Delayed hypersensitivity to mumps antigen in humans

CLin’lCAL

IMMUNOLOGY

Delayed

AND

lMMIJNOPATHOLOGY

Hypersensitivity

26, 102- 110 (1983)

to Mumps

JOHN T. CALLAGHAN,~ BRUCE H. PETERSEN, WARREN W. EPINETTE,* AND ROBERT Lilly

Antigen

in Humans

WENDELL C. SMITH, C. RANSBURC?

Laboratories for Clinical Research. Eli Lilly and Company, Wishard Memorial Hospital, *Department of Dermatology and Pathology and iDepartment of Pathology, Indiana University School of Medicine, Indianapolis, Indiana 46202

and

Sixty-one subjects. preselected for mumps sensitivity, were entered into a doubleblind protocol to standardize Mumps Skin Test Antigen. Four lots of mumps antigen selected on the basis of in vitro potency tests were used. Four other antigens, Histoplasmm, Dermatophytin-0, Dermatophytin, and fluid tetanus toxoid were also tested; positive reactions for these four antigens occurred in 48, 68, 25, and 43% of individuals, respectively. The mumps lots exhibited delayed hypersensitivity with positivity ranging from 63 to 67% at 20 CFUiml and 77 to 84% at 80 CFUiml in the acceptable lots. Side effects were primarily local and minor in nature. Mumps Skin Test Antigen is a useful measure of the integrity of the immune system, but lacks complete specificity because of local dermal factors.

INTRODUCTION

Delayed hypersensitivity was demonstrated by Koch (1889) using tubercle bacilli, but was probably first described by Jenner (1798) using vaccinia. Since then the development of delayed hypersensitivity has been convincingly associated with immunity ( l-3). Skin testing is a widely employed and readily available method for clinically assessing the cellular immune response; a positive skin test reaction indicates previous antigenic exposure, T-cell competance, and an intact inflammatory response and is, therefore, an assessment of the total integrity of the immune response (3-5). A positive reaction is of prognostic significance in many clinical settings (6). Lymphocyte transformation is also a well-established technique for assessing the immune status of a patient. The clinical applications of the assay have been reviewed by Coulson and Inman (7). Lymphocyte transformation in response to antigenic stimulation correlates with both cellular immunity and delayed hypersensitivity (8). Several common antigens, including Mumps Skin Test Antigen (MSTA),2 have been used for skin testing. However, MSTA has not been standardized through clinical trials, and the significance of reactions to this antigen has, therefore, been questioned. The objectives of this study were: (1) to compare the clinical response 1 To whom correspondence should be addressed: Lilly Laboratories for Clinical Research, Eli Lilly and Company, Wishard Memorial Hospital, 1001 West 10th St. Indianapolis, Ind. 46202. ? Abbreviations used: Der, Dermatophytin; Der-0, Dermatophytin-0; DH, delayed hypersensitivity: Hist. Histoplasmin: LT. lymphocyte transformation: MSTA, Mumps Skin Test Antigen: PLl, low-dose MSTA placebo: PL2, high-dose MSTA placebo; TET. fluid tetanus toxoid. 102 0090-1229/83/010102-09$01.50/O Copyright @ 1983 by Academic Press. Inc All rights of reproduction in any form reserved.

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TO

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ANTIGEN

103

of MSTA with in vitro potency tests, (2) to compare dermal delayed hypersensitivity (DH) with lymphocyte transformation, (3) to compare MSTA with other commercially available antigens, (4) to determine the optimum antigen concentration, and (5) to reaffirm the safety of MSTA in clinical practice. METHODS

