Mycobacterial Antigens Exacerbate Disease Manifestations in Mycobacterium tuberculosis-Infected Mice

Abstract

To control tuberculosis worldwide, the burden of adult pulmonary disease must be reduced. Although widely used, Mycobacterium bovis BCG vaccination given at birth does not protect against adult pulmonary disease. Therefore, postexposure vaccination of adults
with mycobacterial antigens is being considered. We examined the effect of various mycobacterial antigens on mice with prior
M. tuberculosis infection. Subcutaneous administration of live or heat-treated BCG with or without lipid adjuvants to infected mice induced
increased antigen-specific T-cell proliferation but did not reduce the bacterial load in the lungs and caused larger lung
granulomas. Similarly, additional mycobacterial antigen delivered directly to the lungs by aerosol infection with viable M. tuberculosis mixed with heat-killed Mycobacterium tuberculosis (1:1) also did not reduce the bacillary load but caused increased expression of tumor necrosis factor alpha (TNF-α) and interleukin
6 (IL-6), which was associated with larger granulomas in the lungs. When M. tuberculosis-infected mice were treated with recombinant BCG that secreted cytokines shown to reduce disease in a preinfection vaccine
model, the BCG secreting TNF-α, and to a lesser extent, IL-2 and gamma interferon (IFN-γ), caused a significant increase in
granuloma size in the lungs. Moreover, treatment of M. tuberculosis-infected mice with recombinant murine TNF-α resulted in increased inflammation in the lungs and accelerated mortality without
affecting the bacillary load. Taken together, these studies suggest that administration of mycobacterial antigens to mice
with prior M. tuberculosis infection leads to immune activation that may exacerbate lung pathology via TNF-α-induced inflammation without reducing the
bacillary load.

Full text

INFECTION AND IMMUNITY, Apr. 2002, p. 2100–2107 Vol. 70, No. 4
0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.4.2100–2107.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Mycobacterial Antigens Exacerbate Disease Manifestations in
Mycobacterium tuberculosis-Infected Mice
Andre L. Moreira,
1
Liana Tsenova,
2
Melles Haile Aman,
3
Linda-Gail Bekker,
4
Sherry Freeman,
2
Bande Mangaliso,
2
Ulf Schro¨der,
3
Jaishree Jagirdar,
1
William N. Rom,
5
Michael G. Tovey,
6
Victoria H. Freedman,
2
and Gilla Kaplan
2
*
Department of Pathology
1
and Division of Pulmonary & Critical Care Medicine,
5
New York University School of Medicine, and
Laboratory of Cellular Physiology & Immunology, The Rockefeller University,
2
New York, New York; Swedish Institute for
Infectious Disease Control, Stockholm, Sweden
3
; Infectious Disease Clinical Research Unit, UCT Lung Institute,
Cape Town, South Africa
4
; and Laboratory of Viral Oncology, CNRS, Villejuif, France
6
Received 2 August 2001/Returned for modification 22 October 2001/Accepted 7 January 2002
To control tuberculosis worldwide, the burden of adult pulmonary disease must be reduced. Although widely
used, Mycobacterium bovis BCG vaccination given at birth does not protect against adult pulmonary disease.
Therefore, postexposure vaccination of adults with mycobacterial antigens is being considered. We examined
the effect of various mycobacterial antigens on mice with prior M. tuberculosis infection. Subcutaneous admin-
istration of live or heat-treated BCG with or without lipid adjuvants to infected mice induced increased
antigen-specific T-cell proliferation but did not reduce the bacterial load in the lungs and caused larger lung
granulomas. Similarly, additional mycobacterial antigen delivered directly to the lungs by aerosol infection
with viable M. tuberculosis mixed with heat-killed Mycobacterium tuberculosis (1:1) also did not reduce the
bacillary load but caused increased expression of tumor necrosis factor alpha (TNF-␣) and interleukin 6
(IL-6), which was associated with larger granulomas in the lungs. When M. tuberculosis-infected mice were
treated with recombinant BCG that secreted cytokines shown to reduce disease in a preinfection vaccine model,
the BCG secreting TNF-␣, and to a lesser extent, IL-2 and gamma interferon (IFN-␥), caused a significant
increase in granuloma size in the lungs. Moreover, treatment of M. tuberculosis-infected mice with recombinant
murine TNF-␣resulted in increased inflammation in the lungs and accelerated mortality without affecting the
bacillary load. Taken together, these studies suggest that administration of mycobacterial antigens to mice with
prior M. tuberculosis infection leads to immune activation that may exacerbate lung pathology via TNF-␣-
induced inflammation without reducing the bacillary load.
