
is the difference in the pattern of curves in the two treatment groups. The polymer group experienced a peak and rapid drop in symptom scores during the season before treatment. This pattern is matched by that of the posttreatment scores. This rapid decline of symptoms may have contributed to the shorter period of symptom score improvement. In contrast to this pattern, the monomer group had a prolonged period of increased symptoms scores for the season prior to treatment Differences between symptom score patterns of the two randomly selected groups may have occurred by chance.
DISCUSSION The purpose of this study was to evaluate polymerized whole ragweedasa potentially improved method of immunotherapy. Polymerized antigen E, which was utilized in previous studies, has certain limitations as opposed to whole ragweed. Extensive fractionation procedures are required to obtain the
large amounts of AgE which would be required for general clinical use.‘I In addition, AgE is but one major allergen in ragweed. Ragweed antigens K, Ra3, Ra4, and Ra5 have been identified. 11, l2 The polymerized whole ragweed preparation has been demonstrated to contain at least AgE, AgK, and Ra3.r3 Polymerized whole ragweed hasbeen shown clinically safe and well tolerated with no systemic reactions and significantly less local reactivity as compared to the monomeric forms. In addition, it has resultedin immunologic changescomparableto those found with standard immunotherapy. Blocking antibody activity rose equally in both treatment groups. Treatment resulted in a blunting of the postseasonal IgE rise in some patients, but no statistically significant trend was noted. Mention has already been madeof the difference in patterns of symptom scores in the two groups. Although the monomer group appearedto have a more
294
Bacal
et al.
prolonged beneficial effect from therapy, there may have been a difference in the two groups of subjects who were selected at random. Overall, treatment with both preparations resulted in symptom amelioration during the ragweed season following therapy. In conclusion, polymerized whole ragweed therapy was found to be safe, as immunogenic as the monomer preparation, and capable of reducing symptoms of ragweed-sensitive individuals. REFERENCES 1. Lieberman, P., and Patterson, R.: Immunotherapy for atopic disease, Adv. Intern. Med. 19:391, 1974. 2. Metzger, W. J., Patterson, R., Zeiss, C. R., et al.: Comparison of polymerized and unpolymerized antigen E in immunotherapy of ragweed allergy, N. Engl. J. Med. 295: 1160, 1976. 3. Patterson, R., Suszko, I. M., Zeiss, C. R., et al.: Comparison of immune reactivity to polyvalent monomeric and polymeric ragweed antigens, J. ALLERGY CLIN. IMMUNOL. 61:28, 1978. 4. Patterson, R., Suszko, I. M., Pruzansky, J. J., et al.: Polymerized ragweed antigen E: In vivo elimination studies and reactivity with IgE antibody systems, J. Immunol. 110:1413, 1973. 5. Patterson, R., and Suszko, I. M.: Polymerized ragweed antigen E. IV. The analysis of the stability of molecular size and antigenic reactivity of the polymer, J. ALLERGY CLIN. IMMUNOL. 57~168, 1976.
J. ALLERGY
CLIN. IMMUNOL. NOVEMBER 1978
6. Zeiss, C. R., Pruzansky, J. J., Patterson, R., et al.: A solid phase radioimmunoassay for the quantitation of human reaginic antibody against ragweed antigen E, J. Immunol. 110:414, 1973. 7. Lidd, D., and Farr, R. S.: Primary interaction between It3’ labeled ragweed pollen and antibodies in the sera of human and rabbits, J. ALLERGY 33:45, 1962. 8. Zeiss, C. R., Metzger, W. J., and Levitz, D.: Quantative relationships between IgE antibody and blocking antibody specific for antigen E in patients given immunotherapy with ragweed antigen E, Clin. Exp. Immunol. 28:250, 1977. 9. Lichtenstein, L. M., Ishizaka, K., Norman, P. S., et al.: IgE antibody measurements in ragweed hayfever. Relationship to clinical severity and the results of immunotherapy, J. Clin. Invest. 52~472, 1973. 10. Levy, D. A., Lichtenstein, L. M., Goldstein, E. O., et al.: Immunologic and cellular changes accompanying the therapy of pollen allergy, J. Clin. Invest. 50:360, 1971. 11. King, T. P., Norman, P. S., and Connell, J. T.: Isolation and characterization of allergens from ragweed pollen. II. Biochemistry 3:458, 1964. 12. Griffiths, B. W., and Brunet, R.: Isolation of basic or protein antigen of low ragweed pollen, Can. J. Biochem. 49:396, 1971. 13. Zeiss, C. R., Ungs, C. J., Levitz, D., Suszko, I. M., and Patterson, R.: The immune response of patients with ragweed hayfever treated with polymerized ragweed antigens. (Submitted for publication in J. ALLERGY CLIN. IMMUNOL.)
