INFECTION AND IMMUNITY, Feb. 1978, p. 391-394 0019-9567/78/0019-0391$02.00/0 Copyright 0 1978 American Society for Microbiology
Vol. 19, No. 2
Printed i U.S.A.
Immunity to Mycobacterium leprae Infections Induced in Mice by BCG Vaccination at Different Times Before or After Challenge C. C. SHEPARD,* L. L. WALKER, AND R. M. VAN LANDINGHAM Center for Disease Control, Atlanta, Georgia 30333 Received for publication 17 October 1977
Viable suspensions of BCG, an attenuated strain of Mycobacterium bovis, have been previously shown to immunize mice against infections with M. keprae. Usually, the mice have been vaccinated about 1 month before challenge. Experiments have now been carried out with single intradermal injections of BCG given before or after the M. Ieprae challenge. Approximately equal immunizing effect was seen in one experiment when the BCG was given at -168, -119, -70, and -28 days relative to challenge. Approximately equal protection was observed in another experiment when the vaccine was given at -28, +28, and +56 days. In the latter experiment, however, vaccine given at +91 days appeared to be somewhat less effective. Enlargement of the lymph nodes regional to the intradermal vaccine site perssted for at least the duration of the experiment, approximately 400 days. Thus, antigenic stimulation appears to have continued throughout the period of observation. The usual incubation period of human leprosy is several years, and that of the experimental infection of mice is 5 to 8 months when the inoculum contains 5,000 Mycobacteriun leprae. In contrast, an immune response can in general be induced in humanst and mice relatively rapidly. Because of the slowness of the M. leprae infection, there are unusual temporal relationships between vaccination and infection. In the experimental infection the inoculation is separated from the later growth of bacilli by a period of months, so the possibility exists that an immune effect can be exerted exclusively against either the inoculated M. Ieprae themselves during the early events of the infection or the new bacillary growth during the later events of the infection. Chronic graft-versus-host reactions, induced in Fl hybrids by injection of parental cells, can exert an effect against the inoculum but not against the later M. leprae growth, especially when the parental cells are injected into the site of M. keprae inoculation (13). This early effect is manifested as simple delay in the appearance of the M. keprae growth; the bacilary growth, nevertheless, takes place at the typical logarithmic rate and terminates at the usual plateau level. Viable suspensions of the BCG strain of M. bovis are effective vaccines against the experimental infection (9), especially when given intradermally (9, 13). Even when given during the incubation period, BCG is active (10),
and this activity is, of course, not exerted against the inoculum itself. Although BCG and M. leprae share some common antigens, as do all mycobacteria, BCG is not closely related to M. Ieprae (6). The specificity of the vaccine activity of BCG against M. leprae is also called into question by its activity against many tumors. BCG apparently shares antigen(s) with some of the tumors against which it is active (3, 4), but whether the activity depends on shared antigen(s), on the adjuvant activity of BCG, or on some other nonspecific immunological activity is not clear. The present experiments on the timing of BCG were undertaken to determine whether special relationships might appear that would clarify the mechanism of action of BCG against M. Ieprae infections. Nonspecific immunity that depends on activation of macrophages is frequently of short duration, since it depends on the persistence of sufficient amounts of antigen to react with specifically sensitized lymphocytes, whereas specific imnunity, even though it may depend on the renewed generation of "mediator" T cells, is longer lasting (3).
MATERIALS AND METHODS The methods have been described elsewhere (9, 12, 13) in detail. In brief, a viable suspension of BCG was injected intradermally in the flank of different groups of mice (strain CFW) at different times in relation to 391
392
INFECTr. IMMUN.