The idea of immune modulation to treat tuberculosis is not
new. In 1890, at the World Congress of Medicine, Robert Koch
announced that he had prepared “substances” that completely
cured guinea pigs in the late stages of tuberculosis (TB) (14).
The substance, later called “old tuberculin,” was a glycerin
extracted filtrate of cultures of the tubercle bacillus. With
much fanfare, patients were treated with this bacterial extract.
Unfortunately, the treatment caused florid local and systemic
reactions in many of the patients with relatively mild disease.
In addition, of the 230 patients with advanced cavitary disease
who received this treatment, 30 died. This intense reaction to
extracts of Mycobacterium tuberculosis and the associated clin-
ical worsening became known as the “Koch phenomenon”
(11).
The practical implications of the Koch phenomenon should
be reconsidered in light of recent efforts to develop a new
anti-TB vaccine. The goal of vaccination is protection against
adult pulmonary TB. Neonatal Mycobacterium bovis BCG vac-
cination, which is widely used throughout the world, does not
protect against adult pulmonary disease, even when it protects
against the more severe forms of childhood TB (8). New strat-
egies have therefore been proposed, including vaccination of
adults with BCG or other mycobacterial preparations (7). The
latter approach may be problematic. In areas of high endemic-
ity, many individuals will have already been infected and may
even have subclinical disease. If they now receive a strong
immunogen, the ensuing host response may result in exacer-
bation of the occult disease leading to severe toxicities (Koch
phenomenon).
The effect of administration of mycobacterial preparations
on the outcome of disease has recently been studied by a
number of investigators (1, 10, 21). Lowrie et al. showed that
postexposure vaccination with either BCG or DNA encoding
the M. tuberculosis heat shock protein Hsp70 or ESAT6 had
little or no effect on bacillary load in the spleens or lungs of
infected mice (16). However, repeated vaccination with DNA
encoding Mycobacterium leprae Hsp65 reduced the number of
bacilli in lungs and spleen by 1 log
10
and 2 log
10
at 2 and 5
months postintervention, respectively. In contrast, Turner and
colleagues explored the use of two other new candidate vac-
cines (a subunit vaccine and a DNA vaccine, both containing
M. tuberculosis Ag85) as immunotherapeutic agents in mice
(23). When administered to mice already infected with M.
tuberculosis, neither BCG nor these two candidate vaccines
caused any improvement in the course of infection. Further-
more, repeated BCG vaccination of infected mice resulted in
exacerbation of the granulomatous response in the lungs (23).
To further examine the effect of mycobacterium-induced
immune activation on the host response during M. tuberculosis
* Corresponding author. Mailing address: Laboratory of Cellular
Physiology & Immunology, The Rockefeller University, 1230 York
Ave., New York, NY 10021. Phone: (212) 327-8375. Fax: (212) 327-
8376. E-mail: kaplang@rockvax.rockefeller.edu.
2100

infection, we infected mice by aerosol with virulent M. tuber-
culosis. Then, 5 weeks later, the infected mice were inoculated
with various preparations containing BCG or with BCG secret-
ing murine cytokines. In addition, to determine whether the
presence of additional antigen in the lungs might have an
impact on outcome, naı¨ve mice were infected by aerosol with
live M. tuberculosis or a mixture of live and dead M. tubercu-
losis. Finally, M. tuberculosis-infected mice were treated di-
rectly with recombinant cytokines via intranasal delivery. The
course of the infection was monitored by enumeration of ba-
cilli in infected tissues, histologic examination of the lungs,
quantitation of cytokine mRNA induced in the lungs, and
peripheral T-cell proliferation in response to mycobacterial
antigens.
MATERIALS AND METHODS
Mice. Seven- to 8-week-old female (B6 ⫻D2)/F
1
mice, free of common
pathogens, were obtained from Charles River Laboratories, Wilmington, Mass.,
and housed in the BSL3 (Biosafety Level 3) Animal Facility of The Rockefeller
University for the duration of these experiments. All protocols were approved by
the Animal Use and Care Committee of The Rockefeller University.
Infection of mice. Aerosol infection was carried out according to a protocol
developed in this laboratory (17). Briefly, mice were inoculated via the respira-
tory route by exposure to an aerosolized suspension of M. tuberculosis (see
below) generated by a Lovelace nebulizer using a nose-only exposure system
(In-Tox Products, Albuquerque, N.M.) (22). For each experiment 24 mice were
exposed for 30 min to the aerosol. This procedure resulted in implantation of
approximately 100 organisms into the lungs of mice, as confirmed by plating lung
homogenates 3 h after infection. In some experiments M. tuberculosis was auto-
claved and then mixed 1:1 with live bacilli prior to aerosol infection.