B.S., Doris
Levitz,
B.S., and
A single-blind study compared the effectiveness qf glutaraldehyde-treated polymerized ragweed with nonpolymerized monomeric ragweed. These studies are an extension of those previously reported for polymerized AgE using a readily available ragweed preparation containing all ragweed antigens. Nineteen ragweed-sensitive patients were randomized into 2 groups: 10 received the polymerized form and 9 received the monomeric form. Four parameters were followed: serum-specijc IgE against antigen E, total blocking antibody against antigen E, local and systemic reactions to injection therapy, and symptom score indices. Pretreatment levels of antigen E-specijic IgE and blocking antibody activity were similar in both groups. Ajier a total of 15,000 protein nitrogen units (PNU) had been given, blocking antibody activity in the monomer group rose from a mean of 173 ng AgE bound per ml to A mean of 2,813. The rise in blocking antibody activity in the polymer group WAS from a mean of 181 ng AgE bound per ml to 1,574. At 15,000 PNU, blocking antibody activity levels were not statistically difSerent in the 2 groups. After I year of treatmenf. no consistent decrease in postseasonal spec$ic IgE rise could be shown in either group. Forty times less erythema and 15 times less induration were found with polymerized ragweed. There were 7 systemic reactions with the monomer and none with the polymer. Both groups experienced symptomatic improvement with treatment.
may be clinically significant and which were not included in the AgE preparationspreviously used. Polymerization of the whole ragweed extract and a comparisonof its immune reactivity in rabbits to nonpolymerized monomer ragweed have been described previously.3 The polymerized preparation consistsof a partially purified, glutaraldehyde-treated whole ragweed extract containing a mixture of ragweed protein polymers with a molecular weight range of 200,000 to 20,000,OOOdaltons. Polymerized whole ragweed had 100 to 1,OOO-foldlessallergenicity than the monomer as shown by end point cutaneoustiters (skin reactivity) in humans.3Despite this decreased allergenicity, animal studiesshowedthat the polymer induced a greater immune responseagainst AgE than was induced by the monomer. This differed from results of studies with polymerized AgE in which a similar responsewas found using the polymeric and monomeric forms. 4 In the current study, polymerized whole ragweed was compared to monomer ragweed in 19 ragweedsensitive patients. The results suggestthat polymerized whole ragweed has increasedsafety becauseof decreased allergenicity. The polymer has retained immunogenicity and clinical efficacy. In this report, allergenicity refers to the ability to elicit an IgE-me-
Immunotherapy has been recognized to be an effective form of treatment of ragweed hay fever.’ A recent clinical and immunologic study utilizing a modified method of immunotherapy with polymerized ragweed antigen E (AgE) showed the polymer to be an improvement over therapy with antigen E.2 The polymer was shown to have equal clinical effectiveness compared to the monomer. In addition, the polymer caused fewer immediate-type reactions and required fewer injections to achieve the sametherapeutic response.The purposeof the current study is to investigate the clinical usefulness of polymerized whole ragweed in immunotherapy. Production of large amountsof polymerized whole ragweed is economically and technically more feasible than that for polymerized AgE. In addition, the whole ragweed mixture contains allergensother than antigen E which From the Section of Allergy-Immunology, Department of Medicine, Northwestern University Medical School. Supported by United States Public Health Service Allergy Diseases Centers Grant AI 11403 and the Ernest S. Bazley Grant. Received for publication Jan. 13, 1978. Accepted far publication July 24, 1978. Reprint requests to: Dr. C. R. Zeiss, Department of Medicine, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, Ill. 6061 I. 0091-6749~78/110289+06$00.60/0
@ 1978
The
C. V. Mosby
Co.
