BCG-induced protection in guinea pigs vaccinated and challenged via the respiratory route

Tuber& and Lung Disease (1993) 74.3846 0 1993 Loneman Grow UK Ltd

BCG-induced protection in guinea pigs vaccinated and challenged via the respiratory route M. Lagranderie*,

I? Ravisse*, G. Marchal”, M. Gheorghiu *, V. Balasubramanian+,

E.H. Weigeshaus+, D. W. Smitht,

*Dkpartement de Physiopathologie Expe’rimentale, Institut Pasteur; Paris, France, Wisconsin-Madison Medical School, USA

‘Department of Microbiology

S U M MA R Z Since studies on cellular immune responses have demonstrated the role of the mucosal lymphoid system of the respiratory tract, we have studied responses obtained from the local respiratory route, compared to the systemic intradermal route, of BCG immunization. Guinea pigs vaccinated with diierent doses of BCG via both routes served to follow lymphoid cell proliferation, hilar lymph node and lung BCG clearance, lung granuloma formation and protection induced after virulent challenge. Results demonstrate that the aerogenic route of vaccination with BCG has no harmful side-effects for the host. In comparison with the intradermal route of vaccination, aerogenic vaccination with lo5 BCG cfu induced higher local cellular immune responses and a substantially improved protective effect. R k S U M k.

Depuis que des etudes sur les responses immunitaires ont montre le r6le du syst&me lympholde muqueux des voies respiratoires, nous avons CtudiCles reponses obtenues par la voie locale, cornparke B la voie systemique intradermique, au tours de l’humunisation par le BCG. Des cobayes vaccines avec differentes doses de BCG via les deux voies ont permis de suivre la proliferation des cellules lymphoides, la clairance du BCG par les ganglions lymphatiques hilaires et pulmonaires, la formation des granulomes dans le poumon et la protection induite apr&sune virulente. Les resultats demontrent que la route aeroge de la vaccination par le BCG n’apporte aucun effet secondaire nocif pour l’h6te. Par comparaison avec la vaccination par voie intradermique, la vaccination par voie aCrog&neavec 10’ BCG ufc (unites formant colonies) a induit des reponses hmuunitaires cellulaires locales plus intenses et a nettement am6liore l’effet protecteur. R E S U M E N. Dado el hecho que estudios sobre la respuesta celular immune han mostrado el rol de1 sistema linfiitico de las mucosas de1tract0 respiratorio, hemos estudiado las respuestas obtenidas despues de inmunizacion con BCG por via respiratoria local, comparada con la via sistemica intrad&mica. Cobayos vacunados con diferentes dosis de BCG por ambas vias permitieron analizar la proliferation de las cClulas linfoides, el clearance de BCG en 10s ganglios linfaticos hiliares y en el pulmon, la formation de granulomas en el pulmon y la protection inducida despues de una provocacidn virulenta. Los resultados muestran que la via aerogena de la vacunacion BCG no tiene efectos colaterales adversos para el huesped. La vacunaciou aer6gena con lo5 ufc de BCG induce una respuesta celular hunune local m&sintensa y uu efecto protector sustancialmente mejor, en comparacidn con la vacunacion por via intradermica.

INTRODUCTION

pulmonary tuberculosis in the Chingleput Trial in South India. These findings indicate the lack of a complete understanding of the mechanism of BCG-induced protection and the need for work to improve knowledge on immunity and pathogenesis of tuberculosis as a step toward obtaining better vaccinesY, Even though it appears that new DNA BCG recombinant vaccines are possible,435 until they actually become available for human use, some of the existing strains of BCG can be utilized to

BCG vaccine, shown to protect against severe forms of tuberculosis,i gave variable results in preventing adult

Correspondence to: Dr Marina Gheorghiu, Laboratoire du BCG, DCpartement de Physiopathologie ExpCrimentale, 25 rue du Dr Roux, 75124 Cedex, Paris, France. Paper received 24 August 1992. Accepted 7 September 1992. 38

