From the National Bacteriological Laboratory, the Hospital for Infectious Diseases, Stockholm, and the Municipal Virological Laboratory, Gothenburg, Sweden

Vaccinia Antigens for Skin Testing. Preparation of Antigens and Quantitative Studies of Skin Reactions

in “Standard Populations”1

LARS N O R ~ N , AKE ESPMARK, ASTRID FAGRAEUS, STIG E. HOLM, JONAS LINDAHL, ERIK LYCKE and JURCEN MARQUARDT

Following the extensive vaccination campaign in Stockholm in 1963 several cases of postvaccinal skin eruptions of suspected allergic nature were encoun- tered (2). This incited the study on the properties of some preparations of vac- cinia antigens in vitro and the ability of these preparations of eliciting skin allergic reactions when tested in some normal populations. In the sequel prep- aration of three different test antigens is briefly described as well as skin tests in animals and man, carried out with the following purposes :

a) To study the relation between the dose of antigen injected and the size of the cutaneous reaction.

b) To study the possible use of this relationship for a quantitative estimation of the allergenic capacity of the vaccinal antigen preparations.

c) To try to estimate quantitatively the “normal” skin sensitivity in some groups of individuals with defined his- tory of vaccination or smallpox.

Material and methods

Preparation and in uitro properties of test antigens Prebaration I contained soluble antigens from the liquid phase of vaccinia virus infected calf kidney cultures. The virus particles were removed almost com- pletely by centrifugation three times at 23,000 g. The last supernatant fluid contained only 3 pock forming units per 0.1 ml as tested on the chorioallantoic membrane. Preparation I tested by the diffusion-in-gel technique against a vac- cinia hyperimmune rabbit serum was shown to contain 4 separate precipitino- genic factors.

Control antigen consisted of the medium from non-infected calf kidney cultures, treated as Preparation I.

Preparation 11 contained concentrated soluble antigens from vaccinia virus in- fected cultures of embryonic calf skin and muscle tissues (13). The material was centrifuged and prepared by pre- cipitation at pH 4.5 principally accord- ing to the technique described by Shedlovsky and Smadel ( 12). When tested by the diffusion-in-gel technique

1 This study was partly supported by a grant from Statens Medicinska Forskningsrhd.

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against a vaccinia hyperimmune rabbit serum, 7 separate (vaccinial) precipita- tion bands were recorded. The prepara- tion contained no infectious virus.

The purijed and inactivated virus prepara- tion (Preparation ZZZ) was produced from rabbit skin adapted vaccinia virus (6). The initial crude suspension was purified by differential centrifugation and treat- ment with fluorocarbon. Virus was separated in a continuous sucrose gra- dient from 20 to 50 per cent and was obtained as a distinct band, which was harvested, washed and inactivated by dialysis in 25-50-100 per cent alcohol- acetic acid mixture and finally subjected to ultrasonic treatment. Electron photo- micrograph of unwashed and unshad- owed material revealed only minor impurities. This preparation contained no infectious virus and was freed from soluble antigens.

Complement fixation titers of the above mentioned three antigen prepara- tions tested against 4 antibody units of a known antivaccinia serum are pre- sented in Table 41.

Perjiirmnnce of skin ttststs and quan:itative esiimation of skin recictions For skin testing 0.1 ml of the antigen preparations was injected intracutaneous- ly. The erythema was recorded after 1- 4 and after 24 and 48 hours and its diameter was measured with a calibrated ruler. (When the erythema was not circular the “average diameter” was calculated as the square root of the product of the short and the long axis.) Induration was also noted and recorded as the increase in thickness of a skinfold at the site of injection as compared to

TABLE 41. Antigenic titers (in CF test against vaccinia-positive serum) of test pre- parations used for skin testing

Complement Antigen preparation fixation titer

Preparation I 1 / 4

Purified virus (Prep. 111) Neg. Control antigen Neg.

Preparation I1 1 /20

the thickness of an adjacent skinfold. The development of induration was essentially similar to the development of erythema.

Intracutaneous tests were performed in unvaccinated and vaccinated guinea pigs, in humans without preceding vac- cination, after uncomplicated vaccina- tion, and in patients recovered from smallpox.

