The relationship of delayed hypersensitivity to acquired antituberculous immunity

CELLULAR

IMMUNOLOGY

1,

266275

The Relationship

(1970)

of Delayed

Hypersensitivity

Antituberculous II. Effect of Adjuvant

Immunity

on the Allergenicity

of Heat-Killed

to Acquired

Tubercle

and lmmunogenicity Bacilli 1

F.M. COLLINSANDC~. B. MACKANESS Trudeau

Imtitute,

Ilzc., Saranac Received

March

Lake,

New

York

12983

2, 1970

Mice were injected subcutaneously on two occasions with a heat-killed, lyophilized suspension of M. tubermlosis H,,R, dispersed in a water-in-oil emulsion or as a saline suspension. The levels of tuberculin sensitivity were followed for 30 days, at which time the mice were infected intravenously with approximately 10s living BCG (Montreal). At intervals thereafter, groups of mice were challenged with 10 LD,,'s Listeria molcocgtogenes so that the development of nonspecific resistance could be followed in relation to the fate of BCG and the state of hypersensitivity in the different treatment groups. Nonvaccinated controls, and mice receiving adjuvant alone, required IO-12 days to respond to the eliciting dose of BCG with the development of detectable tuberculin sensitivity and cellular resistance to the Listeria challenge. They were also unable to limit the growth of BCG in the lung. Mice receiving H,,R, in saline behaved like the normal controls; but animals receiving H,,R,, in adjuvant quickly developed high levels of tuberculin sensitivity. and their resistance to Lisferia and BCG both appeared much sooner. Seven months later. mice of this group showed a marked rise in tuberculin sensitivity and an accelerated onset of cellular immunity in response to the BCG infection. The significance of these findings is discussed in relation to the mechanism of antituberculous immunity and the evaluation of nonliving vaccines.

INTRODUCTION Vaccination

with

living

BCG

results

in the development

of both

tuberculin

sensi-

and acquired resistance to infection by virulent tubercle bacilli (1, 2)) but the relationship of one to the other is still controversial (3, 4). The search for nonallergenic vaccines (5, 6) will continue until the question is resolved. If hypersensitivity proves to be an integral and necessary component of the mechanism of acquired resistance (7), a nonsensitizing vaccine is unlikely to be found. It is this

tivity

1 This work was supported by Grant AI 07809 from the United States-Japan Cooperative Medical Science Program administered by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Department of Health, Education, and Welfare.

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that has prompted an investigation of the conditions necessary for effective immunization (8). Killed tubercle bacilli confer little real immunity (2, 5, 6, 9) unless they are administered in a Freund-type adjuvant (10, 11) or as cell wall preparations in mineral oil (12). Evidence for these conclusions is based largely upon prolonged survival following massive intravenous challenge of mice (2, 6). The fate of challenge organisms in the tissues has seldom been studied sequentially (1, 13-15), and the challenge population has not always been clearly distinguished ( 1, 14) from residual BCG. When they have, slow inactivation of virulent tubercle bacilli could be detected in the lungs of vaccinated mice and guinea pigs (15-17). But the tubercle bacillus is so innately resistant to intracellular inactivation (18, 19) that even this does not represent a wholly satisfactory demonstration of acquired resistance because tubercle bacilli seem to be inhibited (20) rather than killed (15) in animals exhibiting high levels of cellular immunity. List&u wonocytogenes is much more susceptible to the microbicidal properties of activated macrophages so that challenge with this organism can furnish indirect evidence of the state of host resistance following immunization with BCG (21) ; but this device only measures the extent to which host defenses have been activated as a result of efforts to contend with BCG ; it tells nothing about the level of specific, antimycobacterial immunity. The latter requires a specific challenge. To circumvent this problem, BCG was used in the present study to elicit a response from normal and specifically immunized animals, and a Listevia challenge was used to measure its magnitude. In this way the allergenic and immunogenic properties of a saline suspension of heat-killed mycobacteria were compared with those of a water-in-oil emulsion of the same antigen. The former does not favor the development of tuberculin sensitivity or specific immunity, whereas the latter does. MATERIALS AND METHODS