BCG induced protection in guinea pigs vaccinated and challenged via the respiratory route 39

improve knowledge on strain differences, manufacture, dosage and vaccination route, all factors which are known to influence immune responses against tuberculosis. Among the different routes of vaccination (such as oral, scarification, multiple puncture and respiratory), the intradermal (id.) route is the most commonly used. Although, the i.d. route has known efficacy, there are reasons for further investigation of the level of immune responses induced by vaccination via the respiratory route. Epidemiological evidence suggests that inhaled tubercle bacilli induce a superior protective immune response in the host, since of 100 persons infected, only 5-10 will develop disease.6 Studies of cellular and humoral immune responses have demonstrated a contribution of the mucosal lymphoid system of the gut and respiratory tract. Vaccination by the oral or respiratory route with live bacteria or viruses has demonstrated good protective activity with agents other than Mycobacterium tuberculosis.7,* Studies with M. tuberculosis have shown protective responses in primates vaccinated with BCG via the respiratory route.9 These findings motivated us to investigate immune responses elicited in guinea pigs vaccinated by the respiratory route with aerosolized BCG. In recent studies,10 we have shown that activation of alveolar macrophages (M0) is better after inhalation than after intradermal vaccination. In the present study we investigated the lymphoid cell participation in immune responses at different intervals post-immunization, lung granuloma formation, and the extension of lung tissue lesions after BCG inhalation. Finally, the protective response induced in guinea pigs following vaccination and challenge via the respiratory route was examined in an aerosol-challenge guinea pig model.l1912

MATERIAL

AND METHODS

Animals A first group of 44 outbred specific pathogen-free (SPF) Hartley strain female guinea pigs weighing 200-250 g each were used (Charles River Breeding Labs, Portage, Mich). This group of animals served to investigate the

Table 1.

Groups

2 3 4 5 6 7 8 9 10 Placebo (saline)

protection against virulent challenge. They were fed guinea pig chow (Ralston Purina, St Louis, MO) and water ad lib. There was a 2-week adjustment period prior to the initiation of experimental treatment. The principles of randomization were used in the assignment of guinea pigs to cages, order of vaccination, challenge and sacrifice interval. A total of 11 treatment groups were included in the experiment; the groups of primary interest were animals exposed to a high dose of BCG via the respiratory route. The remaining groups, given a low dose of BCG intradennally or by inhalation, were included as controls. A second group of 48 guinea pigs, outbred SPF female weighing 250-300 g each (Charles River, Les Elbeuf, France), were used to follow at different post-immunization time intervals the lymphoid cell immune responses and the lung granuloma formation. The third group of 21 guinea pigs, identical to the second group, were used to follow the toxicity of the BCG vaccine administered aerogenically under a very high dose.

Vaccination BCG was prepared as previously described 13 from Pasteur 1173P2 strain; the dispersed growth was used either as fresh or freeze-dried vaccine. Two routes of vaccination were used for the animals of the first and second groups: i.d. injection and inhalation. For each vaccination route, two doses of BCG were administered: 105 BCG colony forming units (cfu), or 102 BCG cfu. The vaccine was rehydrated in sterile saline, diluted to 10s cfu/ml and lO%fu/ml, and aerosolized using an Ultrasonic nebulizer (De Vilbiss 65 Somerset Pa) as previously described.10 The conditions of exposure permitted only the animal’s nose to be exposed to aerosol flow for 10 min at a rate of 6 ml/min. These conditions resulted in a recovery of 105 cfu and 102 cfu per lung at 24 h after vaccination. These are referred to as the vaccinating doses. The vaccination treatment groups included in this study for protection test are given in Table 1. Another very high dose of 5 x 106 cfu was aerogenitally administered to the third animal group.