Results

Skin tests in guinea pigs Fifty guinea pigs were immunized with smallpox vaccine prepared from vac- cinia virus infected chorioallantoic mem- branes. Another group of 10 animals were not vaccinated but inoculated intra- cutaneously with extract from unin- fected chorioallantoic membranes. The vaccinated as well as the unvaccinated guinea pigs were skin tested, using the test antigens described above (Prepara- tion I was injected in dilution 1/2, 1/20 and 1/200, Preparation I1 in dilution 1/10, 1/100 and 1/1,000, the purified virus preparation in dilution 1/60 and the control antigen in dilution 1 /2). Half

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mm 50

40

30

20

10

m

B 0

a 0

a a cm

n -

16 Dilution of test antigen

Fig. 60. Relationship between dilutions (logarithmic scale) of soluble vaccinia antigen (Prep. I) and diameter of skin reactions 48 hours after intracutaneous injection in adult males (recruits), successfully vaccinated 1 month prior to skin testing. 0 Individual values. Average within dilutions. Regression line fitted to mean values.

of the number of animals were tested 5 days after vaccination, the others 14 days after vaccination.

In the group of vaccinated guinea pigs a positive delayed skin reaction was found when Preparation I1 and Prepara- tion I (the soluble antigens) were used. The diameter of the skin reaction (erythema) seemed to be directly pro- portional to the logarithm of the antigen dilution injected.

In the unvaccinated control group of 10 animals only one developed a weak delayed reaction to Preparation I diluted 1 : 2 (12 x 12 mm after 48 hours) and

another animal a very weak reaction to Preparation I1 diluted 1 : 10 (3 x 3 mm after 48 hours). The other animals of the control group were negative to all the four test antigens.

Antibody response to vaccination was measured by the haemagglutination in- hibition test (HI test) a t the time of skin testing. HI antibodies were demon- strated in the majority of the group tested 5 days after vaccination, and in all vaccinated animals tested after 14 days. No correlation was found between the HI titers and the size of cutaneous reactions.

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m Preparation I 0 Preparation I1 0 Purified virus

(v mean value)

1 I2 ino 1/60 lhOO 1 no00 Dilution of test antigen

Fig. 61. Responses to skin testing in recently vaccinated individuals with different dilutions o soluble vaccinia antigen (mean value) and with one single dilution of purified virus (individua values : open triangles).

Skin tests in humans In a small group of 5 unvaccinated in- dividuals (children aged 5-1 2 years), none displayed any delayed reactions to the test antigens.

Twenty healthy recruits were skin tested one month after successful revac- cination. Each individual was injected with each of three consecutive tenfold dilutions of Preparations I and 11, with purified virus diluted 1 : 60 and with control antigen diluted 1 : 2. The puri- fied virus preparation was calculated to contain about 108 virus particles per injected dose.

Ail injected individuals exhibited de- layed reactions to Preparations I and I1 and weaker reactions to the purified

virus material. One individual in this group developed a weak delayed reac- tion to the control antigen, the others were completely negative for this prep- aration.

Figure 60 shows the size of individual reactions (diameters of erythema after 48 hours) to the three tested dilutions of Preparation I. Open squares represent the individual values, filled squares the mean values, which form a straight line against the logarithms of the antigen dilutions. The mean values of the skin reactions obtained with preparations I and I1 are plotted in the regression diagram of Figure 61 together with in- dividual values obtained with the purified virus preparation. The figure indicates

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TABLE 42. Mean skin titers (dilution estimated to give a 10 mm reaction) obtained with different vaccinia antigens in various groups of individuals differing with respect to history of vac- cination or smallpox

Unvaccinated (5 children)

~ ~~

Purified Control Prep. I Prep. I1 virus antigen

Neg. Neg. Neg. Neg.