SHEPARD, WALKER, AND VAN LANDINGHAM
the M. leprae inoculation. The BCG suspension came from young cultures in Tween-albumin medium; the growth was sedimented by centrifugation, washed, suspended in phosphate-buffered saline (pH 7.3) containing 0.05% Tween 80 (phosphate-buffered saline-Tween) adjusted to a standard concentration, and preserved at -60°C. The administered suspension contained 0.1 1d of bacterial mass (as measured by Hopkins tube) or approximately 107 acid-fast bacteria in the 0.01 ml injected. The M. leprae was a mouse passage strain, and 5,000 acid-fast bacteria were injected into the right hind footpad in a volume of 0.03 ml. At suitable 4-week intervals, the M. leprae in the harvested footpad tissues of mice in control (unvaccinated) and vaccinated groups were counted, so that the growth curves of M. leprae could be compared. Two experiments were carried out-one with BCG given at various times before the challenge with M. leprae up until -28 days, the other with BCG given at -28 days and at various times after the challenge. In both experiments there were two control groups, one inoculated with M. leprae before the vaccinated mice and one inoculated after. Occasionally slightly more growth has been seen with the M. leprae inoculated late in an experiment (9), but in the present work there were no important differences between the two control groups. Each control and vaccinated group contained 30 mice. Earlier results (10) had indicated that part of the effect of BCG vaccine might be a delay in the growth of M. leprae rather than the imposition of a lower plateau level. Therefore, the harvests were carried out at regular intervals so that the growth curve of M. leprae in each mouse group could be characterized. In some cases the size of the regional (inguinal) lymph nodes was measured as an indicator of vaccine activity. For this purpose the fur over the inguinal area was closely clipped, and the skin was wetted with alcohol to render it more transparent. Two diameters at right angles were recorded.
RESULTS Vaccination given before challenge. There were no u-nportant differences among the various groups vaccinated before challenge (Fig. 1). The highest count of M. leprae in the vaccinated mice was 10578, in the harvest at 222 days from the -168-day group. This represented the arithmetic mean of values for individual mice of 10631, 105, and two negative mice. The -110-, -70-, and -28-day vaccinated groups at 222 days had four, four, and two negative mice, respectively; thus, the differences between vaccinated groups were not significant. The growth of M. leprae in the vaccinated mice was obviously suppressed to low plateau levels, but it was not clear from the growth curves in each group whether the growth was also delayed in appearance. The regional inguinal lymph node drains the vaccine site in the flank; the size of the lymph node in vaccinated and unvaccinated animals at
the time of the 222-day and 250-day harvests is shown in Fig. 2. In most vaccinated mice the lymph node was definitely enlarged. For the group vaccinated at -168 days, the period of time that had elapsed since vaccination was 390 and 418 days for the harvests at 222 and 250 70-
BCG vaccine 0 -168D o -119D A -70D
x x
V -28D X Control
X-X
x
60
I
x
~~x
x
502so
40-
Neg
x
100
300
200
Days after challenge
FIG. 1. M. Ieprae harvests with BCG administered before challenge with M. leprae. Each value represents the pooled or averaged results with four mice, with the following exceptions: at 250 days only, each control group had six mice, the -168-day group had two mice, and the -28-day group had three mice. The values represent counts on the pools of footpads of four mice through 188 days and the averages of counts on individual mice at 222 and 250 days. A dot is shown between two symbols having the same value.
* 222 Day Harvest x 250 14
%
x
212 0 .O
i)N o
10
x
. 0 *xx .
0
Z cL
6
E
4
3
2
UK
so0
0
)
ex
.c -
c Time $
of BCG
168-1i9-70 -28 Nil
---
FIG. 2. Average diameter of inguinal lymph node (regional to site of BCG vaccination) for mice harvested at 222 and 250 days.
VOL. 19, 1978
IMMUNITY TO M. LEPRAE INFECTIONS
days, respectively. The relationship between the size of the inguinal lymph node and the harvest of M. leprae was examined. M. leprae were sometimes encountered in mice that had been well vaccinated, as shown by lymph node responses. The one harvest from a vaccinated mouse that was above 1060 was from one with a normal-sized lymph node in the -168-day group at 222 days, so this animal may have been improperly vaccinated. This high harvest was responsible for the high average harvest at 222 days from the -168-day group. Vaccination during the incubation period. The results for mice vaccinated during the incubation period are presented in Fig. 3. The vaccines given at all times were effective, and that given at -28 days was about as effective as the one administered at -28 days in the experiment just described (prechallenge vaccination). The vaccine given at +91 days, however, appears to have been less effective than the others; the harvest at 240 days was higher than even that of the unvaccinated controls, and the harvest at three other intervals was higher than that in the other vaccinated groups. The curve for the +91-day group rises to a maximum at 240 days and then falls, but this may have resulted by chance. In previous work M. keprae has not been observed to be cleared from the
6.0 w
C,,
x Control, (No vaccine) BCG vaccine,ID o -28D +28D A+56D V+91 D
0 w
':1 a.
