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Infection and Immunity, May 2000, p. 2948-2953, Vol. 68, No. 5
0019-9567/00/$04.00+0
CpG Oligodeoxynucleotides and Interleukin-12
Improve the Efficacy of Mycobacterium bovis BCG Vaccination
in Mice Challenged with M. tuberculosis
Brenda L.
Freidag,1
Genevieve B.
Melton,1
Frank
Collins,2
Dennis M.
Klinman,3
Allen
Cheever,4
Laura
Stobie,1
Winnie
Suen,1 and
Robert A.
Seder1,*
Clinical Immunology Section, Laboratory of
Clinical Investigation,1 and
Immunobiology Section, Laboratory of Parasitic
Diseases,4 National Institute of Allergy and
Infectious Diseases, National Institutes of Health, and
Laboratory of Mycobacteria2 and
Section of Retroviral Immunology,3
Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, Maryland
Received 21 December 1999/Returned for modification 24 January
2000/Accepted 5 February 2000
 |
ABSTRACT |
Mycobacterium bovis bacillus Calmette-Guérin
(BCG) is the only vaccine approved for prevention of tuberculosis. It
has been postulated that serial passage of BCG over the years may have resulted in attenuation of its effectiveness. Because interleukin-12 (IL-12) and oligodeoxynucleotides (ODN) containing cytidine phosphate guanosine (CpG) motifs have been shown to enhance Th1 responses in
vivo, they were chosen as adjuvants to increase the effectiveness of
BCG vaccination. In this report, mice were vaccinated with BCG with or
without IL-12 or CpG ODN and then challenged 6 weeks later via the
aerosol route with the Erdman strain of M. tuberculosis. Mice vaccinated with BCG alone showed a 1- to 2-log reduction in
bacterial load compared with control mice that did not receive any
vaccination prior to M. tuberculosis challenge. Moreover, the bacterial loads of mice vaccinated with BCG plus IL-12 or CpG ODN
were a further two- to fivefold lower than those of mice vaccinated
with BCG alone. As an immune correlate, the antigen-specific production
IFN-
and mRNA expression in spleen cells prior to challenge were
evaluated. Mice vaccinated with BCG plus IL-12 or CpG ODN showed
enhanced production of IFN- compared with mice vaccinated with BCG
alone. Finally, granulomas in BCG-vaccinated mice were smaller and more
lymphocyte rich than those in unvaccinated mice; however, the addition
of IL-12 or CpG ODN to BCG vaccination did not alter granuloma
formation or result in added pulmonary damage. These observations
support a role for immune adjuvants given with BCG vaccination to
enhance its biologic efficacy.
| |
INTRODUCTION |
Mycobacterium
tuberculosis infection is one of the most significant causes of
mortality worldwide. Since the onset of the human immunodeficiency
virus-AIDS epidemic, awareness of M. tuberculosis infection
has resurfaced, as it is now a major cause of mortality for human
immunodeficiency virus-infected individuals. Furthermore, the evolution
of multidrug-resistant strains represents a significant health problem
among normal, nonimmunocompromised individuals. The World Health
Organization has recently declared the current situation to be a global
emergency and has made it a priority to develop more effective vaccines
against M. tuberculosis.
The efficacy of M. bovis bacillus Calmette-Guérin
(BCG) as a vaccine is dependent on many factors and determined
clinically by its ability to prevent pulmonary or systemic disease
following exposure to M. tuberculosis. Because the
protective efficacy of BCG may range from 0 to 80% worldwide
(18), it is necessary to determine the correlates of
protection and exploit these issues in future vaccination attempts. For
example, it was recently reported that the continued propagation of BCG
over the past 60 years has lead to an evolution of BCG substrains
marked by deletions in the parental M. bovis genome
(1). Although the deletions do not include known bacterial
virulence factors, the strain diversity may account for the attenuation
or relative lack of efficacy against pulmonary disease. Identification
of these missing genes may be an essential component of improvement of
BCG efficacy.
Other vaccine approaches for tuberculosis prevention include
vaccination of mice with plasmid DNA encoding specific M. tuberculosis antigen. These vaccines can substantially reduce the
mycobacterial load following infectious challenge; however, the
protection achieved by this type of vaccination has not proven any more
effective than that achieved by vaccination with BCG alone. Thus, BCG
must be considered a standard against which other vaccines should be tested in the murine model. Due to its longstanding safety profile in
humans, it is likely that a future large-scale clinical efficacy trial
for vaccination against M. tuberculosis will include BCG in
some capacity as a control.
It is well established that the type 1 cytokines interleukin-12 (IL-12)
and gamma interferon (IFN-) are essential for control of
mycobacterial infection in both mice and humans (3, 5, 6).
Although BCG can elicit type 1 immune responses, it is possible that
the magnitude of this response has diminished over the years. To
address this concern, we attempted to enhance the host immune response
against M. tuberculosis by delivering immune adjuvants with
a given strain of BCG. In this report, both IL-12 and synthetic
oligodeoxynucleotides (ODN) containing cytidine phosphate guanosine
(CpG) motifs were examined for the ability to enhance the efficacy of
BCG vaccination by increasing the magnitude of the Th1 response prior
to infectious challenge.
| |
MATERIALS AND METHODS |
Bacteria.