Twenty recruits (successfully revaccinated 1 month earlier) 1 / S O 1 /220 1 / S O Neg. Often vaccinated group (six lab. staff members) 3 months after the last vaccina- tion 1 /80 1/140 1 /30 Neg. Often vaccinated group (four lab. staff members) 6 months after the last vaccina- tion 1/10 1 /40 1 /30 Not done Eight smallpox patients 5 months after recovery 1/10 1 /20 1/30 Neg.

that Preparation I1 contained about 5 times as much “allergen” as Preparation I and the purified virus preparation. The latter contained, however, a much larger amount of elementary bodies than Preparation I, and therefore it is reason- able to assume that the elementary bodies played a less active role as an “allergen”. Accordingly most of the allergen seemed to be present as soluble antigens,

The straight course of the lines in Figure 61 permits an estimation of the “skin titer” of the different antigens. “Skin titers” were arbitrarily defined as the dilution of antigen preparaticin corresponding to a skin reaction with a diameter of 10 mm. They will be used below to estimate the relative sensitivity of different populations. Some of the “skin titers” were obtained by extra- polation.

Antibody response to the preceding vaccination could be demonstrated in

all the skin tested individuals, but there was no correlation between the anti- body titers in serum (HI and CF titers) and the size of the cutaneous reactions.

Eight patients, recovered from variola 5 months prior to this study were skin tested in the same way as the groups described above. The cutaneous reac- tions in this group were weaker for Preparations I and I1 and slightly weaker for the purified virus preparation than the reactions recorded among the re- cruits. The relatively weak reactions in the variola group was unexpected. This group was, however, tested 5 months after recovery from smallpox, i. e. rela- tively later than the recruit group. Therefore it was considered that the dif- ference might be due to time. This assumption was corroborated by com- parison of repeated skin tests 3 and 6 months after vaccination in an often vaccinated group of individuals, sug- gesting that “skin titers” decrease signif-

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icantly in the course of a few months. The “skin titers” obtained in the dif- ferent tested groups are summarized in Table 42.

Discussion

Delayed skin reactions to vaccinia an- tigen preparations were shown to be significantly dose-dependent. A linear relation was obtained between the diameter of the erythema and the logarithm of the injected dose of antigen. The results from the skin tests in the guinea pigs were essentially similar to those in human beings, although the reactions in the animals were weaker. This is not unexpected, as the delayed type of hypersensitivity in general is known to be particularly pronounced in man (4).

In humans a tenfold increase of the dose corresponded to about 10 mm increase of the diameter of the reaction.

However, the variation in individual sensitivity within a seemingly homo- genous group of individuals was very pronounced. In the example of Figure 60 the mean diameters with the two highest doses were 25 and 14.5 mm and the standard deviation of individual values was of the magnitude 14 and 9 mm respectively. Hence, the dose- dependence of reactions may be revealed only if relatively large groups are tested.

Obviously the large individual varia- tion will cause similar difficulties when comparing the sensitivity of different categories of individuals. For instance, a difference between the mean diameter of skin reactions in two groups of 20

persons each has to exceed 7.5 mm in order to be revealed with 95 yo con- fidence with regard to the variation men- tioned above.

Evidently the use of only ungraded response (+ or -) would be an even less favourable method for detecting differences between groups.

In the comparison between the test antigens it was found that the prepara- tions of soluble antigens were more ac- tive as allergens than that of purified virus particles. This may be partly due to differences in diffusibility, and partly to differences in antigenic composition. Vaccinia virus materials have been found to contain several immunopre- cipitating antigens (1 1, 7). Among the soluble antigen preparations I and 11, which both contained several antigenic components, the latter was on the average 5 times more potent as an “allergen” than the former.

The difference in antigenicity between the preparations was also reflected in the diffusion-in-gel tests as only 4 pre- cipitinogens were detected in Prepara- tion I while 7 were demonstrable in Preparation 11.

However, a difference in antigenic potency as demonstrable e. g. in pre- cipitation analyses must not necessarily correspond to a difference in the ability to provoke allergic reactions. Prepara- tion I11 contained no soluble antigens functioning as precipitinogens but could nevertheless provoke skin reactions. Yet unpublished results have shown that most if not all the precipitinogens among the soluble antigens correspond to pre- cipitinogenic factors in the virus particles (8 ) *

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It seems reasonable to assume that the intact virus particles were not par- ticularly active as allergens. The skin reactions observed with Preparation 111 might be caused by allergens released by degradation of the virus particles intra- cutaneously.