5.0
-J 0
i0 4.0 Neg.
x
x
x.o
200 100 DAYS AFTER CHALLENGE
300
FIG. 3. M. leprae harvests with BCG administered during the incubation period. Each point represents the results on a pool offour mice, with the following exceptions: at 300 day8 only, one contol group had one mowuse, and the +56-day group had three mice. A dot is shown between two symbols having the same value.
393
footpad with such rapidity. The inguinal lymph nodes were not measured in this group, but other observations with the same vaccine have shown that this dose produces definite lymph node enlargement as early as 1 week.
DISCUSSION In the two experiments reported, the BCG was administered from -168 days to +91 days relative to the M. leprae challenge. The resultant immunity, as measured by counts of M. leprae at various times after challenge, was approximately the same for all groups vaccinated from -168 to +56 days. For unknown reasons, the temporal changes in immunity that were observed in a previous experiment on vaccine given during the incubation period (10) were not seen this time. Instead there was a simple picture of a vaccine-induced immunity that persisted unchanged over a long period, and the results with vaccine given after infection were similar to those with vaccine given before infection. These findings suggest that the immunity was exerted against the new growth of M. leprae in the mice and that it did not simply eliminate part of the inoculated M. leprae or temporarily restrain multiplication of M. leprae. Growth of M. leprae became detectable in the controls at about 100 days after inoculation, or 270 days after vaccination in the -168-day group. In the +91-day group the new growth of M. leprae probably appeared before the mice had completed their immune response. Lymph node measurements provided useful information. Preliminary histological observations indicated that the enlargement was caused by increased numbers of lymphocytes chiefly in the paracortex, although small islands of macrophages were sometimes present. No increased follicular activity and no necrosis were observed. The enlarged lymph nodes seem to depend upon the persistence of antigen and sensitized T-lymphocytes. Thus, the lymph node enlargements observed here indicate that both BCG antigen and lymphocyte sensitivity to BCG antigen persisted for at least 400 days after immunization. Continuing lymphocyte stimulation may be necessary for vaccine activity in mice (5). We have observed little, if any, vaccine protection provided by cultivable mycobacteria that do not produce lymph node enlargement after intradermal injection. So far, 13 other cultures (8 species) have been tried. The only suspensions that have given distinct protection have been living BCG and living and heat-killed M. leprae, and only these suspensions have produced distinct regional lymph node enlargement (unpublished data).
INFECT. IMMUN. 394 SHEPARD, WALKER, AND VAN LANDINGHAM As stated, the experiments were undertaken initiate the chain of events that would result in to determine whether temporal changes in the the early interruption of infection. The differentiation between specific and noneffect of BCG vaccination might clarify the mechanism of its action against M. leprae. In specific immunity is important in considerations many models, specific immunity against a bac- of the applicability of the experimental results terial infection is long lasting, whereas nonspe- to humans. In vaccination experiments the chalcific immunity (against antigenically unrelated lenge is usually given soon after the vaccination, organisms) is of short duration (reviewed in ref- in our work with M. leprae normally at 28 days. erence 5). The most widely accepted theoretical If BCG is to be useful prophylactically in huexplanation for this difference in the duration mans, it must provide protection for years. The efficacy of BCG vaccine against leprosy of specific and nonspecific immunity is as follows: Although in both cases the bacteria are in humans remains unsettled because of the eliminated by activated macrophages, the mac- difference in the results obtained in three large, rophages are activated only as a result of the well-conducted field trials (1, 2, 8). Recent rereaction between antigen and lymphocytes that sults in mice offer promise that a vaccine can are sensitized to the