Vol.
62, No.
5, pp. 289-294
290
Bacal
J. ALLERGY
et al.
TABLE 1. Comparison
of local and systemic Total
Antigen
Monomer (9 pt .) Polymer (10 pt.)
Total
PNU
254,500 438,000
reactions
occurring
with polymerized
CLIN. IMMUNOL. NOVEMBER 1978
and monomeric
ragweed
AgE
(cc91
Erythema*
Induration*
360 9
60 4
916 1,577
Generalized
reaction
7 0
*Mean cm of induration and erythema for entire immunotherapy treatment program. PNU: protein nitrogen unit; AgE: antigen E, pt.: patient.
TABLE II. Patients’
blocking
antibody
response
Pretreatment
Monomer group Polymer group
173* (l-600) 181* (9-700)
to immunotherapy 100 PNU RWP I
(6wg
420 (l-1,000) 325 (10-700)
1,000 PNU (60 fig RWP 1
584 (l-1,600) 346 (lo-1,600)
15,ooO PNU ( 900 pg RWP )
2,813 (l-5,400) 1,574 (88-5,000)
*Mean ng of AgE bound per ml serum; range in brackets.
diated response and immunogenicity refers to the ability to induce an IgG-mediated responsein animals or humans. MATERIALS AND METHODS Selection of subjects Nineteen atopic patients with a history of seasonal rhinitis compatible with ragweed pollinosis and marked skin reactivity to short ragweed (Ambrosia eliator) (1 : 20 Hollister-Stier Laboratories, Spokane, Wash.) were selected for study. Eleven patients had received prior immunotherapy which had been discontinued for at least three years. Of those who had previous immunotherapy, there were 6 in the monomer group and 5 in the polymer group. Patients with asthma or other significant medical illnesses were excluded. In addition, all patients underwent an initial screening hemogram and urinalysis which were normal in all cases. The patient population consisted of I3 men and 6 women ranging in age from 22 to 53 years. The subjects were randomized into 2 groups, one to receive polymerized ragweed and one to receive monomer ragweed. There were 10 patients in the polymer group and 9 in the monomer group.
Methods of therapy and clinical evaluation Treatment was conducted in a single-blind fashion with either the monomer or the polymer. The polymerized antigen was made as previously described by polymerization of monomer antigen (obtained by the extraction of giant and short ragweed pollen) with glutaraldehyde treatment.3 The stock monomer and polymer ragweed preparations contained 0.3 mg ragweed protein (P)/ml. The monomer preparation contained 18 pg AgE/ml. These preparations approximated a 1: 100 preparation of crude ragweed extract based on the starting preparation of ragweed and the pro-
tein content. In order to record doses as positive numbers, I ml of the polymer and monomer preparation was considered to contain 5,000 protein nitrogen units (PNU). Each preparation was filtered through a 0.20-p bacterial filter (Nalge Filter Unit, Nalge Sybron Corp., Rochester, N. Y.) and cultured for bacteria and fungi. Fresh polymer and monomer extracts were prepared every 6 months because previous studies showed the polymer to be stable for at least 6 months.” Both were diluted in a solution of phosphate-buffered 0.15 M NaCI, pH 7.35, containing 0.5% phenol and stored at 4” C. The schedule of immunization was comparable to that used at the Northwestern University Allergy Service for routine pollen immunotherapy. One injection per week was given until a maintenance dose of 0.5 ml of 1: 100 (0. I.5 mg ragweed P) was achieved. Maintenance dosage should be achieved in 27 wk. Once a maintenance dose was reached, the frequency of injections was reduced to once every four weeks. Therapy was started in November, 1976. The patients completed symptom score sheets for the 6-wk ragweed season (August I4 to September 30) of 1976 prior to onset of therapy and for the same period during the ragweed season in 1977 after approximately 10 months of treatment. The daily symptom scores were derived from weighted total scores recorded by the patient for symptoms experienced and the amount of medication needed to control his symptoms. A daily record was kept by the patient of the following symptoms: sneezing, nasal congestion, nasal discharge, red pruritic eyes, cough, and wheezing. The severity was graded on a scale of 0 to 3, the former having no symptoms and the latter severe symptoms. In addition, the amounts of antihistamines, bronchodilators, and nasal spray were recorded and given a standard score. Local reactions at injection sites were determined by measuring the diameters of erythema and wheals by a nurse who did not know which of the two types of immunotherapy
VOLUME NUMBER
Polymerized
62 5
ragweed
immunotherapy
291
MONOMER
POLYMER PRE TREATMENT
whole
POST TREATMENT
PRE TREATMENT
POST TREATMENT
10000
*
1ooc
E & c ” P z
z a
100
W o! a
1c
FIG. 1. Antibody activity against ragweed AgE. Pretreatment sera are compared with posttreatment sera in the polymer and monomer groups after 15,000 PNU had been given. Note log scale. Values for two patients in the monomer group (---) are not reported at 15,000 because one patient was able to reach only 6,000 PNU after 1 year of treatment. The other patient had no response at 15,000 PNU and the response at 45,000 is shown.