S~lbjecls. This was a doubie-blind randomized clinical trial in 61 preselected volunteers who were in apparent good health. A threefold rise in the lymphocyte activation index following MSTA stimulation was used as a criterion for selection. Informed consent was obtained from each volunteer. Mumps Skin Test Antigen. Four lots of MSTA concentrate each diluted to three or four concentrations were compared to MSTA placebo and control skin test antigens. Concentrations of 5, 20, 80, and 320 complement fixing units/ml (CFU/ml) were prepared from Lots 1 and 4, and concentrations of 5, 20, and 80 CFU/ml were prepared from Lots 2 and 3. Lots 1, 2, and 4 passed in vitro direct and indirect potency and safety testing, while Lot 3 only passed indirect potency and safety testing. Lot 3 was selected purposely in order to establish the validity of direct potency testing. CFU and direct and indirect titers were determined by laboratory branch complement fixation tests (9). Control skin fesf antigens. Skin test antigens employed in addition to MSTA included Histoplasmin (Hist) (Parke Davis and Co., Detroit, Mich.) and fluid tetanus toxoid (TET), 1:lO (Lederle Laboratories, Division of American Cyanamid Co., Pearl River, N.Y.); Dermatophytin-0 (Der-O), 1: 100 (Hollister-Stier Laboratories, Spokane, Wash.), and Dermatophytin (Der), I:30 (Hollister-Stier Laboratories). Der-0 and Der are antigens prepared from Cundida and Trichophyton species, respectively. These four antigens were administered on study Day 1. One week later, the first two concentrations of MSTA (5 and 20 CFU/ml) in duplicate and a single injection of MSTA placebo (PLl) were administered. If the subjects were unreactive to the low concentrations of MSTA, two higher MSTA concentrations (80 and 3203 CFU/ml) in duplicate and MSTA placebo (PL2) were administered 48 hr later. All skin tests were performed using disposable plastic syringes with 27-gauge needles. As intradermal injection of 0.1 ml of each antigen was made into the buttocks or volar surface of the forearms. The buttocks were used to prevent obvious cosmetic injuries, if ulcers developed from skin testing. The study was divided into two phases. In phase A, MSTA and MSTA placebo sites were biopsied with a 3-mm high-speed rotary punch (Model 5B, Robbins Instruments, Inc.); 11 subjects participated in this phase. In Phase B, no biopsies were obtained. The response to each dose of MSTA was tested using duplicate injection sites for each subject. Measures of erythema and induration at each site were recorded by two blinded observers at 20 min and 6, 24, and 48 hr after injection. Positive immunoreactivity to Der-0 and Der, Hist, TET, and MSTA is given in ” Lots 2 and 3 lacked

320 CFU/ml

concentrations.

104

CALLAGHAN

ET

TABLE

AL.

1

CLINICAL CRITERIA FOR A POSITIVE DERMAL RESPONSE -Antigen Der-0” Der” Hist TET MSTA”,”

Erythema 5 5

70 mm’

Size (mm)

Induration 5 5 10 10 30 mm2

0 Positivity occurs when the criteria for either erythema or induration are met. D This is a two-dimensional measurement based on the formula for an ellipse, Areae,,ivse = abni4. where a and 6 are the diameters of the ellipse. As a simplification for clinical use, ?r/4 is a constant which could be omitted; the area criteria (u x b ) for erythema and induration would then be 90 and 40 mm2, respectively.

Table 1. Single-dimensional measurements of the longest axis of the dermal reaction was used for Der-0, Der, Hist, and TET, while two-dimensional criteria were employed for MSTA. Skin biopsies. Punch biopsy specimens (3 mm) were obtained with a high-speed rotary punch from randomly selected 6- and 48-hr MSTA sites as well as 48-hr placebo sites in 11 subjects. All biopsies were obtained from buttock skin test sites. The specimens were processed in routine fashion, stained with hematoxylin and eosin, and read by two observers independently. Lymphocyte activation. Lymphocyte transformation (LT) assays were performed by using a microassay procedure similar to that of Oppenheim and Schecter (10). Microcultures contained 1 x lo6 (0.1 ml) peripheral blood lymphocytes prepared by separation on a Ficoll-Hypaque gradient (11). Cultures were stimulated with MSTA (0.05 ml) at protein concentrations ranging from 0.01 to 1.0 &ml. Cultures were incubated for 5 days with MSTA. Tritiated thymidine was added and the culture incubated an additional 24 hr before harvesting. A threefold increase in counts per minute over background was considered evidence of stimulation. RESULTS