antigen. When the animal be prepared from heat-killed M. leprae (11). eliminates the infecting bacterium and recovers ACKNOWLEDGMENTS from the infection, "memory" lymphocytes perTbis work was supported by the United States-Japan Cosist for a long time, perhaps in small numbers, Medical Science Program administered by the Naand when they encounter the same bacterium operative tional Institute of Allergy and Infectious Diseases by means in the second challenge, the increase in numbers of an interagency agreement between the National Institute of sensitized lymphocytes is so accelerated that of Allergy and Infectious Diseases and the Center for Disease the macrophage activation and consequent elim- Control. ination of the challenge bacterium come at an LITERATURE CITED early stage and abort the infection. In contrast 1. Bechelli, L M., A. Garbajosa, K. Uemura, V. Engler, to memory lymphocytes, activated macrophages V. Martinez Dominguez, L. Paredes, T. Sundaresoon disappear after the elimination of the insan, G. Koch, and M. Matejka. 1970. BCG vaccination fecting bacterium, and as a result they are found of children in leprosy. Preliminary findings of the WHOonly for short periods after the encounters becontrolled trial in Burma. Bull. W.H.O. 42:235-281. tween antigen and specific lymphocytes. Thus, 2. Brown, J. A. K., M. M. Stone, and I. Sutherland. 1969. Trial of BCG vaccination against leprosy in the temporal dissociation between specific and Uganda. Lep. Rev. 40:3-7. nonspecific immunity depends upon the elimi- 3. Bucana, C., and M. G. Hanna, Jr. 1974. Immunoelecnation of the specific bacterium and consequent tronmicroscopic analysis of surface antigens common cessation of macrophage activation. In our to Myocbacterium bovis (BCG) and tumor cells. J. Natl. Cancer Inst. 63:1313-1323. model, however, the continuing lymph node enP., J. K. McClatchy, M. Wainberg, and D. largements showed that the BCG antigen per- 4. Minden, W. Weiss. 1974. Shared antigens between Mycobactesisted throughout the period of observation. rium bovis (BCG) and neoplastic cells. J. Natl. Cancer Therefore, because macrophage activation conInst. 53:1325-1331. 5. North, R. J. 1974. Cell mediated immunity and the retinued without interruption, there may have sponse to infection, p. 185-220. In R. T. McCluskey been no temporal dissociation of specific and and S. Cohen (ed.), Mechanism of cell-mediated imnonspecific immunity. munity. John Wiley and Sons, Inc., New York. Nevertheless, it seems unlikely that the pro6. Paul, R. C., J. L. Stanford, and J. W. Carswell. 1975. tection afforded by BCG vaccination against M. Multiple skin testing in leprosy. J. Hyg. 76:57-68. G. H., M. E. Smith, and W. L. Ford. 1977. leprae infection was nonspecific, i.e., that it de- 7. Rannie, Lymphocyte migration into cell-mediated immune lepended upon activation of macrophages as a sions is inhibited by trypsin. Nature (London) result of the reaction between BCG-sensitized 267:520-522. lymphocytes and BCG. Macrophages are 8. Russell, D. A., G. C. Scott, and S. C. Wigley. 1968. BCG and prophylaxis. Int. J. Lepr. 36:618. thought to be activated only locally and, even 9. Shepard, C. C. 1965. Vaccination against experimental if they gain access to the circulation, to be ininfection with Mycobacterium leprae. Am. J. Epidecapable of leaving the circulation to enter an miol. 81:150-163. extravascular scene of infection. In our model 10. Shepard, C. C. 1966. Vaccination against human leprosy bacillus infections of mice: protection by BCG during the BCG antigen was concentrated largely at the incubation period. J. Immunol. 96:279-283. the intradermal site of vaccination and in the 11. Shepard, C. C. 1976. Heat stability of M. Leprae's imlymph nodes draining the site and was therefore munogenicity. Int. J. Lepr. 44:554. unlikely to reach the footpad in critical quanti- 12. Shepard, C. C., and D. H. McRae. 1968. A method for counting acid-fast bacteria. Int. J. Lepr. 36:78-82. ties. In contrast, lymphocytes course normally C. C., R. Van Landingham, and L. L. through peripheral, nonlymphoid tissues (7); in 13. Shepard, Walker. 1976. Immunity to Mycobacteriun leprae inthis case BCG-sensitized lymphocytes could enfections in mice stimulated by M. leprae, BCG, and counter cross-reacting M. leprae antigens and graft-versus-host reactions. Infect. Immun. 14:919-928.