BCG treatment groups and number of primary lesions (PL) after virulent challenge Strain

Culture

Preparation

Route

BCG dose (cfu)

Number of PL

Pasteur Pasteur Pasteur Pasteur Pasteui Pasteur Pasteur Pasteur Danish Danish

Dispersed Dispersed Dispersed Dispersed Dispersed Dispersed Veils Veils Veils Veils

Fresh Freeze-dried Freeze-dried Fresh Freeze-dried Freeze-dried Freeze-dried Freeze-dried Freeze-dried Freeze-dried

Aerosol Aerosol Aerosol Intradermal Intradermal Intradermal Intradermal Intradermal Intradermal Intradermal Intradermal

IO5

2.25 0.75 2.00 4.75 3.25 3.25 3.00 3.50 3.75 2.25 6.25

10” lo2 lo5 105 K? lo5 IO2 IO” IO2

40 Tubercle and Lung Disease

Histologic examination Groups of guinea pigs vaccinated via the respiratory route with two BCG doses (105 and 5 x 106 cfu) were examined at regular intervals after immunization (1, 7, 14, 28, 70 days) for lung tissue granulomas and fibrosis formation. The recovery of BCG from the lungs and satellite lymph nodes was investigated in animals receiving the very high dose of 5 x 106cfu. Three animals were used for each dose and each time interval. Organs were fixed, paraffin embedded, and lung sections stained with hematein eosin saffron for histologic examination. To determine the number of BCG in the lungs and lymph nodes, after organ homogenization, suitable dilutions were plated on agar-based medium as previously described.11

Proliferative responses to PPD At 14, 28 and 70 days post-immunization, 3 guinea pigs from each group were killed. These animals were used to test the proliferative capacity of satellite lymph nodes (LN) and spleen cells. At each time interval after vaccination via the i.d. and respiratory routes the draining lymph nodes, respectively inguinal and hilar, as well as the spleen were removed, and cell suspensions were prepared14 in RPM1 1640 (Seromed) supplemented with 2mM L- Glutamine, gentamicin 50 ug/ml, 5 x 10-s M 2 Mercaptoethanol and fetal calf serum 10% (FCS). Cells were cultured in 0.2 ml of medium at 5 x 105 cells per well in 96-well flat bottom culture plates (Coming) in the presence of PPD (10 ug/ml) as specific antigen and Concanavalin A(Con A)(2.5 ug/ml) as the positive control for cell reactivity. Each test was performed in triplicate. Cells were incubated for 5 days at 37°C in humidified air 7% CO2; during the last 20 h, 0.4 @/well of [3H] dThd [(lmCi/ml); Amersham] were added. The cells were harvested on glass fibre filters, washed with Automash 2000 Dynatech (Bioblock-France) and the incorporated radioactivity was measured in a liquid scintillation counter (Beckman). The results are expressed (with the background subtracted) as geometric means. Standard deviations of triplicate culture were less than 15% of the mean. Background values of control cultures were less than 104 cpm.

were challenged via the respiratory route under conditions described elsewhere.11 Briefly, the H37Rv suspension was diluted at 5 x 104 cfu/ml and the animals exposed according to procedures described earlier.” Based on counts of primary lesions at autopsy, conditions for exposure resulted in the inhalation and retention by each animal of 5-7 cfu. Necropsy procedure

and culture

On 2 successive days, 5 weeks post-challenge, the 4 animals representing each of the 11 treatment groups were exposed to an aerosol of histamine and then killed by exposure to CO2.t2 The histamine aerosol inflated the lung lobes for X-ray examination. The lung lobes of a given animal were exposed in a Faxitron X-ray system (HewlettPackard Co. McMinnville, OR) at 18 Kv for 45 s using X-Omat Ma-2 film (Eastman Kodak, Rochester, NY). Examination of X-ray films facilitated the detection of primary lesions and the identification of primary lesionfree lung (PLF) lobes.l2%1s The 3 largest primary lesions, spleen and a sample of a PLF lobe were excised, individually homogenized, diluted appropriately and plated on Middlebrook 7H10 agar as previously described.t1,12 Analysis of protection data