Two of the tested recruits reacted much more strongly to Preparation I and they also had the two largest reac- tions noted with the purified virus (cf., Figure 61). This may suggest that in- dividuals may differ in their hyper- sensitivity to different antigenic com- ponents. This question should be further elucidated by testing purified separate antigen components.

The antigen preparations were ap- plied also in a small group of often vac- cinated individuals and in eight persons recovered from smallpox. On account of the large variation already noted it was expected that only large differences would be detected by these tests. In the former group identical tests 3 and 6 months after vaccination indicated that skin sensitivity to soluble vaccinia anti- gens is rapidly reduced (4-8 times within 3 months), whereas reactions to purified virus did not change signif- icantly during the time of observation. Reactions in patients recovered from smallpox were similar in size to those seen in the often vaccinated group.

The elements of the positive skin reaction are probably similar in nature to the induration and erythema seen after revaccination and in the later course of the primary reaction (10, 1). From the above dose-response rela- tionship it may be assumed that a large local reaction around a vaccination pock

on the average signifies a large amount of antigen produced, although this would be difficult to prove by direct means in practise.

Similar dose-response relationship have been demonstrated also with tuberculin ( 5 ) and the same regression technique has been used for the assay of diphtheria toxin (9) and even live vaccinia virus (3).

The relation between skin sensitivity as determined by intracutaneous testing and the tendency of developing gem eralized postvaccinal rashes still remains to be investigated.

Summary Cutaneous reactions were studied after injection of three vaccinia antigen prep- arations in unvaccinated and vaccinat- ed guinea pigs, in humans without pre- ceding vaccination, after uncomplicated vaccination, and in patients recovered from smallpox. The elementary body preparation proved to be less active as an “allergen” than the soluble antigen preparations. A linear relation was found between the diameter of the reac- tion and the log-dose of the injected antigens. The cutaneous reactions to the soluble antigen preparations decreased significantly during a 3 month period. The results revealed no difference in reaction between normally vaccinated persons and patients recovered fmm smallpox.

On the basis of the estimated magni- tude of variation among the skin reac- tions some considerations are presented for evaluation of the possibility of verify- ing sensitivity differences of various populations.

References 1.

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4.

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6.

BALDRIGE, G. D., and KLIGMAN, A. M.: The relationship between hypersensitivity and immunity to vaccinia. J. Invest. Derm. 18: 205, 1952. RAJKA, G. and NILZ~N, A.: Postvaccinal Dermatoses. Acta Med. Scand. Suppl. 464, 158, 1966. FISEK, N. H.: Standardization of smallpox vaccine. Working paper: WHO/BS/381, 16 August, 1957. HUMPHREY, J. H., and WHITE, R. G.: Im- munology for Students of Medicine, p. 345, Blackwell Scientific Publications, Oxford, 1963. LONG, D. A., and MILES, A. A.: Opposite actions of thyroid and adrenal hormones in allergic hypersensitivity. Lancet I : 492, 1950. MARQIJARDT, J., HOLM, S. E., and LYCKE, E.: Preparation of a purified vaccinia virus freed from soluble antigens. Proc. SOC. Exp. Biol. Med. 116: 112, 1964.

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MARQUARDT, J., HOLM, S. E., and LYCKE, E. : Immunoprecipitating soluble vaccinia antigens. Arch. ges. Virusforsch., in press. MARQUARDT, J., HOLM, S. E., and LYCKE, E. : Immunoprecipitating factors of vaccinia virus, to be published. MILES, A. A.: The fixation of diphtheria toxin to skin tissue with special reference to the action of circulating antitoxin. Brit. J. Exp. Path., 30: 319, 1949. VON PIRQUET, C.: uber die verschiedenen Formen der allergischen Reaktion bei der Revaccination. 2. Immunitatsforsch., 10: 1, 1911. RONDLE, C. J., and DUMBELL, K. R.: Antigens ofcowpox virus. J. Hyg. (London), 60: 41, 1962. SHEDLOVSKY, TH., and SMADEL, J. E.: The LS-antigen of vaccinia. 11. Isolation of a single substance containing both L- and S-activity. J. Exp. Med., 75: 165, 1942. WESSLEN, T.: The production of smallpox vaccine in tissue culture of bovine embryonic skin. Arch. ges. Virusforsch., 6: 430, 1956.