Vol. 19, No. 2
Printed i U.S.A.
Immunity to Mycobacterium leprae Infections Induced in Mice by BCG Vaccination at Different Times Before or After Challenge C. C. SHEPARD,* L. L. WALKER, AND R. M. VAN LANDINGHAM Center for Disease Control, Atlanta, Georgia 30333 Received for publication 17 October 1977
Viable suspensions of BCG, an attenuated strain of Mycobacterium bovis, have been previously shown to immunize mice against infections with M. keprae. Usually, the mice have been vaccinated about 1 month before challenge. Experiments have now been carried out with single intradermal injections of BCG given before or after the M. Ieprae challenge. Approximately equal immunizing effect was seen in one experiment when the BCG was given at -168, -119, -70, and -28 days relative to challenge. Approximately equal protection was observed in another experiment when the vaccine was given at -28, +28, and +56 days. In the latter experiment, however, vaccine given at +91 days appeared to be somewhat less effective. Enlargement of the lymph nodes regional to the intradermal vaccine site perssted for at least the duration of the experiment, approximately 400 days. Thus, antigenic stimulation appears to have continued throughout the period of observation. The usual incubation period of human leprosy is several years, and that of the experimental infection of mice is 5 to 8 months when the inoculum contains 5,000 Mycobacteriun leprae. In contrast, an immune response can in general be induced in humanst and mice relatively rapidly. Because of the slowness of the M. leprae infection, there are unusual temporal relationships between vaccination and infection. In the experimental infection the inoculation is separated from the later growth of bacilli by a period of months, so the possibility exists that an immune effect can be exerted exclusively against either the inoculated M. Ieprae themselves during the early events of the infection or the new bacillary growth during the later events of the infection. Chronic graft-versus-host reactions, induced in Fl hybrids by injection of parental cells, can exert an effect against the inoculum but not against the later M. leprae growth, especially when the parental cells are injected into the site of M. keprae inoculation (13). This early effect is manifested as simple delay in the appearance of the M. keprae growth; the bacilary growth, nevertheless, takes place at the typical logarithmic rate and terminates at the usual plateau level. Viable suspensions of the BCG strain of M. bovis are effective vaccines against the experimental infection (9), especially when given intradermally (9, 13). Even when given during the incubation period, BCG is active (10),
and this activity is, of course, not exerted against the inoculum itself. Although BCG and M. leprae share some common antigens, as do all mycobacteria, BCG is not closely related to M. Ieprae (6). The specificity of the vaccine activity of BCG against M. leprae is also called into question by its activity against many tumors. BCG apparently shares antigen(s) with some of the tumors against which it is active (3, 4), but whether the activity depends on shared antigen(s), on the adjuvant activity of BCG, or on some other nonspecific immunological activity is not clear. The present experiments on the timing of BCG were undertaken to determine whether special relationships might appear that would clarify the mechanism of action of BCG against M. Ieprae infections. Nonspecific immunity that depends on activation of macrophages is frequently of short duration, since it depends on the persistence of sufficient amounts of antigen to react with specifically sensitized lymphocytes, whereas specific imnunity, even though it may depend on the renewed generation of "mediator" T cells, is longer lasting (3).
MATERIALS AND METHODS The methods have been described elsewhere (9, 12, 13) in detail. In brief, a viable suspension of BCG was injected intradermally in the flank of different groups of mice (strain CFW) at different times in relation to 391
392
INFECTr. IMMUN.