M. tuberculosis and M. bovis strains. M. tuberculosis strain Erdman was pro-
vided as multiple stock vials by J. Belisle, Colorado State University (Fort
Collins, Colo.); M. tuberculosis H37Rv was from the Trudeau Mycobacterial
Culture Collection (Trudeau Institute, Saranac Lake, N.Y.). The M. tuberculosis
clinical isolate HN878 was provided by J. M. Musser (20). BCG was obtained
from Statens Serum Institute, Copenhagen, Denmark (BCG vaccine SSI, batch
9854). BCG strain Montreal, which was used to generate recombinant BCG
expressing murine cytokines, including BCG expressing gamma interferon
(BCG–IFN-␥), tumor necrosis factor alpha (BCG–⌻NF-␣), and interleukin 2
(BCG–IL-2) and the control strain of BCG, carrying the plasmid vector only
(BCG vector), was obtained from Richard Young, The Whitehead Institute,
Cambridge, Mass. (18). All mycobacteria were grown on Middlebrook 7H9
medium (Difco, Detroit, Mich.), and bacillary stocks were stored at 10
7
to 10
8
bacilli/ml and kept at ⫺70°C until use.
BCG preparations for postinfection inoculation. Lyophilized BCG (SSI) (1.5
mg) was reconstituted in 2 ml of diluent (Sauton; Statens Serum Institute),
according to the manufacturer’s instructions (viable BCG). In addition, BCG was
reconstituted in 200 ␮l of diluent as described above and was heated in a water
bath at 60°C for 10 min. No growth of heat-treated BCG was noted using culture
on egg-based solid media (Lowenstein-Jensen) or 4 weeks of growth in the
BACTEC system (Bactec 4600; Becton Dickinson, Sparks, Md.). L3 adjuvant was
prepared using 0.62 mg of solid monooleate (analytic grade; Kebo AB, Stock-
holm, Sweden), 0.48 mg of oleic acid (analytic grade; Kebo AB), and 180 mg of
soybean oil (pharmaceutical grade; Karlshamn AB, Karlshamn, Sweden) and was
liquefied by gentle heat at 30°C (manuscript in preparation). For injection, 200
␮l of the heat-treated BCG was mixed in the L3 adjuvant, probe sonicated for 10
to 15 s, and brought to volume (2.25 ml) with 0.1 M Tris buffer, pH 7.5 (heat-
treated BCG). For boosting of mice, 200 ␮l of heat-treated BCG was mixed in
half the amount of L3 adjuvant (see above) and brought to volume (2.25 ml) with
0.1 M Tris buffer, pH 7.5.
Vaccination of infected mice with BCG preparations. Five weeks after the
initial infection with M. tuberculosis H37Rv, mice were divided into four groups.
Groups 1 and 2 received 0.1 ml of viable BCG subcutaneously (s.c.). Group 3
received 0.1 ml of heat-treated BCG in L3 adjuvant s.c., while group 4 received
no treatment. Three weeks later, groups 2 and 3 were boosted with heat-treated
BCG in L3 adjuvant by intranasal administration of 10 ␮l(5␮l per nostril). Mice
were evaluated at 12 weeks. In another experiment, either 6 weeks before or 5
weeks after the aerosol infection, mice were vaccinated s.c. with one of the
recombinant BCG strains (BCG–IFN-␥, BCG–TNF-␣, BCG–IL-2, or BCG vec-
tor) (18) at a dose of 10
6
organisms per mouse. Three weeks after the initial
vaccination, the mice received a boost with the same dose of recombinant BCG.
A control group of mice received no BCG. Mice were evaluated at 12 and 20
weeks postchallenge with M. tuberculosis.
Intranasal treatment of infected mice. Recombinant murine TNF-␣was ob-
tained from Endogen (Boston, Mass.), reconstituted in 1% bovine serum albu-
min–phosphate-buffered saline to a final concentration of 2 ⫻10
5
IU/ml, and
kept at 4°C. The cytokine was administered intranasally for 5 consecutive days
per week for 4 weeks (6). Five microliters was applied directly to the nostrils of
each mouse (final dose of TNF-␣,10
3
IU per mouse per day). Recombinant
murine IFN-␥was obtained from Valbiotech (Paris, France) and reconstituted in
1% bovine serum albumin–phosphate-buffered saline to a final concentration of
10
6
IU/ml. Ten microliters was applied into the nostrils of each mouse as
described above (final dose of IFN-␥,10
4
IU per mouse per day). Control mice
were untreated.