Sixty-one subjects randomly selected from preselected candidates were admitted to the study. Three subjects withdrew before completing the study. The 35 female and 26 male subjects, all Caucasians, ranged in age from 21 to 39 years. Thirty-five were nonsmokers. Sixty subjects had a history of tetanus immunization; 35, measles vaccinations: and 2 1, influenza vaccinations. One volun11 candidiasis, 14 athletes foot, and 34 teer had had clinical histoplasmosis, mumps infection (one had mumps 1 month before the study). The area of the dermal response for the battery antigens did not differ among groups given each MSTA lot. Immediate reactions were prominent in all but Hist. The mean 6- and 24-hr areas generally were less than the 20-min and 48-hr mean

DELAYED

OBSERVER

HYPERSENSITIVITY

AGREEMENT

TO MUMPS

2 TABLE FOR BATTERY

ANTIGEN

105

ANTIGEN

READINGS

Agreement Antigen

No. of subjects

Der-0 Der Hist TET

60 60 61 61

Pos.

Neg.

39 12 24 21

19 43 27 28

No. in disagreement 2 5 10 12

Proportion in agreement 0.97 0.92 0.84 0.80

Intraclass correlation 0.92 0.77 0.67 0.60

areas. Der was least frequently positive (25%) although its criteria were less stringent. The frequency of positivity for TET, Hist, and Der-0 were 43, 48, and 68%, respectively. Two measures of observer agreement are provided in Table 2 for each battery antigen. The proportion of agreement is a crude index which is easily interpeted, but it does not adjust for chance agreement between observers. The intraclass correlation proposed by Cohen (12) corrects for chance-expected agreement. It assumes the value + 1 when there is complete agreement between observers and the value 0 when agreement is precisely that expected by chance. Justification for using Cohen’s K statistic as a measure of intraclass correlation is discussed by Fleiss (13). The correlation between observers for the four antigens ranged between 0.60 and 0.92. The highest correlation occurred for Der-0 which used both erythema and induration criteria. Der frequently gave no reaction, which explains the high proportion of agreement (0.92) but lower intraclass correlation (0.77). Hist and TET readings resulted in greater numbers of disagreement with TET having the poorest correlation. Descriptive statistics for MSTA reactions are summarized in Table 3. In Lot 1, the placebo mean area was increased inordinately by the reaction of one subject. The size of the reactions among lots was in reasonable agreement, except for Lot 3 which had the smallest areas (P < 0.05). Several dimensional criteria were analyzed in an effort to standardize MSTA positivity. The criteria finally selected were erythema equal to or greater than 70 mm*, or induration equal to or greater than 30 mm2. Dose-response curves reflecting percentage dermal positivity were generated (Fig. 1). No difference existed between the 80 and 320 CFUiml concentrations. At 20 CFU/ml more than half of the subjects exhibited immunoreactivity in the acceptable lots. Measures of positive and negative agreement between observers were computed for MSTA readings at 48 hr (Table 4). For each observer positivity was determined using the mean reaction of duplicate sites at each dose level (single site for placebo). Placebo results were highly correlated, with disagreement on only one subject, and there was complete agreement for the 16 subjects given 320 CFU/ml injections. Correlations for other MSTA dose levels ranged between 0.42 and 0.49, somewhat lower than for the battery antigens. Observer disaccord was due largely to differences in induration readings.