An%&. Specific pathogen-free CD-l female mice (Charles River Farms, Inc.) were used throughout the present study (8). Organiswzzs. Mycobacteria were obtained from the Trudeau Mycobacterial Collection (TMC), Saranac Lake, N.Y. M. twbcrculosis HZ7Rv (TMC No. 102) was grown as a surface pellicle on Proskauer-Beck synthetic liquid medium. It was transferred at 14-day intervals (22). Pellicle was removed by filtration, suspended in saline, and heated in a boiling water bath for 10 min. Inoculation of aliquots of the heated suspension onto 7HlO agar plates failed to show evidence of viable organisms. The heated suspension was lyophilized and stored in ZYZCUO at room temperature. M. bovis BCG strain Montreal (TMC No. 1012) was cultured on Sauton medium for 10 days, homogenized in buffered gelatin, and stored at -70” in fresh Sauton. Vials were rapidly thawed and homogenized as described elsewhere (8). Mice were injected intravenously with 105-lo6 organisms. The viability of the in. oculating suspension was always checked by plating suitable serial dilutions on 7HlO agar plates. Listed rnonocytogenes (21) was maintained by serial passage in CD-1 mice. It has an intravenous LD,, of 5 X lo3 viable organisms . A fresh isolate was obtained

268

COLLINS

AND

MACKANESS

from an infected spleen, grown once in tryptone soy broth (Difco, Detroit), and discarded immediately after use. Vaccination procedure-Saline suspension. The lyophilized, heat-killed M. tuberculosis H,,R, was suspended in sterile saline at a concentration of 0.5 mg/ml (dry wt) and homogenized thoroughly (15). The suspension (100 pg in 0.2 ml) was injected subcutaneously into four sites across the back of the mouse. After 2 or 4 weeks the injections were repeated on the ventral surface. Adjuvant vaccine. The lyophilized H,,R, was suspended at a concentration of 1 mg/ml of sterile mineral oil-Arlacel A (17:3, v/v) and dispersed with a Teflon homogenizer (23). The oil-Arlacel A-mycobacterial mixture was then emulsified thoroughly with an equal volume of sterile saline. A drop of the resulting emulsion remained stable on the surface of water for at least 24 hr. Mice were injected subcutaneously at four sites with a total of 0.2 ml emulsion, and 2 weeks later the injections were repeated on the ventral body surface. Tuberculin tests were carried out periodically by injecting 2.0 pg PPD (Parke-Davis) into the right hind footpad as described elsewhere (8, 15). Assessment of cellular resistance. Groups of vaccinated and normal mice were infected intravenously with approximately 104 (5-10 LD,,,‘s) viable L. wzonocytogenes in 0.1 ml saline. After 30 min, 24 hr, and 48 hr, groups of five randomly selected mice were sacrificed and the number of viable Listeria determined in homogenates of the livers and spleens. The index of resistance was estimated as the difference between the logarithm of the organ counts of vaccinated and control mice (21). Enumeration of viable BCG in lung, liver, and spleen. Viable counts were made by plating suitable serial saline dilutions of organ homogenates (15) on 7HlO agar containing 2 pg penicillin G/ml to inhibit the growth of the Listeria. Counts were made at approximately 3-day intervals for up to 15 days. The standard error for the mycobacterial counts was similar to that reported earlier (15, 22). RESULTS Comparative allergenicity of heat-hilled vant. Mice were injected subcutaneously

H,,R,

in saline and Freund-type

adju-

with a single, lOO-pig dose of heat-killed H,,R, in a water-in-oil emulsion or as a saline suspension. Controls were injected with saline or with adjuvant alone. Representatives of each group were tested at intervals for tuberculin sensitivity, readings being made at 3 and 24 hr. The findings, (Table 1) showed that little if any hypersensitivity of delayed type developed in either of the immunized groups. Although the reactions obtained at 24 hr were statistically significant at the 5% level, they may have been residual Arthus reactions rather than true reactions of delayed type (24). It seemed from the foregoing experiment that a single exposure to antigen was not sufficient to produce significant levels of tuberculin sensitivity. Two more groups of mice, and their appropriate controls, were therefore sensitized with two similar injections of heat-killed Ha7Rv suspended in a saline or in Freund-type adjuvant. The injections were given at an interval of 14 days. The levels of hypersensitivity developed in the immunized mice are recorded in Table 2. Comparable levels of Arthus (3 hr) sensitivity developed in both groups, but tuberculin sensi-