CFU assay. The number of viable mycobacteria in lungs, livers, and spleens of
infected mice were evaluated at designated time points. Tenfold serial dilutions
of organ homogenates were plated onto 7H11 agar (Becton Dickinson) and were
incubated at 37°C. The number of viable bacilli was evaluated by counting
individual colonies after 2 to 3 weeks of growth.
Cytokine mRNA levels in the infected lung. Total cellular RNA from lungs of
infected mice was obtained at designated time points following aerosol infection.
Tissues were homogenized in 3 ml of RNAzolB (Cinna/Biotcx Lab. Inc., Hous-
ton, Tex.), and RNA was extracted according to the manufacturer’s instructions.
The reverse transcription-PCR was carried out as previously described (15).
Briefly,1␮g of RNA was reverse transcribed using a Moloney murine leukemia
virus reverse transcriptase and was amplified with Taq polymerase according to
procedures given in the GeneAmp RNA PCR kit (Perkin-Elmer, Branchburg,
N.J.). Primers for cytokines and ␤-actin were used as described (17). Densitom-
etry of the amplified bands was carried out using a PhosphorImager (Molecular
Dynamics, Sunnyvale, Calif.). Results were normalized to the density of ␤-actin.
Lymphocyte proliferation assay. Spleen and draining lymph nodes were re-
moved at each time point and were processed as described (4). Briefly, cells
isolated from spleen and lymph nodes were cultured at 2 ⫻10
5
cells/100 ␮lin
96-well U-bottom plates in RPMI 1640 medium (Gibco BRL, Gaithersburg,
Md.) and were supplemented with 10% fetal bovine serum, penicillin, and strep-
tomycin (final concentration, 50 ␮g/ml) (all obtained from Gibco BRL). The cells
were incubated with concanavalin A (final concentration, 5 ␮g/ml) (Sigma, St.
Louis, Mo.) or M. tuberculosis H37Ra sonicate (final concentration, 50 ␮g/ml) at
37°C for 3 or 5 days, respectively, and were then pulsed with [H
3
]thymidine
(1 ␮Ci/well) for an additional 18 h. Incorporation of [H
3
]thymidine was mea-
sured by ␤-scintillation counting. Values were expressed as mean counts per
minute in the cultures.
Histopathology. At various time points after infection, lungs were fixed in 10%
neutral buffered formalin, embedded in paraffin, and processed for histology.
Sections were stained with hematoxylin and eosin and Ziehl-Neelsen for histo-
logic evaluation and photography.
Morphometric evaluation of granuloma size. Morphometry of the lesions was
performed using Microcomp, a computer-based image analysis system (Southern
Micro Institute, Atlanta, Ga.) and/or Sigmascan Pro 5:0 (SPSS Science Inc.,
Chicago, Ill.). A calibration micrometer (in square micrometers) slide was used
to determine the area evaluated.
Statistical analysis. Data were analyzed using an independent Student ttest.
APof ⬍0.05 was considered statistically significant. Kaplan-Meier analysis was
used to determine statistical significance of the differences in survival time of
mice.
RESULTS
The effect of postinfection exposure to mycobacterial anti-
gens. To determine whether the course of established disease
is altered by immune stimulation with mycobacterial antigens,
mice were first infected by aerosol with 100 CFU of M. tuber-
culosis H37Rv. At 5 weeks after infection, prior to any addi-
tional treatment, the number of CFU in the lungs was approx-
imately 6.6 log
10
. At this time, infected mice were split into
four groups, and mice in groups 1 and 2 were vaccinated with
viable BCG while mice in group 3 received heat-treated BCG
in L3 adjuvant. Three weeks later mice in groups 2 and 3 were
boosted with heat-treated BCG in L3 adjuvant, as described
VOL. 70, 2002 MYCOBACTERIUM-INDUCED PATHOLOGY IN MURINE TUBERCULOSIS 2101

above (Materials and Methods). Four weeks later (at 12 weeks
after the initial infection), the numbers of CFU in lungs, livers,
and spleens of mice in all four groups were evaluated and
found to be similar (Fig. 1A). Thus, postinfection immunother-
apy with these BCG preparations had no effect on the bacillary
loads in the organs of any of the infected mice.