were administered. Late local reactions occurring after several hours or systemic symptoms were reported and evaluated by the physician.
Immunologic
studies
Blood specimens were drawn from each patient before and after the 1976 ragweed season after 100, I ,000, 15,000 PNU had been given and at the end of the 1977 ragweed season. The serum obtained at the end of both ragweed seasons was collected in late October or early November. The sera were analyzed for antigen E specific IgE and total serum antigen E binding capacity. Measurement of&E specific IgE. IgE specific for antigen E was measured by the polystyrene tube radioimmunoassay technique previously described.‘j Briefly, polystyrene tubes were coated with purified myeloma IgEps. Anti-IgE (Behring Diagnostics, Sommerville, N. J.) was added in excess to form an IgE immunoabsorbent. A I : 10 dilution of the patients’ serum was added to the tubes and incubated in the cold for 48 hr, during which time a known fraction of the IgE in the serum was bound to the tube. After discarding the unreacted serum, the tubes were washed, and ‘? AgE was added in excess. The tubes were incubated for 48 hr in the cold. Unbound AgE was removed by washing
and bound counts were determined. The ng of AgE bound by specific IgE in I ml of serum was determined and the ng of IgE antibody specific for AgE per ml calculated.” Measurement of total antibody against AgE. Total serum AgE binding capacity was measured by a modification of the ammonium sulfate technique of Lidd and Farr.7, x Briefly, 0.1 ml aliquots of sera were incubated with 100 and 1,000 ng of ‘%I AgE. After appropriate incubation, the antibody-bound AgE was separated from the free AgE by precipitation with 40% saturated ammonium sulfate. The ng AgE bound by 1 ml serum in antigen excess was then calculated and the results expressed as ng AgE bound/ml serum.
RESULTS Therapeutic
safety
All patients in both treatment groups were monitored with hemograms and urinalyses. No abnormalities were noted in these laboratory teststhroughout one year of treatment. After approximately one year of treatment, the total amount of polymerized ragweed injected was approximately twice the amount of monomer. Despite the fact that the number of injections in both groups was
292
J. ALLERGY
Bacal et al.