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IMMUNITY

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AND

MACKANESS

tivity (24 hr) was always higher in animals immunized with the adjuvant mixture. At 28 days the tuberculin reactions obtained in animals immunized with saline-suspended organisms were extremely variable (Table 2) ; some showed good reactions while others were quite unreactive. Despite this variability, a comparison was made of the responses of the four groups of mice to a small intravenous infection with BCG. Comparative imwmnogenicity of heat-killed H,,RV in saline and Freund-type adjuvant. Mice of the three groups shown in Table 2, and of a group of untreated controls, were injected intravenously with 4 X lo5 viable BCG (Montreal). Representatives of each group were tested for tuberculin sensitivity at intervals during the next 15 days ; others were challenged on the same days with a standard inoculum of L. nzonocytogenes (S-10 LD,,‘s). The populations of Listeriu found 30 min and 24 hr later in spleens and livers were used to calculate an index of nonspecific resistance (21). The results recorded in Fig. 1 show that the two groups of immunized animals differed markedly in their responses to BCG. The level of tuberculin sensitivity, which was initially higher in the adjuvant-immunized animals, rose much more steeply in these mice. They also showed a more abrupt increase in resistance to challenge with L. rtzonocytogenes. Corresponding differences were seen in the growth patterns of BCG in spleen, liver, and lung. Only in animals immunized with HZ7Rv in adjuvant was the growth of BCG effectively inhibited. The foregoing experiment was repeated in animals which had been more recently

FIG. 1. Effect of a BCG (Montreal) infection on mice vaccinated with two dose,s of 100 pg of heat-killed H,,R, suspended in Freund’s adjuvant or in saline. Tuberculin sensitivity is represented by the histogram in the bottom section. The index of resistance to a Listeria challenge in spleen (H) and liver (0) and the behavior of the BCG population in the lung A,, liver 0, and spleen W are also shown. The arrow represents the size of the intravenous dose of BCG. Mice immunized with adjuvant or with saline alone are included as controls.

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derived from germ-free stock. The same doses of H,?R, were given, this time at an interval of 28 days. When tested 28 days after the second immunizing injection,. the animals of the two vaccinated groups differed markedly in their sensitivity to, tuberculin. Those immunized with H,,R, in saline gave a weak tuberculin reactionl (0.06 mm compared with 0.44 mm in the adjuvant-immunized mice). The twos groups of vaccinated mice and a group of normal controls were then challenged with 2 X 10” viable BCG (Montreal). The results recorded in Fig. 2 show consistent differences between the two immunized groups in respect to the growth of BCG in lung, liver, and spleen, the rate of increase in tuberculin sensitivity, and especially in the development of nonspecific resistance to the Listeria challenge. The adjuvant-immunized animals were again the only ones which effectively controlled the growth of the BCG in z&o. In a further experiment, the persistence of reactivity following immunization with two doses of 0.1 mg dead organisms in saline or in adjuvant was assessed 7 and 12 months after immunization. The results were similar; those recorded in Fig. 3 show that only the adjuvant-immunized animals remained specifically reactive to BCG as evidenced by the rate of development of nonspecific resistance to a Listeria challenge. The effect of antigen dose. The effects observed in the foregoing experiment with H,,R, in adjuvant were shown to be dose dependent. Three groups of mice were

FIG. 2. Effect of a BCG infection on mice vaccinated 1 month previously with two doses of 100 ,ug of heat-killed H,,R., in adjuvant or saline. An adjuvant control gave results similar to those shown in Fig. 1 and is therefore not included. See legend to Fig. 1 for the key to the curves.

272

COLLINS

0

5

AND

10

0

MACKANESS

5

10

0

5

1015

II'.'E Ih W,YS

FIG. 3. Effect of a BCG infection on mice vaccinated 7 months previously with two doses of 100 gg of heat-killed H,,R, in adjuvant or in saline. See legend to Fig. 1 for the key to the curves. Similar data were obtained 12 months after vaccination.

injected twice at an interval of 1 month with 500, 100, or 10 pg dry weight of heat-killed H,,R, in adjuvant. Twenty-eight days after the second injection, mice of all groups were challenged with BCG and tested for a response at intervals thereafter. The results showed (Fig. 4) that the two larger doses of HS7Rv were almost equivalent in their ability to presensitize the host for an accelerated response to infection with BCG. The highest dose was clearly the most effective in controlling the growth of BCG in lung, liver, and spleen ; but the animals were no better prepared for an anamnestic response to BCG, perhaps because they were already maximally reactive at a dose of 100 pg. Although smaller than in the other two groups, the response of mice which received the smallest dose of antigen was still substantial (cf. Fig. 1). DISCUSSION