An evaluation of the systemic T-cell response of the infected
mice was also carried out. Cells were isolated from the draining
lymph nodes of infected and vaccinated mice at 12 weeks and
stimulated in vitro with M. tuberculosis H37Ra sonicate. Com-
pared to the control infected mice (group 4), an increased
proliferative response was noted in all treatment groups and
was significant for group 3 (Fig. 1B). Lymph node cells from
infected animals that received heat-treated BCG in L3 adju-
vant followed by intranasal boosting with heat-treated BCG in
L3 adjuvant (group 3) showed the largest increase in prolifer-
ative responses in vitro compared to the other treatment group
and the unvaccinated controls (P⫽0.03). Cells isolated from
the spleens of the infected, vaccinated animals showed similar
patterns of proliferative responses, although these were much
lower (not shown). To examine the immune stimulation in the
lungs of infected, vaccinated animals, cytokine mRNA was
prepared from the lungs at 12 weeks. When compared to the
infected, unvaccinated controls (group 4), the vaccinated, in-
fected animals (groups 1 to 3) had only slightly higher levels of
TNF-␣mRNA in the lungs (Fig. 1C). Animals in group 1 also
had somewhat higher levels of mRNAs for IL-12 and IFN-␥;
all these differences were not statistically significant.
When the lungs of the mice in the four treatment groups
were examined histologically at week 12, granulomas were
larger in the infected animals that had received heat-treated
BCG in L3 adjuvant and were then boosted intranasally (group
3). Morphometric analysis revealed that 69% of the lung pa-
renchyma was occupied by granulomas, compared to 56% in
group 1, 47% in group 2, and 36% in the control animals
(group 4) (Fig. 1D). Thus, although the levels of systemic
immune activation as measured by antigen-specific T-cell pro-
liferation increased, the vaccine-induced immune stimulation
did not affect the number of CFU in the lungs. However, the
immune activation was associated with an exacerbation of pa-
thology, as indicated by the size of the granulomas in the lungs
(compare Fig. 1B and D).
Effect of additional antigen on the host response to M.
tuberculosis infection. To examine whether the presence of
additional mycobacterial antigen in the infected lung changed
the course of infection, mice were infected with either viable
M. tuberculosis strain Erdman (100 CFU) or the same dose of
viable bacilli (100 CFU) mixed with an equivalent dose of
heat-killed organisms (1:1). The presence of additional heat-
killed organisms in the infecting inoculum had no significant
effect on the subsequent bacillary load (CFU) in the lung,
spleen, and liver of infected mice (Table 1). In mice that
FIG. 1. Effect of postinfection vaccination on bacillary load (CFU) (A), lymph node (LN) T-cell proliferation (B), lung cytokine mRNA levels
(C), and area of lung occupied by granulomas (D). (A and C) M. tuberculosis-infected mice were vaccinated according to the protocol described
in Materials and Methods. Group 1, viable BCG (black bar); group 2, viable BCG followed by boost with heat-treated BCG in L3 adjuvant (gray
bar); group 3, heat-treated BCG in L3 adjuvant and boosted as above (hatched bar); group 4, no vaccination (empty bar). (B) Lymph node cells
stimulated with H37Ra sonicate (gray bars) or unstimulated control (empty bar). (D) Percentage of lung parenchyma occupied by granuloma.
Results are from four animals per group at 12 weeks post-initial infection, expressed as means ⫾standard deviation. ⴱ,Pcompared to unvaccinated
controls (group 4).
2102 MOREIRA ET AL. INFECT.IMMUN.

received the live infection only, levels of mRNA for TNF-␣and
for IL-6 increased over the first 3 weeks of infection (Table 1).
When mice were infected with the mixture of live plus dead M.
tuberculosis, even higher expression of cytokine mRNA for
TNF-␣and for IL-6 was noted. The difference was most pro-
nounced at week 3 (P⬍0.05); by week 4 the difference was no
longer seen (Table 1). In the lungs of mice infected with the
mixture of viable and heat-killed bacilli, the higher cytokine
mRNA levels were associated with larger granulomas (Fig. 2).
In the mice infected with live plus dead organisms, the mean
size of lesions at 4 weeks postinfection was 6.17 mm
3
⫾1.3
mm
3
, compared to 4.38 mm
3
⫾0.64 mm
3
in those infected with
live organisms only. Distended alveolar spaces (edematic lung)
were also seen, suggesting a relative loss of lung function (Fig.
2). These results indicate that the additional mycobacterial
antigen in the lungs of mice receiving the mixed infection
induces a stronger local inflammatory response (increased
TNF-␣and IL-6 production; Table 1), leading to increased
lung pathology without altering the bacillary burden.