TABLE III. Percent
change in postseasonal and after treatment
specific
CLIN. IMMUNOL. NOVEMBER 1978
total of 15,000 PNU had been given, blocking antibody activity in the monomergroup rose from a mean of 173 ng AgE bound per ml to a mean of 2,8 13. Patient Pretreatment* Patient Posttreetmentt Excluded from these averages was one patient who was unable to reach 15,000 PNU. In the polymerized Monomer + 439% + 87% ragweed group, the rise was from a mean of 181 ng 2 + 189% + 83% AgE bound per ml to 1,574. Blocking antibody activ3 +lOO% + 100% ity at all levels of treatment was not statistically dif4 + 81% - 24% ferent for the 2 groups(analysiswith Student’st test). 5 + 49% + 59% As can be seen in Fig. 1, the pattern of rise in 6 + 9% + 44% blocking antibody activity for individuals in both 7 0% 0% groups was similar. Each individual’s rise in antibody 8 28% + 35% activity (value at 15,000 PNU divided by pretreat9 35% + 42% ment value) was calculated, the individual rises addPolymer 1 +l,loo% + 84% ed, and divided by the number of individuals in each 2 + 111% 2 0% group reaching 15,000 PNU. The average individual 3 + 264% 3 + 87% rise in the monomer group was l&fold (O-56) and in 4 + 220% 4 + 32% the polymer group 18-fold (3-100). It is evident that 5 + 700% 5 0% both groups had low-, mid-, and high-responding in6 + 62% 6 + 81% dividuals. 7 + 41% 7 + 58% Before initiation of therapy, 66% of the monomer 8 + 20% 8 + 173% patients and 80% of the polymer patients showed a 9 36% 9 - 11% rise in postseasonalspecific IgE. In evaluating the 10 58% 10 - 20% decreasedor blunted postseasonalIgE rise after im*Percent increase in AgE-specific IgE after1976ragweed season munotherapy describedby previous investigators,g, lo withnoimmunotherapy. certain patterns could be noted (Table III). In the $-Percent increase in AgE-specific IgE after 1977ragweed season monomergroup, 3 patients out of 9 showeda blunted with ragweed immunotherapy. rise. In the polymer group, 5 out of 10 showed a blunting of the rise in IgE. nearly the same,the polymer group reachedthe maintenancedosagein the preplanned27 injections, while Clinical efficacy the monomer group, becauseof significant local or The majority of patients in both treatment groups systemic reactions, had to advance more slowly and reported subjective clinical improvement during the received less total antigen per injection. All patients ragweed season following treatment. Assessment in the polymer group received 15,000 PNU in 6 basedon analysis of their weighted symptom scores months. In the monomer group it took an average of revealed that 8 out of 9, or 88% of patients in the 8.1 months (6 to 11 months) to receive 15,000 PNU. monomer group, had a decreasein symptom scores Although a greater total amount of antigen was given after treatment. Seven out of 10 patientsor 70% of the to the polymer group, there was 40 times as much polymer group had a decrease in symptoms. The erythema and 15 times as much induration in the groups were not statistically different by this analysis monomer group as compared to the polymer group of their symptom scores(chi square). (Table I). Seven systemic reactions occurred in the Ragweedcounts were similar for seasonspreceding monomer group while none occurred in the polymer and following treatment. Fig. 2 compares symptom group. The systemic reactions consisted of genscores for 1976 and 1977 ragweed seasonfor both eralized urticaria, pruritus with throat tightness, or monomer and polymer groups. In both treatment nasalsymptomswith or without asthma.These sympgroups, the mean symptom score after treatment was toms were reversed by subcutaneousepinephrine and oral antihistaminesand did not progressto any serious less than pretreatment. The statistical significance varies on individual days (statistical analysis by Stusequelae. dent’s t test). Averages of symptom scoreswere taken Immunologic results for 2-day periods and the number of days demonstrating statistical significance was totaled. The monomer Blocking antibody activity against AgE rose in all group had approximately two weeks of statistically subjects treated (Table II and Fig. 1). Pretreatment significant decrease in symptoms scores while the levels of blocking antibody were not statistically difpolymer group had approximately one week. Of note ferent in the monomer and polymer groups. After a IgE before
VOLUME NUMBER
Polymerized
62 5
POLYMER
whole
ragweed
immunotherapy
293
-1976 ---*
1077 +
Pd.05
50,
40 30
20 10 it 0
0
:: MONOMER
I 0 i
l
l
50
l
@ 40
30 20 10 0 I
18
I
2’2
J
2’6’dOl
I
AUGUST
I
b
’
‘e’,‘3’?7’2: SEPTEMBER
‘?I5’29
I
FIG. 2. Mean symptom score indices for polymer and monomer groups for the 1976 (-) and 1977 (---) ragweed seasons. Symptom scores are averaged for two days at a time. Means and standard deviation for selected days are shown by vertical lines. Levels of significance less than 0.05 for selected days are shown by an asterisk.
is the difference in the pattern of curves in the two treatment groups. The polymer group experienced a peak and rapid drop in symptom scores during the season before treatment. This pattern is matched by that of the posttreatment scores. This rapid decline of symptoms may have contributed to the shorter period of symptom score improvement. In contrast to this pattern, the monomer group had a prolonged period of increased symptoms scores for the season prior to treatment Differences between symptom score patterns of the two randomly selected groups may have occurred by chance.