Analysis of variance was performed on the data for all 11 treatment groups. All treatments contained 4 animals per group. Protection was defined as a difference in the number of tubercle bacilli recovered from the spleen (BE-spleen), from excised primary lung lesions (BE-primary) and from PLF lung lobes (BE- PLF) of vaccinated vs unvaccinated guinea pigs for each treatment group. Analyses of variance were carried out on the data for each of the measured responses. The high F values for the several analyses allowed comparison of the protection data as the individual confidence intervals based on pooled standard deviation.16

RESULTS Lung granuloma formation

Assay of protective response The protection test was the result of the cooperation of two groups, BCG department, Institut Pasteur, Paris and the Tuberculosis Research group, Department of Medical Microbiology and Immunology, University of WisconsinMadison. The vaccination was carried out in Madison by the Institut Pasteur group. The challenge and assessment of the protective response was carried out by both groups following procedures previously reported11312 and described briefly below. Challenge The challenge suspension was prepared from M. tuberculosis strain H37Rv. 11 weeks post-vaccination, all animals

In the present study, we investigated the dose of aerosolized BCG needed to produce beneficial granulomas but without harmful tissue lesions. Therefore, we investigated the lung histology of animals exposed to aerosols containing a dose of 10s cfu and the larger dose, 5 x 106 cfu. As shown in Figure 1 both doses of aerosolized BCG led to the formation of numerous microgranulomas in the lung tissue rarely centered by a small bronchi. These granulomas contained histiocytes, lymphocytes and rare neutrophils. No giant cells and no tissue necrosis were observed. A few microgranulomas appeared at day 7 post-immunization. Microgranulomas became numerous at day 28, decreased and disappeared at day 70 after immunization. Fibrotic lesions were not observed during the experimen-

BCG - induced protection in guinea pigs vaccinated and challenged via the respiratory route 41

E

F

Fig. l- Different granulomas in the lung tissue : microgranulomas at 14 days post-immunization with BCG dose of 105cfu (A) HES x 30, (B) HES x 500; at 28 days post-immunization, numerous microgranulomas with predominantly histyocytes and few lymphocytes, no giant cells nor necrosis is observed (C) HES x 30, (D) HES x 1200. The same aspect and number of granulomas is seen after immunization with high BCG dose of 5 x 106cfu after (E)14 days and (F)28 days. (HES = hematein eosin saffron staining)

tal period. The histological aspects were similar in the groups vaccinated with 105 cfu and the higher dose, 5 x 106 cfu. The highest dose applied via the respiratory route to test its toxicity, has shown (Fig. 2) a low number of BCG cfu recovered from the cervical and hilar lymph nodes. These lymph nodes were small (3-5 mm diameter) and rare (2-3 per animal). They never suppurated and had completely disappeared by 70 days. Clearance of BCG from the lungs was also similar to preceding findingsto decreasing to no more than 1 log after 28 days. This was

associated with the highest number of microgranulomas the lung tissue.

in

Delayed type hypersensitivity The degree of delayed-type hypersensitivity, tested in the animal group followed for protection against virulent challenge, was similar for both intradermal and aerosol routes of vaccination, as shown in Figure 3. The diameter of induration induced by PPD was related only to the

42 Tubercle and Lune Disease

Protection potency of the several vaccination treatment groups

1

14

28

days

Fig. 2-BCG colonies recovered from lung ( B ), cervical ( m[ ) and hilar ( Bm ) lymph nodes after aerogenic vaccination with 5 x 106BCG dose.

10

cfu

10

cfu BCQi do8.8

Fig. 3-Delayed-type hypersensitivity values obtained 28 days post-immunization by intradermal ( B ) and aerogenic ( m ) routes with two doses of BCG.

vaccine

dose (20 mm for 105 cfu dose and 17 mm for 102

cfu dose).