M. tuberculosis Erdman (TMC 107) and
M. bovis BCG Pasteur strain (TMC 1011) were obtained from
the Trudeau Mycobacterial Culture Collection. They were grown in
Middlebrook 7H9 broth (Difco) enriched with 10% ADC additive (Becton
Dickinson) and 0.05% Tween 80 (Sigma Chemical Co.) in roller bottles
rotating at 4 rpm for 8 days at 37°C (4). The
logarithmic-phase growth was enriched with 10% glycerol and stored at
70°C (12). The viability of the suspension was checked
24 h after freezing by plating on Middlebrook 7H11 agar and
incubation at 37°C for 21 days in sealed plastic bags, at which time
CFU were counted.
Vaccination.
BALB/c mice were vaccinated via the
subcutaneous s.c., intranasal (i.n.), or intravenous i.v. route with
various amounts of BCG (Pasteur strain). The method used for i.n.
delivery of fluid has been previously described (16). At the
same time, groups of mice were given a single intraperitoneal injection
of IL-12 protein (500 ng) (Genetics Institute) i.p. or 50 µg of CpG
ODN. (The route of delivery of CpG ODN varied in different experiments as outlined in the figure legends.) Mice were housed for 6 weeks until
administration of the infectious challenge.
Oligonucleotides.
Two immunostimulatory CpG-containing ODN
with the sequences GCTAGACGTTAGCGT and TCAACGTT,
as well as control ODN from which the CpG motifs were eliminated
by inversion (GCTAGAGCTTAGGCT and TCAAGCTT), were
synthesized as previously described (13). All ODN were
produced on the same synthesizer and purified by extraction with
phenol-chloroform-isoamyl alcohol (25:24:1), followed by ethanol
precipitation. All were endotoxin free. All ODN were administered at a
dose of 50 µg per mouse.
Cytokine analysis by ELISA.
Spleen cells were harvested from
vaccinated animals (6 weeks postvaccination) prior to infectious
challenge. Cells (3 × 105/200 µl) were cultured in
the presence of M. tuberculosis (H37Ra strain, 1 × 105/200 µl) or heat-killed BCG (1 × 105/200 µl) for 48 h at 37°C. Antigen-specific
production of IFN- was measured in culture supernatants by specific
enzyme-linked immunosorbent assay (ELISA) methods.
Cytokine analysis by reverse transcription-PCR.
Cytokine
mRNA levels were determined by semiquantitative reverse
transcription-PCR techniques. In brief, total RNA was isolated from
spleen cells using Trizol methods (Gibco-BRL). Total RNA was reverse
transcribed by avian myeloblastosis virus reverse transcriptase
(Promega). Transcribed RNA (250 µg) was used for specific
semiquantitative amplification of cytokine mRNA with Taq DNA
polymerase (Promega) and specific cytokine sense and antisense primers
as previously described (8). Southern transfers of PCR
products were subsequently probed with internal cytokine-specific oligonucleotides and visualized using the ECL chemiluminescence detection system (Amersham Corp.).
Infectious challenge.
A frozen ampoule of M. tuberculosis (Erdman strain) was thawed and shaken on a Vortex
shaker for 10 s. The suspension was diluted in 0.05% Tween saline
to 106 CFU/ml, which previous studies had shown to infect
mice, with 100 to 500 CFU of strain Erdman using a Middlebrook chamber
(Glas-Col) as previously described (4). In some experiments,
mice were challenged with 105 CFU of strain Erdman by tail
vein injection.
Quantitation of M. tuberculosis.
Lungs were harvested
6 weeks following the infectious challenge with M. tuberculosis. Tissue was homogenized in cold 0.05% Tween saline
in a Seward Stomacher 80 blender (Tekmar) and plated onto Middlebrook
agar containing 2.0 µg of thiophene carboxamic hydrazide per ml to
inhibit the grown of BCG. The plates were incubated in sealed plastic
bags for 21 days at 37°C, after which the CFU were counted. In each
experiment, quantitation of M. tuberculosis was performed on
lung homogenates from at least six mice per vaccination group.
Pathology stains.
The left lung was inflated with 10%
formalin injected through a no. 26 needle. Routine paraffin sections
were prepared and stained with hematoxylin and eosin of Kinyoun's
acid-fast stain. The number of granulomas per low-power (4× objective)
field, granuloma size, and the qualitative appearance of granulomas
were evaluated.
Error and statistical analysis.
A two-tailed, two-sample,
equal-variance Student t test was applied to determine
statistical significance between groups of vaccinated mice following
infection with M. tuberculosis. Error bars represent the
standard error of the mean.
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RESULTS |
Mice vaccinated with BCG plus IL-12 or CpG ODN have enhanced
IFN- production compared with mice vaccinated with BCG alone.