DISCUSSION The purpose of this study was to evaluate polymerized whole ragweedasa potentially improved method of immunotherapy. Polymerized antigen E, which was utilized in previous studies, has certain limitations as opposed to whole ragweed. Extensive fractionation procedures are required to obtain the
large amounts of AgE which would be required for general clinical use.‘I In addition, AgE is but one major allergen in ragweed. Ragweed antigens K, Ra3, Ra4, and Ra5 have been identified. 11, l2 The polymerized whole ragweed preparation has been demonstrated to contain at least AgE, AgK, and Ra3.r3 Polymerized whole ragweed hasbeen shown clinically safe and well tolerated with no systemic reactions and significantly less local reactivity as compared to the monomeric forms. In addition, it has resultedin immunologic changescomparableto those found with standard immunotherapy. Blocking antibody activity rose equally in both treatment groups. Treatment resulted in a blunting of the postseasonal IgE rise in some patients, but no statistically significant trend was noted. Mention has already been madeof the difference in patterns of symptom scores in the two groups. Although the monomer group appearedto have a more
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et al.
prolonged beneficial effect from therapy, there may have been a difference in the two groups of subjects who were selected at random. Overall, treatment with both preparations resulted in symptom amelioration during the ragweed season following therapy. In conclusion, polymerized whole ragweed therapy was found to be safe, as immunogenic as the monomer preparation, and capable of reducing symptoms of ragweed-sensitive individuals. REFERENCES 1. Lieberman, P., and Patterson, R.: Immunotherapy for atopic disease, Adv. Intern. Med. 19:391, 1974. 2. Metzger, W. J., Patterson, R., Zeiss, C. R., et al.: Comparison of polymerized and unpolymerized antigen E in immunotherapy of ragweed allergy, N. Engl. J. Med. 295: 1160, 1976. 3. Patterson, R., Suszko, I. M., Zeiss, C. R., et al.: Comparison of immune reactivity to polyvalent monomeric and polymeric ragweed antigens, J. ALLERGY CLIN. IMMUNOL. 61:28, 1978. 4. Patterson, R., Suszko, I. M., Pruzansky, J. J., et al.: Polymerized ragweed antigen E: In vivo elimination studies and reactivity with IgE antibody systems, J. Immunol. 110:1413, 1973. 5. Patterson, R., and Suszko, I. M.: Polymerized ragweed antigen E. IV. The analysis of the stability of molecular size and antigenic reactivity of the polymer, J. ALLERGY CLIN. IMMUNOL. 57~168, 1976.
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6. Zeiss, C. R., Pruzansky, J. J., Patterson, R., et al.: A solid phase radioimmunoassay for the quantitation of human reaginic antibody against ragweed antigen E, J. Immunol. 110:414, 1973. 7. Lidd, D., and Farr, R. S.: Primary interaction between It3’ labeled ragweed pollen and antibodies in the sera of human and rabbits, J. ALLERGY 33:45, 1962. 8. Zeiss, C. R., Metzger, W. J., and Levitz, D.: Quantative relationships between IgE antibody and blocking antibody specific for antigen E in patients given immunotherapy with ragweed antigen E, Clin. Exp. Immunol. 28:250, 1977. 9. Lichtenstein, L. M., Ishizaka, K., Norman, P. S., et al.: IgE antibody measurements in ragweed hayfever. Relationship to clinical severity and the results of immunotherapy, J. Clin. Invest. 52~472, 1973. 10. Levy, D. A., Lichtenstein, L. M., Goldstein, E. O., et al.: Immunologic and cellular changes accompanying the therapy of pollen allergy, J. Clin. Invest. 50:360, 1971. 11. King, T. P., Norman, P. S., and Connell, J. T.: Isolation and characterization of allergens from ragweed pollen. II. Biochemistry 3:458, 1964. 12. Griffiths, B. W., and Brunet, R.: Isolation of basic or protein antigen of low ragweed pollen, Can. J. Biochem. 49:396, 1971. 13. Zeiss, C. R., Ungs, C. J., Levitz, D., Suszko, I. M., and Patterson, R.: The immune response of patients with ragweed hayfever treated with polymerized ragweed antigens. (Submitted for publication in J. ALLERGY CLIN. IMMUNOL.)