Traditional knowledge of the protective effect of BCG vaccination is that BCG influences the bacillemic phase of first infection with virulent tubercle bacilli. The extent to which BCG inhibits the bacillemia is reflected in the number of virulent bacilli recovered from the spleen and also in the number of bacilli recovered from lung lobes which did not receive a primary lesion, i.e. the PLF lobes. According to these traditional measures of the protective effect of BCG vaccination (Table 2 for BE-spleen and Table 3 for BE-PLF), highly significant differences existed between all BCG treatment groups and the placebo group. Although each BCG vaccinated group differed significantly from the control, group 8 and group 10 (vaccinated i.d. with 102 cfu of 1173P2 or 1331 BCG strains) showed a slightly reduced level of protection as judged by BEspleen compared to the other vaccine groups. With respect to BE-PLF, the confidence intervals are wider and the vaccine groups which differed most are group 3 (aerosol vaccinated with 102 cfu), group 4 (vaccinated i.d. with 105 cfu) and group 6 (vaccinated i.d. with 102 cfu). Even so, the contrast with the placebo reveals a high degree of protection in comparison to the other vaccine groups. Because of the high dose aerosol vaccine treatment groups included in this study, we were permitted to observe a new parameter in BCG-induced protection; namely the effect of vaccination on the number of primary lesions (Table 1) and the number of bacilli recovered from primary lesions developing in the lungs after challenge with virulent tubercle bacilli. The data presented in Table 4 shows the number of bacilli developing in primary lesions in the lungs. Whereas the mean log10 number of bacilli recovered from BCG groups 10-3 ranged from 2.3 to 2.9, groups 2 and 1 (aerosol vaccinated with 105 cfu) showed significantly lower mean log10 recovery of bacilli from primary lesions, ranging from 1 to 1.7. These findings are in agreement with the significantly reduced number of primary lesions (Table 1) observed in groups 2 and 3.

Lymph node and splenocyte cells specific proliferative responses

DISCUSSION

T cell proliferation from LN and from the spleen was observed for both routes of immunization. The values varied significantly as a function of the vaccinating dose. The 105 cfu dose gave a IO-fold higher cpm than 102 cfu dose as shown in Figure 4 for spleen cells, and was independent of the vaccination route. Differences between the routes of vaccination for the same does used were 4 times higher in the inguinal LN group vaccinated intradermally than in the hilar node of the group vaccinated by aerosol. Spleen cell proliferation was highest in the group vaccinated aerogenically with 105 cfu at 70 days post-immunization.

Earlier studies, beginning with those of Gardner Middlebrook, employing a small number of guinea pigs, demonstrated that vaccination via aerosol was as protective as that following subcutaneous or intravenous vaccination.17 Thereafter various investigators using one or another animal species (guinea pig, mouse, monkey) demonstrated that a good level of protection was conferred by vaccination with BCG by the aerosol route against aerosol challenge with virulent strains of M. tuberculosis. 18-20The administration of BCG to human volunteers by aerosol was not followed up by an evaluation of the degree of protection, but the authors did conclude that there were no

BCG - induced protection in guinea pigs vaccinated and challenged via the respiratory route 43

1

14

28

70

days

14

20

70

1

14

28

70

days

14

28

70

0

Fig. 4-Specific proliferation of satellite LN ( B immunization with two doses of BCG.

adverse respiratory

effects

administration

of BCG via the

route.21

The present laboratory

following

) and spleen ( T;rTT

studies

animals

confirm

our previous

and demonstrate

findings

a relationship

in

to our

preceding work related to the improved alveolar macrophages activation after vaccination with aerosolized BCG. 10 This improved local activation of macrophages in tuberculosis infection was earlier emphasized by Dannenberg.22 He suggested that at the local site, products of bacilli and products from sensitized lymphocytes are at a higher concentration than that found systemically, and he therefore suggests that macrophages in the local lesion achieve much higher antimicrobial resistance than that observed for systemic macrophages. Recent observations concerning the role of cellular immune response against M. tuberculosis infection have demonstrated the importance of sensitized T lymphocytes in activating macrophages in order to render them more efficient against phagocytized mycobacteria.23-25 The increased number of lymphocytes and their increased proliferative response observed in this study in the hilar lymph node cells after aerosol vaccination suggests a key role of the homing of T lymphocytes in the improved protective response following vaccination via the respiratory route. On the other hand, the very strong spleen cell proliferation after aerosol vaccination (118 000 cpm),