A positive tuberculin reaction is accepted as evidence of successful vaccination with BCG, since it is statistically associated with an increased resistance to clinical tuberculosis in man and experimental animals (25, 26). Yet it has been argued that the mechanism of antituberculous resistance and the phenomenon of tuberculin skin sensitivity are not causally related (4), hypersensitivity and immunity being regarded as independent expressions of the immune state (3, 27). The evidence which would deny a role for hypersensitivity in acquired resistance assumes that delayed skin or footpad sensitivity is commensurate with other expressions of cellular hypersensitivity. The ability of BCG to produce tuberculin sensitivity is even thought by some to be an undesirable quality in a prophylactic agent because it

ANTITLJBERCULOUS

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5

10

lh!EMUNITY

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15

T, 2 E IN IMS

FIG. 4. Effect of a BCG infection in mice vaccinated twice with SO& 100, or 10 pg of heat-killed Hs7RV in Freund’s adjuvant at l-month intervals. Controls injected with adjuvant or saline alone have been omitted since they gave curves similar to those already shown in Figs. 1 and 2. Seelegend to Fig. 1 for the key to the curves. eliminates ~t~ber~ul~uconversion as an epidemiolog~ca~ and diagnostic tool (5). The accompanying paper (8) shows, however, that lymphoid cells from animals with a negative tuberculin reaction can sensitize normal recipients adoptivdy. Looked at from the opposing viewpoint, the present study provides positive evidence that hypersensitivity and resistance are related by focusing attention upon a fact that has been known for many years; namely, that the effectiveness of an inactivated antituberculous vaccine depends critically upon the manner of its presentation to the host (28). A subcutaneous injection of lyophi~ized organisms caused humoral antibody production, as evidenced by intense Arthus reactivity following the injection of PPD into the footpad. Much of this immediate hypersensitivity was presumably due to polysaccharide antigens, both in the vaccine and in the commercial PPD preparation (29). There was also an indication that saline-suspended organisms caused a minor degree of DTH; but the rate at which tnberculin sensitivity developed in response to RCG suggests that these were residual Arthus reactions (24). Animals immunized with the same preparation of antigen in adjuvant developed a minor degree of DTH, the importance of which became apparent only after infection with living BCG. A number of workers have also claimed that killed mycobacterial cells, cell walls, and cell extracts incorporated into a water-in-oil emulsion will induce DTH and/or increased resistance to tuberculous infection (1 I, 28). But tests employing a tuberculous challenge are inherently difficult to interpret because the level of a&tuberculous immunity at the time of challenge is inevitably obscured (6). In particular,

274

COLLINS

AND

MACKANESS

protection experiments employing a massive intravenous challenge with virulent tubercle bacilli are open to the criticism that they do not necessarily measure the level of antibacterial immunity in the vaccinated host at the time of challenge because the host response to a large dose of mycobacteria can be extremely rapid

(21). The present study represents an attempt to circumvent the foregoing difficulty by a method which is based upon the observation that mice which have been presensitized with a small dose of BCG display a dramatic recall of tuberculin sensitivity and nonspecific resistance following injection of a second dose of BCG (21). Much of the resistance observed in vaccinated animals could thus be generated in response to the challenge infection itself, aided no doubt by the extent to which immunization had prepared them to respond. It is this state of preparedness that the present test aims to measure. It has been reported (21) that heat-killed BCG, and BCG which has been inhibited by isoniazid treatment of the host, fail to sensitize or to raise resistance to a nonspecific challenge. This seems to indicate that the immunogenicity of living organisms is qualitatively different from that of dead bacilli. It has been argued, however, that the antigenic stimulus provided by a living vaccine is potentially much greater than that of an inactivated one, or that new antigens are elaborated in vivo. The present experiments take these objections into account, for the same suspension of heat-killed organisms was used for both groups of immunized animals. A response resembling that of an active infection occurred only in animals which received their dose of antigen in a sensitizing vehicle. The implication is that the development of cell-mediated reactivity to the tubercle bacillus is a critical step in the acquisition of specific resistance. It is unlikely that conventional methods would have detected the subtle, but crucially important, differences that characterize the host’s response to the same antigen in these two different forms. Each of three aspects of the host’s response to a small eliciting dose of living BCG revealed a marked inequality in the degree to which the two methods of immunization had prepared the host for an effective response to a mycobacterial infection. It is true that one of the parameters used to measure this response was nonspecific. But it should be remembered that nonspecific resistance is due to activation of macrophages (21), and that the enhanced antimicrobial properties of such cells are presumably available for expression against specific and nonspecific agents alike (30, 31). This view is consistent with the fact that high levels of nonspecific resistance to Listeria were always accompanied by a corresponding expression of antimycobacterial resistance against the eliciting dose of BCG itself. The results of the present study make it unlikely that a nonallergenic vaccine (5) can ever be developed for prophylactic use against tuberculosis. Apparently, effective antituberculous immunity depends upon the presentation of bacterial antigens in a manner that ensures the induction of cell-mediated reactivity. It follows that tuberculin sensitivity is an inevitable accompaniment of antituberculous immunity even though the host may at times be unable to express all of its various manifestations (8). It should be mentioned in support of this view that DTH and acquired resistance to other facultative intracellular parasites can be adoptively transferred with immunologically committed lymphocytes (32). Since these modali-