SHEPARD, WALKER, AND VAN LANDINGHAM
the M. leprae inoculation. The BCG suspension came from young cultures in Tween-albumin medium; the growth was sedimented by centrifugation, washed, suspended in phosphate-buffered saline (pH 7.3) containing 0.05% Tween 80 (phosphate-buffered saline-Tween) adjusted to a standard concentration, and preserved at -60°C. The administered suspension contained 0.1 1d of bacterial mass (as measured by Hopkins tube) or approximately 107 acid-fast bacteria in the 0.01 ml injected. The M. leprae was a mouse passage strain, and 5,000 acid-fast bacteria were injected into the right hind footpad in a volume of 0.03 ml. At suitable 4-week intervals, the M. leprae in the harvested footpad tissues of mice in control (unvaccinated) and vaccinated groups were counted, so that the growth curves of M. leprae could be compared. Two experiments were carried out-one with BCG given at various times before the challenge with M. leprae up until -28 days, the other with BCG given at -28 days and at various times after the challenge. In both experiments there were two control groups, one inoculated with M. leprae before the vaccinated mice and one inoculated after. Occasionally slightly more growth has been seen with the M. leprae inoculated late in an experiment (9), but in the present work there were no important differences between the two control groups. Each control and vaccinated group contained 30 mice. Earlier results (10) had indicated that part of the effect of BCG vaccine might be a delay in the growth of M. leprae rather than the imposition of a lower plateau level. Therefore, the harvests were carried out at regular intervals so that the growth curve of M. leprae in each mouse group could be characterized. In some cases the size of the regional (inguinal) lymph nodes was measured as an indicator of vaccine activity. For this purpose the fur over the inguinal area was closely clipped, and the skin was wetted with alcohol to render it more transparent. Two diameters at right angles were recorded.
RESULTS Vaccination given before challenge. There were no u-nportant differences among the various groups vaccinated before challenge (Fig. 1). The highest count of M. leprae in the vaccinated mice was 10578, in the harvest at 222 days from the -168-day group. This represented the arithmetic mean of values for individual mice of 10631, 105, and two negative mice. The -110-, -70-, and -28-day vaccinated groups at 222 days had four, four, and two negative mice, respectively; thus, the differences between vaccinated groups were not significant. The growth of M. leprae in the vaccinated mice was obviously suppressed to low plateau levels, but it was not clear from the growth curves in each group whether the growth was also delayed in appearance. The regional inguinal lymph node drains the vaccine site in the flank; the size of the lymph node in vaccinated and unvaccinated animals at
the time of the 222-day and 250-day harvests is shown in Fig. 2. In most vaccinated mice the lymph node was definitely enlarged. For the group vaccinated at -168 days, the period of time that had elapsed since vaccination was 390 and 418 days for the harvests at 222 and 250 70-
BCG vaccine 0 -168D o -119D A -70D
x x
V -28D X Control
X-X
x
60
I
x
~~x
x
502so
40-
Neg
x
100
300
200
Days after challenge
FIG. 1. M. Ieprae harvests with BCG administered before challenge with M. leprae. Each value represents the pooled or averaged results with four mice, with the following exceptions: at 250 days only, each control group had six mice, the -168-day group had two mice, and the -28-day group had three mice. The values represent counts on the pools of footpads of four mice through 188 days and the averages of counts on individual mice at 222 and 250 days. A dot is shown between two symbols having the same value.
* 222 Day Harvest x 250 14
%
x
212 0 .O
i)N o
10
x
. 0 *xx .
0
Z cL
6
E
4
3
2
UK
so0
0
)
ex
.c -
c Time $
of BCG
168-1i9-70 -28 Nil
---
FIG. 2. Average diameter of inguinal lymph node (regional to site of BCG vaccination) for mice harvested at 222 and 250 days.
VOL. 19, 1978
IMMUNITY TO M. LEPRAE INFECTIONS
days, respectively. The relationship between the size of the inguinal lymph node and the harvest of M. leprae was examined. M. leprae were sometimes encountered in mice that had been well vaccinated, as shown by lymph node responses. The one harvest from a vaccinated mouse that was above 1060 was from one with a normal-sized lymph node in the -168-day group at 222 days, so this animal may have been improperly vaccinated. This high harvest was responsible for the high average harvest at 222 days from the -168-day group. Vaccination during the incubation period. The results for mice vaccinated during the incubation period are presented in Fig. 3. The vaccines given at all times were effective, and that given at -28 days was about as effective as the one administered at -28 days in the experiment just described (prechallenge vaccination). The vaccine given at +91 days, however, appears to have been less effective than the others; the harvest at 240 days was higher than even that of the unvaccinated controls, and the harvest at three other intervals was higher than that in the other vaccinated groups. The curve for the +91-day group rises to a maximum at 240 days and then falls, but this may have resulted by chance. In previous work M. keprae has not been observed to be cleared from the
6.0 w
C,,
x Control, (No vaccine) BCG vaccine,ID o -28D +28D A+56D V+91 D
0 w
':1 a.