) cells after intradennal ( upper

days

days

) and aerogenic ( lower )

compared to intradermal vaccination (53 000 cpm), at day 70 after immunization demonstrates a long lasting, memory T cell response which is higher in aerosol vaccinated animals. In contrast, the unvaccinated animals demonstrated only a non-specific proliferative response. These results imply that local immunization may prove superior to systemic immunization. Recent results concerning lymphocyte homing have shown the major role of tissue specificity for the recirculation and the distribution of sensitized lymphocytes into different organs. An essential point of this defense mechanism is the continuous recirculation of lymphocytes from the blood into lymphoid organs and back again. The lymphocyte recirculation serves to increase the probability of an interaction between a given antigen sequestered in a particular tissue and the typically rare lymphocytes capable of responding to that epitope. After priming and clonal expansion in the lymphoid tissue (hilar nodes, bronchi and alveolar lymphoid aggregates) the sensitized memory lymphocytes preferentially recognize the organ to which they home to receive their priming. Other investigators have substantiated the preferential homing to the lung of lymphocytes obtained from lung or from lung draining lymph node. Two recent reviews on this aspect of the ability of lung lymphocytes to return to this organ have been published.26.27 One purpose of this investigation with the highest dose of BCG

44

Tubercle and Lung Disease

Table 2.

Analysis of variance

: one-way analysis of variance of the logto number of bacilli recovered from spleen (using Minitab)

Source Factor Error Total

DF 10 33 43

ss

Level

n

Mean

SD

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group I Group 8 Group 9 Group 10 Placebo

4 4 4 4 4 4 4 4 4 4 4

1.0325 1.OlOo 1.0100 1.OlOO 1.1075 1.0100 1.3250 2.1425 1.OlOo 1.6350 5.8975

0.2000 0.0000 0.0000 0.0000 0.3500 0.0000 0.5447 0.9631 0.0000 0.0100 0.8398

P 0.000

F 40.12

MS 8.445 0.210

85.248 6.946 91.399

Individual 95% Cls for mean based on pooled SD l____i____l ____ I ____ I _-_- I ----

I____1

(...*_.)

I ____ I ____ ~____‘____~____l____i--_-I 1.6

3.2

4.8

6.0

Pooled SD = 0.5391

Table 3. Minitab)

Analysis of variance

: one-way analysis of variance of the loglo number of bacilli recovered from primary lesion-free lung lobes (using

Source Factor Error Total

DF 10 33 43

ss 3.5856 1.2836 4.8692

MS 0.3586 0.0389

Level

n

Mean

SD

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Group 10 Placebo

4 4 4 4 4 4 4 4 4 4 4 4

1.OlOO 1.0100 1.2375 1.1500 l.OlOil 1.1275 0.9450 1.OlOO 1.0100 1.0100 2.0075

o.oooo

P 0.000

F 9.22

0.0000 0.4550 0.2800 0.0000 0.2350 0.075 1 0.0000 0.0000 O.C0OO 0.2821

Individual 95% Cls for mean based on pooled SD I _--I ---_ I ---I ---l____l____l____l (....... * ........ (....... *. .... ..)

(....... *. .... ..) (....... *. .... ..) (....... *. .... ..) (....... *. .... ..) (....... *. .... ..) (....... *. .... ..) (....... * ........ (....... *. .... ..)

I ---0.8

I ----

I ---1.2

I ----

I ---1.6

(....... *. .... ..) I ---I ---2.0

I

Pooled SD = 0.1972.