Effect of vaccination with recombinant BCG secreting mu-
rine cytokines on the bacillary load and granuloma size in
infected organs. To determine whether particular cytokines
could enhance the BCG-induced protective immune response
to M. tuberculosis, mice were vaccinated s.c. with recombinant
BCG secreting murine cytokines (BCG vector, BCG–TNF-␣,
BCG–IFN-␥,orBCG–IL-2) followed by M. tuberculosis
H37Rv challenge, as described (Materials and Methods). In
these experiments, the recombinant BCG used was prepared
from BCG strain Montreal, a known weak immunogen for
mice (19). By 12 weeks after M. tuberculosis challenge, the
bacilli in the lungs had grown to a concentration of 3 ⫻10
7
CFU in control (nonvaccinated) mice (Fig. 3, top). Vaccina-
tion with BCG vector resulted in a bacillary load reduced by
about fourfold. Cytokine secretion by the recombinant BCG
TABLE 1. Infection with viable and viable plus heat-killed M. tuberculosis
Type of infection and no. of wk No. of CFU
a
(log
10
) in: Amt of lung cytokine mRNA
a
(units) for:
Lung Spleen Liver TNF-␣IL-6
Viable bacilli
1 3.7 ⫾0.1 0 1.4 ⫾1.2 63 ⫾22 46 ⫾10
2 5.2 ⫾0.3 1.6 ⫾1.5 1.9 ⫾1.6 69 ⫾30 174 ⫾17
3 5.5 ⫾0.1 3.9 ⫾0.2 3.2 ⫾0.6 144 ⫾53 233 ⫾33
4 6.0 ⫾0.3 4.1 ⫾0.1 2.6 ⫾0.3 151 ⫾51 161 ⫾15
Viable ⫹heat-killed bacilli
1 3.5 ⫾0.4 0 1.8 ⫾1.7 33 ⫾546⫾5
2 5.1 ⫾0.2 0.9 ⫾0.5 2.8 ⫾0.8 111 ⫾9 243 ⫾25
3 5.5 ⫾0.1 3.6 ⫾0.2 3.2 ⫾0.2 269 ⫾24
b
384 ⫾37
b
4 5.8 ⫾0.2 3.9 ⫾0.6 3.4 ⫾0.2 132 ⫾81 149 ⫾27
a
Number of mice per group per time point (n⫽5).
b
P⬍0.05 compared to infection with viable bacilli only.
FIG. 2. Effect of additional antigen on the granulomatous response in the lungs of mice at 3 weeks post-aerosol infection with viable M.
tuberculosis (live) or viable plus heat-killed M. tuberculosis (live ⫹dead, 1:1). G, granuloma; ⴱ, distended alveolar spaces (edematic lung).
Ziehl-Neelsen stain, magnification, ⫻10.
VOL. 70, 2002 MYCOBACTERIUM-INDUCED PATHOLOGY IN MURINE TUBERCULOSIS 2103

did not improve the control of the bacillary growth in the lungs.
At 12 weeks postinfection, the lungs of unvaccinated controls
showed an extensive inflammatory infiltrate composed of mac-
rophages, lymphocytes, and neutrophils within the alveolar
space consistent with a pneumonic process in addition to large,
coalescent granulomas. The size of the granulomas was re-
duced by about 50% in BCG vector-vaccinated mice compared
to unvaccinated controls (P⬍0.05) (Fig. 3, top). Vaccination
with BCG secreting IL-2 or BCG secreting IFN-␥resulted in
even smaller granulomas than the BCG vector-vaccinated mice
(P⬍0.05). However, BCG–TNF-␣vaccination resulted in
larger granulomas than did vaccination with BCG vector (P⬍
0.05), though the granulomas were still smaller than in non-
vaccinated mice (P⬍0.05). Thus, of the cytokines tested, IL-2
together with BCG appeared to impart the most protection in
terms of limiting immunopathology in the lungs; the presence
of TNF-␣appeared to exacerbate the pathology.
Effect of postinfection vaccination with cytokine-producing
BCG. To determine whether particular cytokines could render
BCG vaccination more effective against ongoing M. tuberculo-
sis infection, recombinant strains of BCG producing various
murine cytokines were used to treat infected mice. Mice in-
fected with H37Rv were vaccinated twice (at 5 and 8 weeks)
postinfection with 10
6
organisms of viable BCG–TNF-␣, BCG–
IL-2, BCG–IFN-␥, or BCG vector. No effect on the number of
CFU in the lungs or spleens was observed at weeks 12 and 20
postinfection compared to in the organs of unvaccinated, in-
fected mice (Fig. 3, bottom). Postinfection administration of
recombinant BCG secreting cytokines did, however, affect the
size of the granulomas. Larger granulomas involving 56.2% of
the lung were observed in the infected mice that had received
recombinant BCG–TNF-␣, compared to 29.6% in the control
unvaccinated mice (P⫽0.02) (Fig. 3, bottom). Infected mice
treated with BCG–IL-2 or BCG–IFN-␥had intermediate-sized
granulomas (50 or 46.4%, respectively). Thus, the combination
of the immune-stimulatory cytokines with BCG resulted in
exacerbation of lung pathology; the worst effect was seen when
BCG was combined with TNF-␣.