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ties of the immune state are transferred concurrently and proportionately, there can be little doubt that cell-mediated hypersensitivity is directly involved in the mechanism of host resistance to parasites of this type. It may not be essential, however, to maintain high and potentially dangerous levels of hypersensitivity in order to provide protection. Immunization which results in the creation of a specific memory from which the cells which mediate hypersensitivity and immunity can be rapidly regenerated may be all that is needed. It is quite possible, in fact, that memory and sensitivity are vested in different cell populations. In this circumstance, it would be possible for animals to possess most of the benefits of hypersensitivity without its inherent risks. This proposition deserves exploration. ACKNOWLEDGMENTS The ruff.

writers are grateful

for the technical

assistance of Oliver

Duprey,

and William

Wood-

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Ill. 1961. Uhr, J. W., Physiol. Rev. 46, 359, 1966. Weiss, D. W., Amer. Rev. Resp. Dis. 80, 340, 1959. Smith, D. W., Grover, A. A., and Wiegeshaus, E., Advalz. Tuberc. Res. 16, 191, 1968. Mackaness, G. B., Amer. Rev. Resp. Dis. 97, 337, 1969. Collins, F. M., and Mackaness, G. B., Cell. Immunol. 1, 253, 1970. Dubos, R. J., Schaefer, W. B., and Pierce, C. H., J. Exp. Med. 97, 221, 1953. Weiss, D. W., Amer. Rev. Tuberc. 77, 719, 1958. Youmans, G. P., and Youmans, A. S., J. Bacferiol. 97, 107, 1969. Ribi, E., Anacker, R. L., Brehmer, W., Goode, G., Larson, C. L., List, R. H., Milne, K. C., and Wicht, W. C., J. Bacterial. 92, 869, 1966. Sever, J. L., and Youmans, G. P., Amer. Rev. Tuberc. 76, 616, 1957. Hobby, G. L., Lenert, T. F., Maier-Engallena, J., Wakely, C., Keblish, M., Manty, A., and Auerbach, O., Amer. Rev. Resp. Dis. 93, 396, 1966. Collins, F. M., and Miller, T. E., J. Infec. Dis. 120, 517, 1969. Kanai, K., and Yanagisawa, K., Jab. J. Med. Sci. Biol. 8, 115, 1955. Dubos, R. J., and Schaefer, W. B., Amer. Rev. Tuberc. 74, 541, 1956. Mackaness, G. B., Smith, N., and Wells, A. Q., Amer. Rev. Tuberc. 69, 479, 1954. Steenken, W., Jr., Amer. Rev. Resp. Dis. 83, 550, 1961. Levy, F. M., Conge, G. A., Pasquier, J. F., Mauss, H., Dubos, R. J., and Schaedler, R. W., Amer. Rev. Resp. Dis. 84, 28, 1961. Blanden, R. V., Lefford, M. J., and Mackaness, G. B., J. Exfi. Med. 129, 1079, 1969. Collins, F. M., and Smith, M. M., Amer. Rev. Resp. Dis. 100, 631, 1969. Freund, J., Advalz. Tuberc. Res. 1, 130, 1956. Collins, F. M., Volkman, A., and McGregor, D. D., Inzmunology 19, 1970, in press. Cohn, M. L., Davis, C. L., and Middlebrook, G., Science 128, 1282, 1958. Springett, V. H., Tubercle 46, 76, 1965. Arnason, G. B., and Waksman, B. H., Advan. Tuberc. Res. 13, 1, 1964. Weiss, D. W., and Dubos, R. J., J. Exp. Med. 103, 73, 1956. Seibert, F. B., Amer. Rev. Tubeuc. 59, 86, 1949. Mackaness, G. B., J. Exp. Med. 116. 381, 1962. Mackaness, G. B., and Blanden, R. V., Progr. Allergy 11, 89, 1967. Mackaness, G. B., J. E.Yfi. Med. 129, 973, 1969.