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TABLE DESCRIPTIVE

MSTA dose

STATISTICS

FOR

3 MSTA REACTIONS

AT

Erythema

48-hr Induration

Statistics

Lot 1

Lot 2

-~~____ Lot 3 Lot 4

SE II

100.5 90.3 1.5

21.9 9.4 I5

22.5 7.0 IS

20.2 9.3 15

23.9 21.9 15

12.2 8.8 I5

9.9 6.5 IS

9.0 7.8 I5

SE ‘I

72.8 21.1 15

73.5 15.1 I5

37.0 Il.4 I5

59.5 II.8 IS

34.7 16.0 15

33.5 6.7 is

18.6 6.0 I5

35.5 8.1 I5

SE ,I

114.4 24.6 I5

136.5 33.3 15

69.9 18.9 IS

118.6 27.4 IS

43.3 16.3 I5

47.1 9.7 15

36.3 7.0 I5

58.7 1 I.1 15

SE 12

119.9 41.5 7

131.6 37.1 7

86.7 IS.3 10

116.1 23.7 9

28.7 8.1 7

40.9 11.2 7

30.0 5.4 10

49.4 11.9 9

SE II

139.6 53.7 7

-

-

240.7 76.7 9

27.6 5.7 7

Placebo

5 CFU/ml

20 CFUiml

80 CFU/ml

320 CFUiml

Lot 1

Lot 2

Lot 3

Lot 4

-

-

73.3 18.6 9

Positive and negative agreement between duplicate sites was generally better than between observers. At the lower two dose levels, intraclass correlations were near 0.70 (Table 5). Induration values for duplicate sites were in better accord than readings of two observers for the same site. In addition to mumps antigen ovalbumin is present in both MSTA and MSTA placebo. The 24-hr placebo reaction usually was larger than the 48 hr reaction. Enders et al. (14) also observed a similar decrease in reactivity from 24 to 48 hr

1

i :. t

i 0

20

40

60

CFU

I ML

*LOT A--LOT . ..__._LOT

1 2 3

G--

4

LOT

80”

320

1. Percentage dermai positivity according to mumps antigen lot. Individuals were given one of four lots of MSTA. Lot 3 failed in vitro potency tests: Lots 2 and 3 lacked a 320 CFU/ml concentration. The percentage of individuals exhibiting positive skin tests is shown for each of MSTA concentrations and for placebo which has been equated in the figure to zero. FIG.

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HYPERSENSITIVITY

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TABLE OBSERVER

MSTA dose (CFUlml) 0.0 5.0 20.0 80.0 320.0

AGREEMENT

MUMPS

4

FOR 48-hr

MSTA

READINGS

Agreement No. of subjects 60 60 55 32 16

Pos.

Neg.

5 16 27 19 12

54 29 13 6 4

107

ANTIGEN

No. in disagreement

Proportion in agreement

1 15 I5 1 0

0.98 0.75 0.73 0.78 1.00

Intraclass correlation 0.90 0.49 0.42 0.48 1.00

when monkey parotid gland extracts were used as placebo. The response to MSTA placebo did not correlate with previous exposure to egg-containing vaccinations. Thirty-two percent and thirty-nine percent of subjects who received influenza or measles vaccinations, respectively, had greater than 20 mm2 induration with placebo. Sixteen percent of subjects who denied vaccination with either agent had similar reactions with placebo. No subject had a history of egg allergy. Overall, 17% of our subjects demonstrated positive reactions at placebo sites: this frequency compares with a 5-- 10% reported incidence (15, 16). Side effects were documented with a written questionnaire. Most side effects were local and dose related (Table 6). Vesicle formation was more frequent with the antigen battery (mainly with Hist) than with MSTA. Other complaints included mild-to-moderate nausea, altered appetite, headache, unsteadiness, and drowsiness. Skin rash developed after receiving battery antigens: one of these subjects had a contact dermatitis resulting from the alcohol skin preparation. None of the observed reactions were considered serious. One subject complained of tender inguinal lymphadenopathy and another of nontender adenopathy during the middle of the study. Consequently, 22 of 23 subjects in the last part of the study were systematically evaluated for inguinal adenopathy. Seventeen subjects had palpable adenopathy; of these 6 noted nodal tenderness, Adenopathy usually disappeared after a week. Results of the skin biopsies obtained from 11 of the subjects were consonant with published data from tetanus, tuberculin, and other studies of histological

AGREEMENT MSTA dose (CFUlml) 5.0 20.0 80.0 320.0

BETWEEN

TABLE 5 DUPLICATE SITES FOR 48-hr

Agreement

No. of subjects

Pos.

Neg.

60 55 32 16

16 31 17 10

36 16 7 3

disagreement 8 8 8 3

MSTA

READINGS

Proportion in agreement 0.87 0.85 0.75 0.81

Intraclass correlation 0.70 0.69 0.46 0.54

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TABLE OBSERVED

SIDE

EFFECTS

WITH

No.