in guinea pigs (5 x l@), was to answer the question whether very high dose vaccination would lead to suppurative tracheobroncheal adenitis. Our observations were that even the largest dose of BCG did not lead to suppuration of the tracheobronchial nodes or to enlarged cervical and hilar lymph nodes. The extend of lymphadeand accompanied by moderate nitis was small multiplication (104 cfu) of BCG. The lowest vaccinating dose (102 cfu) produced no lymph node enlargement following either route of immunization. Typical lung granulomas with epitheloid, lymphoid giant cells were not found in guinea pigs in the present study. They have been demonstrated in other studies in rats or mice.28.29 As demonstrated by Lowrie,3u whereas living cells alone fail to kill bacilli, the accumulated toxic products of dead ones

may account for the extracellular killing that occurs in granulomas in vivo. It was shown in our study that specific numerous microgranulomas were present; they then increased in number at 28 days, a finding which mirrors the lung clearance of BCG. These granulomas had completely disappeared by 70 days post-immunization. These results suggest that BCG aerosol administration is not harmful towards the lung tissue and presumably its function. The protective response induced in guinea pigs vaccinated and challenged via the respiratory route, is one of the most conclusive lines of evidence of whether immunization with BCG via the respiratory route is more efficacious than traditional i.d. immunization. The importance of the animal model test system used to detect the protective effect of BCG has been discussed previously.31

BCG Table 4. Source Factor Error Total

Analysis of variance DF 10 33 43

Level Group I Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Group 10 Placebo

3

4 4 4 4 4 4 4 4 4 4

inducedprotectionin guinea pigs vaccinated and challenged via the respiratory route

4.5

: one-way analysis of variance of the log10 number of bacilli recovered from primary lesions (using Minitab) SS 38.549 21.064 59.614

MS 3.855 0.638

Mean

SD

1.7350 1.0100 2.9750 3.1575 2.7000 2.9375 2.9050 2.3100 2.3200 2.3050 4.9425

0.9618 0.0000 0.6504 0.1889 0.8943 0.8514 1.5018 0.9918 0.6845 0.6396 0.2371

F 6.04

P

O.OilO

Individual 95% Cls for mean based on pooled SD l____l____l____l____l__~._l____

(,...... * ... ....)

l____l____l____l____l____l I .6 3.2

I 4.8

Pooled SD = 0.7989

In the present studies, in comparison with the placebo group challenged with H37Rv, the control groups vaccinated with traditional dosages of BCG showed a substantial level of protection based on recovery of bacilli from spleen or PLF (Tables 2 and 3). These protective responses measure the well-known capacity of BCG to inhibit the bacillemia associated with primary infection with virulent bacilli. They compare favourably with data from earlier publications with the aerosol challenge in guinea pig model.31 In contrast, the protective response induced in groups 1 and 2, the groups of primary interest in the experiment. indicates a degree of protection as assessed by two parameters not previously reported in the aerosol challenge guinea pig model. One parameter involved the number of primary lesions per animal, while the other included the number of bacilli recovered per primary lung lesion as the relevant host response. These two parameters are independent of each other. Whereas the number of primary lesions measures the infectivity of the challenge strain, the number of bacilli per primary lesion measures the survival of the challenge strain after establishing a primary implant. The significantly reduced number of primary lesions (Table 1) as well as the significantly lower number of bacilli recovered from primary lesions in the lungs of guinea pigs in treatment groups 1 and 2 (Table 4) in comparison to other groups, indicate that the response to aerosol vaccination not only reduced the bacillemic spread but also affected the development of primary lesions. Our interpretation of these findings is that high dose vaccination, delivered directly into the lungs, induced cellular events which caused earlier activation of macrophages at the site of deposition of the 5-7 virulent bacilli administered via the respiratory route at the time of challenge with H37Rv. Whether the reduced number of primary lesions (Table 1) which developed resulted from foci which were eliminated immediately or only after a brief period of multiplication, will require further experimentation. It is clear from the data in Table 4 that some of

the effect of vaccination came from the earlier activation of macrophages which restricted the development of bacilli. In either case, the present findings with high dose aerosol vaccination with BCG add a new dimension to the known protective effects of BCG, namely, the potential for BCG vaccination to affect the growth of virulent bacilli at the sites of their implantation in the lungs. This new dimension for a protective effect of BCG suggests that BCG vaccination could affect not only endogenous reactivation, but also exogenous tuberculosis reinfection.32 Acknowledgements We acknowledge help.

Pasteur-MCrieux

SCrums et Vaccine for its financial

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