5.0
-J 0
i0 4.0 Neg.
x
x
x.o
200 100 DAYS AFTER CHALLENGE
300
FIG. 3. M. leprae harvests with BCG administered during the incubation period. Each point represents the results on a pool offour mice, with the following exceptions: at 300 day8 only, one contol group had one mowuse, and the +56-day group had three mice. A dot is shown between two symbols having the same value.
393
footpad with such rapidity. The inguinal lymph nodes were not measured in this group, but other observations with the same vaccine have shown that this dose produces definite lymph node enlargement as early as 1 week.
DISCUSSION In the two experiments reported, the BCG was administered from -168 days to +91 days relative to the M. leprae challenge. The resultant immunity, as measured by counts of M. leprae at various times after challenge, was approximately the same for all groups vaccinated from -168 to +56 days. For unknown reasons, the temporal changes in immunity that were observed in a previous experiment on vaccine given during the incubation period (10) were not seen this time. Instead there was a simple picture of a vaccine-induced immunity that persisted unchanged over a long period, and the results with vaccine given after infection were similar to those with vaccine given before infection. These findings suggest that the immunity was exerted against the new growth of M. leprae in the mice and that it did not simply eliminate part of the inoculated M. leprae or temporarily restrain multiplication of M. leprae. Growth of M. leprae became detectable in the controls at about 100 days after inoculation, or 270 days after vaccination in the -168-day group. In the +91-day group the new growth of M. leprae probably appeared before the mice had completed their immune response. Lymph node measurements provided useful information. Preliminary histological observations indicated that the enlargement was caused by increased numbers of lymphocytes chiefly in the paracortex, although small islands of macrophages were sometimes present. No increased follicular activity and no necrosis were observed. The enlarged lymph nodes seem to depend upon the persistence of antigen and sensitized T-lymphocytes. Thus, the lymph node enlargements observed here indicate that both BCG antigen and lymphocyte sensitivity to BCG antigen persisted for at least 400 days after immunization. Continuing lymphocyte stimulation may be necessary for vaccine activity in mice (5). We have observed little, if any, vaccine protection provided by cultivable mycobacteria that do not produce lymph node enlargement after intradermal injection. So far, 13 other cultures (8 species) have been tried. The only suspensions that have given distinct protection have been living BCG and living and heat-killed M. leprae, and only these suspensions have produced distinct regional lymph node enlargement (unpublished data).
INFECT. IMMUN. 394 SHEPARD, WALKER, AND VAN LANDINGHAM As stated, the experiments were undertaken initiate the chain of events that would result in to determine whether temporal changes in the the early interruption of infection. The differentiation between specific and noneffect of BCG vaccination might clarify the mechanism of its action against M. leprae. In specific immunity is important in considerations many models, specific immunity against a bac- of the applicability of the experimental results terial infection is long lasting, whereas nonspe- to humans. In vaccination experiments the chalcific immunity (against antigenically unrelated lenge is usually given soon after the vaccination, organisms) is of short duration (reviewed in ref- in our work with M. leprae normally at 28 days. erence 5). The most widely accepted theoretical If BCG is to be useful prophylactically in huexplanation for this difference in the duration mans, it must provide protection for years. The efficacy of BCG vaccine against leprosy of specific and nonspecific immunity is as follows: Although in both cases the bacteria are in humans remains unsettled because of the eliminated by activated macrophages, the mac- difference in the results obtained in three large, rophages are activated only as a result of the well-conducted field trials (1, 2, 8). Recent rereaction between antigen and lymphocytes that sults in mice offer promise that a vaccine can are sensitized to the