Effect of exogenous TNF-␣on course of M. tuberculosis in-
fection. The previous experiments suggested that the deleteri-
ous component of the immune response to mycobacterial an-
tigens is excess TNF-␣. To directly examine the effect of excess
TNF-␣on lung pathology, mice infected with 80 CFU of M.
tuberculosis were treated intranasally with recombinant murine
TNF-␣for the first 4 weeks of infection. At 4 weeks and then
8 weeks after infection and treatment, the numbers of CFU in
lungs and spleens were similar (Fig. 4, top). However, survival
of the mice was substantially altered. Mice infected with M.
FIG. 3. Effect of vaccination (preinfection, top) and immunotherapy (postinfection, bottom) with cytokine-secreting BCG on number of CFU
in lung at 12 and 20 weeks post-initial infection (left panels) and on granuloma size at 12 weeks (right panels). Results are from four animals per
group per time point, expressed as means ⫾standard deviation. a, P⬍0.05 compared to unvaccinated controls; b, P⬍0.05, higher than for BCG
vector-vaccinated mice; c, P⬍0.05, lower than for BCG vector-vaccinated mice.
2104 MOREIRA ET AL. INFECT.IMMUN.

tuberculosis and treated with TNF-␣succumbed to the infec-
tion significantly earlier than mice that had not received the
cytokine treatment (P⫽0.008) (Fig. 4, top). In contrast, when
infected mice (100 CFU at baseline) were treated intranasally
with IFN-␥, no effect on survival or number of CFU in lungs
and spleens was noted (Fig. 4, bottom).
TNF-␣treatment of infected mice was associated with in-
creased cellularity of the lungs, including higher numbers of
polymorphonuclear leukocytes in the granulomas and larger
numbers of foamy macrophages in the alveolar spaces at 4
weeks (Fig. 5). The macrophages did not appear to be more
activated, as there was no increase in the mRNA levels of
inflammatory cytokines in the lungs (IL-6, IL-10, IL-12, and
TNF-␣) (not shown). The lungs of the infected, untreated mice
appeared to have higher numbers of small lymphocytes (Fig.
5). Again, the lymphocytes did not appear to be more acti-
vated, since the IFN-␥mRNA levels were similar or even
somewhat lower (about 40%) in the lungs of TNF-␣-treated
mice at 4 and 8 weeks (not shown). Thus, TNF-␣treatment
appeared to result in the exacerbation of lung pathology with-
out significant changes in the bacillus burden. On the other
hand, IFN-␥treatment did not affect these parameters.
DISCUSSION
Our results, as well as those of others, demonstrate how
difficult it is to change the course of existing M. tuberculosis
infection in the lungs by immunotherapy with vaccines. Indeed,
Koch himself did not claim that his treatment with “tuberculin
lymph”killed the bacteria (5). In our postinfection experi-
ments, vaccination with BCG, BCG plus other antigens, re-
combinant BCG secreting murine cytokines, or heat-killed M.
tuberculosis did not reduce the bacterial load in the lungs of
infected mice. Even those vaccines that were shown to be
effective as preexposure vaccines (Fig. 3) failed to reduce the
number of CFU when administerred as postinfection vaccines.
Analogous results have been obtained by other investigators.
Turner et al. tested three different vaccines in infected mice
and guinea pigs as immunotherapy: (i) BCG Pasteur, (ii) a
subunit vaccine containing purified culture filtrate proteins of
M. tuberculosis emulsified in an adjuvant together with recom-
binant murine IL-2, and (iii) a DNA vaccine consisting of a
vector expressing the gene for M. tuberculosis Ag85A (23). The
candidate vaccines were selected because they had shown ef-
ficacy as preexposure vaccines in protecting naı¨ve animals
against challenge with M. tuberculosis (1, 10). However, all
three of these vaccines, although protective as preexposure
vaccines, failed to reduce the bacterial load in the lungs of mice
with prior tuberculosis infection. Similarly, Lowrie et al. tested
a number of DNA vaccines encoding M. leprae Hsp65 or M.
tuberculosis Hsp70 or ESAT-6 as postinfection treatments (16).
Only the DNA vaccine encoding M. leprae Hsp65 resulted in
some reduction in bacterial load in the lungs of infected mice.