Side effects

Battery antigens

Local (a) Tenderness (b) Pruritus (c) Vesiculation (d) Lymphadenopathy”

6

MSTA

AND

of subjects

BATTERY

experiencing

MSTA (5 and 20 CFU/ml)

31 50 16

ANTIGENS

side effects MSTA (80 and 320 CFUlml)

34 28 2

32 21 3 17

Systemic (a) Nausea (b) Body rash (c) Headache (d) Hot feeling (e) Sweating @I Anorexia I’ This finding developed.

was evaluated

in only

22 subjects

and only

for MSTA.

Only

inguinal

adenopathy

reactions to intradermal antigens (17, 18). Forty-eight-hour MSTA biopsies were characterized by a mixed PMN/mononuclear cell infiltrate which was distributed both perivascularly and diffusely in the dermis, whereas 4%hr placebo biopsy sections demonstrated predominantly perivascular superficial and deep mononuclear reactions. PMN leukocytes were more common in 6-hr infiltrates. Endothelial swelling, infiltation of vessel walls, occasional microthrombi and rare necrosis were more prominent in the MSTA-injected 6- and 48-hr sites. DISCUSSION

Skin testing effectively probes the integrity of the immune system providing both diagnostic and prognostic information to the clinician. To be useful, antigens must elicit an anamnestic response in much of the population. Mumps antigen is frequently used for this purpose (15, 16, 19). However, no standardized preparation of Mumps Skin Test Antigen has been marketed. This study was undertaken to determine optimum concentration, the criteria of positivity, and the efficacy of MSTA. Immunocompetent subjects were screened for exposure to mumps by a threefold rise in lymphocyte activation. Several studies (20-22) indicate that a correlation exists between lymphocyte transformation, cellular immunity, and cutaneous DH. Correlation between lymphocyte activation by MSTA and cutaneous DH reactions was observed in these studies (23). Of the battery antigens, Hist gave the greatest mean area, and was most frequently associated with vesiculation. A histoplasmosis epidemic occurred in this area in the months preceding study, and evidently many subjects had had recent exposure (24). Despite this, Hist was not as frequently positive as was Der-0,

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109

ANTIGEN

perhaps in part because the accepted clinical criteria for Hist reactions are more stringent. Der was least likely to give a reaction in our subjects; another study using Der reported positivity in 23 to 36% of subjects with clinical dermatophytosis depending on the antigen lot (25). The selected MSTA criteria (70 mm2 erythema or 30 mm2 induration) maximized positive response, while keeping the placebo response low. These criteria may still be too stringent since some placebo responses were histologically DH reactions. Enders et al. (14) showed that 90% specificity for previous mumps infection was obtained if the erythematous reaction was about 78 mm2. Thus, decreasing the size criteria further seems inappropriate. In vitro potency testing of MSTA concentrate positivity correlated with the skin test response. Lot 3, which failed direct titer in vitro testing, gave the poorest clinical response of the four lots. Lots 1, 2, and 4 had acceptable dose-response characteristics and showed similar clinical reactivity. The 5 CFU/ml concentration of all lots gave an unacceptable dermal response, whereas the response was nearly maximum at 20 CFU/ml and maximum at 80 CFU/ml for these three lots, and these concentrations are recommended for clinical testing. Side effects from MSTA injection were minimal, and should not preculde clinical use. The major side effects included localized pruritus and tenderness. MSTA injections caused more burning sensations than any other antigen injection. This may have been due to the fact that MSTA was injected into the buttocks, or to the formulation itself. No ulceration occurred at any antigen test site. MSTA commonly induced inguinal lymphadenopathy, which was probably the result of the large antigen burden (5 to 10 injections in the buttocks over 96 hr). We were unable to clearly show sensitization to these injections by LT; in the few individuals tested for antibody, titers to MSTA increased markedly. Enders et al. (14, 26) found that mumps antigen was capable of stimulating antibody production chiefly after previous infection. It is apparent from our study that side effects with MSTA are not significantly greater than those with the commercial battery antigens. The results of these studies support the conclusion that MSTA is a valid skin test but, as with other commonly used antigens, lacks complete specificity because of local dermal factors. ACKNOWLEDGMENTS The authors their invaluable

thank Ms. assistance

Mary Collins, in the study

Mrs. Gwen Hamilton, Rita Root, and in preparing the manuscript.

and Mr.

Larry

Steimel

for

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ET AL.

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