antigen. When the animal be prepared from heat-killed M. leprae (11). eliminates the infecting bacterium and recovers ACKNOWLEDGMENTS from the infection, "memory" lymphocytes perTbis work was supported by the United States-Japan Cosist for a long time, perhaps in small numbers, Medical Science Program administered by the Naand when they encounter the same bacterium operative tional Institute of Allergy and Infectious Diseases by means in the second challenge, the increase in numbers of an interagency agreement between the National Institute of sensitized lymphocytes is so accelerated that of Allergy and Infectious Diseases and the Center for Disease the macrophage activation and consequent elim- Control. ination of the challenge bacterium come at an LITERATURE CITED early stage and abort the infection. In contrast 1. Bechelli, L M., A. Garbajosa, K. Uemura, V. Engler, to memory lymphocytes, activated macrophages V. Martinez Dominguez, L. Paredes, T. Sundaresoon disappear after the elimination of the insan, G. Koch, and M. Matejka. 1970. BCG vaccination fecting bacterium, and as a result they are found of children in leprosy. Preliminary findings of the WHOonly for short periods after the encounters becontrolled trial in Burma. Bull. W.H.O. 42:235-281. tween antigen and specific lymphocytes. Thus, 2. Brown, J. A. K., M. M. Stone, and I. Sutherland. 1969. Trial of BCG vaccination against leprosy in the temporal dissociation between specific and Uganda. Lep. Rev. 40:3-7. nonspecific immunity depends upon the elimi- 3. Bucana, C., and M. G. Hanna, Jr. 1974. Immunoelecnation of the specific bacterium and consequent tronmicroscopic analysis of surface antigens common cessation of macrophage activation. In our to Myocbacterium bovis (BCG) and tumor cells. J. Natl. Cancer Inst. 63:1313-1323. model, however, the continuing lymph node enP., J. K. McClatchy, M. Wainberg, and D. largements showed that the BCG antigen per- 4. Minden, W. Weiss. 1974. Shared antigens between Mycobactesisted throughout the period of observation. rium bovis (BCG) and neoplastic cells. J. Natl. Cancer Therefore, because macrophage activation conInst. 53:1325-1331. 5. North, R. J. 1974. Cell mediated immunity and the retinued without interruption, there may have sponse to infection, p. 185-220. In R. T. McCluskey been no temporal dissociation of specific and and S. Cohen (ed.), Mechanism of cell-mediated imnonspecific immunity. munity. John Wiley and Sons, Inc., New York. Nevertheless, it seems unlikely that the pro6. Paul, R. C., J. L. Stanford, and J. W. Carswell. 1975. tection afforded by BCG vaccination against M. Multiple skin testing in leprosy. J. Hyg. 76:57-68. G. H., M. E. Smith, and W. L. Ford. 1977. leprae infection was nonspecific, i.e., that it de- 7. Rannie, Lymphocyte migration into cell-mediated immune lepended upon activation of macrophages as a sions is inhibited by trypsin. Nature (London) result of the reaction between BCG-sensitized 267:520-522. lymphocytes and BCG. Macrophages are 8. Russell, D. A., G. C. Scott, and S. C. Wigley. 1968. BCG and prophylaxis. Int. J. Lepr. 36:618. thought to be activated only locally and, even 9. Shepard, C. C. 1965. Vaccination against experimental if they gain access to the circulation, to be ininfection with Mycobacterium leprae. Am. J. Epidecapable of leaving the circulation to enter an miol. 81:150-163. extravascular scene of infection. In our model 10. Shepard, C. C. 1966. Vaccination against human leprosy bacillus infections of mice: protection by BCG during the BCG antigen was concentrated largely at the incubation period. J. Immunol. 96:279-283. the intradermal site of vaccination and in the 11. Shepard, C. C. 1976. Heat stability of M. Leprae's imlymph nodes draining the site and was therefore munogenicity. Int. J. Lepr. 44:554. unlikely to reach the footpad in critical quanti- 12. Shepard, C. C., and D. H. McRae. 1968. A method for counting acid-fast bacteria. Int. J. Lepr. 36:78-82. ties. In contrast, lymphocytes course normally C. C., R. Van Landingham, and L. L. through peripheral, nonlymphoid tissues (7); in 13. Shepard, Walker. 1976. Immunity to Mycobacteriun leprae inthis case BCG-sensitized lymphocytes could enfections in mice stimulated by M. leprae, BCG, and counter cross-reacting M. leprae antigens and graft-versus-host reactions. Infect. Immun. 14:919-928.