FIG. 4. Effect of exogenous recombinant cytokines on the bacillary load (right panels) and survival of infected mice (left panels). Mice were
infected with M. tuberculosis and treated intranasally with recombinant murine TNF-␣(top panels) or IFN-␥(bottom panels). Solid squares,
cytokine-treated mice; empty squares, control untreated mice. Results for survival are from 11 mice per group and for numbers of CFU from four
mice per group per time point.
VOL. 70, 2002 MYCOBACTERIUM-INDUCED PATHOLOGY IN MURINE TUBERCULOSIS 2105

Interestingly, when preexisting infection had been elimi-
nated with antibiotic therapy, repeated postexposure vaccina-
tion did protect against reactivation of disease (16). Thus, it is
possible that immune modulation in the presence of antituber-
culous therapy may facilitate bacillary clearance in the lungs
(9). Indeed, in a previous pilot study of patients with multi-
drug-resistant pulmonary TB, we observed that administration
of recombinant IL-2 as adjunctive therapy combined with an-
ti-TB chemotherapy resulted in accelerated sputum clearance
and improved radiographic appearance of the lungs (12).
As we have clearly shown here, increased immune activation
is associated with exacerbation of pathology at the sites of
preexisting TB. This had originally been noted by Koch. In
response to the treatment with “tuberculin lymph,”infected
tissues became inflamed and necrotic and partially sloughed
often in association with clinical worsening. The English phy-
sician and writer A. Conan Doyle wrote about Koch’s treat-
ment, “It may also be remarked that the fever after the injec-
tion is in some cases so very high (41°C) that it is hardly safe to
use in the case of a debilitated patient”(in Review of Reviews,
December 1890) (5). Our experiments in mice suggest that the
cause of the worsening pathology and fever observed by Koch
in patients may be excess TNF-␣at the site of infection. In our
murine experiments, the increased size and cellularity of the
granulomas were accompanied by increased expression of
TNF-␣mRNA in the lung. Consistent with this, vaccination of
mice with BCG–TNF-␣resulted in an increase in the size of
lung granulomas. Also, treatment of infected mice with recom-
binant murine TNF-␣resulted in increased inflammation in
the lung and accelerated death. In a previous study, in which
mice were infected with BCG–TNF-␣, we noted that high
levels of TNF-␣at the BCG infection site resulted in much
severer lung pathology and decreased survival (2). Further-
more, TNF-␣-associated clinical worsening has been observed
in patients with severe TB upon initiation of anti-TB chemo-
therapy (3). This was probably due to death of the organisms
and release of mycobacterial components. Mycobacteria and
their products have previously been shown to induce TNF-␣
production through signaling of the macrophage Toll-like re-
ceptors (24, 25).
In addition to the safety issues associated with postinfection
vaccination and the Koch phenomenon, there is also an oper-
FIG. 5. Effect of exogenous TNF-␣on the granulomatous response in the lungs. Mice were infected with M. tuberculosis and treated intranasally
with recombinant murine TNF-␣, and the lungs were examined at 4 weeks postinfection. (A to C) Untreated mice. (D to F) TNF-␣-treated mice.
Note extensive lymphocyte infiltration in arrows (A) and Ly (C). In response to TNF-␣treatment (E), large macrophages are present in the
alveolar spaces (arrowheads). Hematoxylin and eosin stain. (A and D) Magnification, ⫻4. (B, C, E, and F) Magnification, ⫻40.
2106 MOREIRA ET AL. INFECT.IMMUN.

ational consideration for any trial designed to test postexpo-
sure vaccine. Vaccination of adults in an area of high ende-
micity might reveal occult disease, which might confound
interpretation of the results, since the number of TB cases in
the vaccine arm would be selectively increased compared to
that in the untreated arm. This may have been the case in the
BCG vaccine trial carried out in the Karonga region of Malawi,
where significantly higher rates of pulmonary TB were re-
ported among scar-positive persons who had received a second
dose of BCG (13). Thus, for both safety (immune-mediated
pathology) and study design (subclinical disease scored as a
newly acquired TB), any study of candidate postexposure vac-
cines must ensure that recruited participants do not have un-
diagnosed disease.
ACKNOWLEDGMENTS
These studies were supported by NIH grants AI22616 and AI42056
(to G.K.) and AITRP TW00231 (to L.-G.B.) and by Direct Effect (New
York, N.Y.). A.L.M. was supported by a grant from the Pott’s Foun-
dation.
We thank Judy Adams for preparation of the figures and Marguerite
Nulty for secretarial assistance.
L.T. and A.L.M. contributed equally to the paper.
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Editor: S. H. E. Kaufmann
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