Because the magnitude of the Th1 response is critical in mediating
protection against a variety of intracellular pathogens, we sought to
optimize BCG vaccination by using immune adjuvants to enhance the Th1
response. BALB/c mice were vaccinated s.c. with BCG, BCG plus IL-12, or
BCG plus CpG ODN. Six weeks later, antigen-specific production of
IFN- was determined in cultured spleen cells. As shown in Fig.
1A, the addition of IL-12 to BCG caused a
two- to fivefold increase in IFN- production following in vitro
stimulation with either an avirulent strain of M. tuberculosis (H37Ra) or heat-killed BCG. Similarly, enhanced
production of IFN- following vaccination with BCG plus IL-12 protein
was seen as early as 1 week postvaccination (Fig. 1C). Finally, as seen in Fig. 1B, the addition of CpG ODN to BCG vaccination also increased the antigen-specific production of IFN- compared with that of mice
receiving BCG alone, although this effect was less dramatic than that
achieved by the addition of IL-12 protein.
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FIG. 1.
IL-12 protein and CpG ODN enhance the in vitro
production of IFN- induced by BCG vaccination. We pooled spleen
cells (3 × 105/200 µl) from at least three
individual mice vaccinated with BCG (s.c.) or BCG (s.c.) plus rIL-12
(i.p.) (A) or with BCG (s.c.) or BCG (s.c.) plus CpG ODNs (i.p.) (B)
and cultured them in the presence of H37Ra (an avirulent strain of
M. tuberculosis) or heat-killed BCG and determined the
antigen-specific production of IFN- by a specific ELISA. (C) Spleen
cells (3 × 105/200 µl) were taken from mice
sacrificed at 2, 7, and 21 days postvaccination with BCG or BCG plus
IL-12 and assessed for antigen-specific production of IFN- following
in vitro stimulation with H37Ra. Data are shown after subtraction of
the IFN- production from splenocytes cultured in medium alone (<100
pg/ml). *, P < 0.02; **, P < 0.05 compared with BCG alone.
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As an in vivo correlate, we also examined the effect of IL-12 and CpG
ODN on the mRNA expression of various cytokines. As
shown in Fig.
2, the addition of IL-12 or CpG ODN to
BCG vaccination
increased mRNA expression for IFN-
but had little
effect on IL-10
or tumor necrosis factor alpha. In addition, there was
no difference
in the expression of mRNA for IL-12 p40 (data not shown).
Taken
together, these data suggest that IL-12 or CpG ODN can enhance
the magnitude of Th1 priming induced by BCG vaccination prior
to
challenge with
M. tuberculosis.
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FIG. 2.
Expression of mRNA for cytokines following vaccination
with BCG plus IL-12 or CPG ODN. mRNA was isolated from pooled
splenocytes (combined from at least three individual mice) at 6 weeks
postvaccination of mice with BCG (s.c.), BCG (s.c.) plus IL-12 (i.p.),
or BCG (s.c.) plus CpG ODN (i.p.). As a control, mRNA from unvaccinated
mice was also prepared. Cytokine mRNA was subsequently determined for
IFN-, IL-10, and tumor necrosis factor alpha (TNF- ) by
semiquantitative reverse transcription (RT)-PCR as described in
Materials and Methods. HPRT, hypoxanthine phosphoribosyltransferase.
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Vaccination with BCG plus IL-12 reduced the mycobacterial burden in
lungs of mice following aerosol challenge with virulent M. tuberculosis.
Because the magnitude of the IFN- response often
correlates with immunity against intracellular infection, the previous
figures suggest that mice vaccinated with BCG plus IL-12 or CpG ODN may have increased protection following infectious challenge. To determine the biologic relevance of increased IFN- production prior to infection, mice were challenged with live, virulent M. tuberculosis via the aerosol route 6 weeks after vaccination. The
mycobacterial burden in the lungs 6 weeks after challenge was then
assessed. Consistent with previous data (10, 21), mice
vaccinated i.v. or s.c. with BCG had a 1- to 2-log bacterial load
decrease compared with unvaccinated mice. Moreover, mice vaccinated
with BCG plus IL-12 showed a two- to fivefold further bacterial load
reduction compared with mice vaccinated with BCG alone (Fig.
3). This trend was seen in other
experiments as early as 4 weeks postinfection (data not shown).
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FIG. 3.
Mice vaccinated with BCG plus IL-12 protein have
decreased mycobacterial loads following an infectious challenge. BALB/c
mice were initially vaccinated (n = 6 to 10/group) with
BCG (106) either i.v. or s.c., with or without a single
i.p. injection of IL-12 (500 ng). Six weeks later, mice were challenged
with 500 virulent M. tuberculosis bacteria via the aerosol
route. Six weeks postinfection, lungs were harvested from individual
mice (n = 6) and numbers of CFU per lung were
quantitated as described in Materials and Methods. These results were
obtained in at least five different experiments. *, P < 0.03 compared with BCG alone.
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The ability of IL-12 protein to enhance immunity against
M. tuberculosis when given with BCG vaccination is in contrast to
the
results obtained in a previous study (
7). That study
differed
from ours in that the infectious challenge was given i.v.
rather
than aerogenically and repeated doses of IL-12 were given with
BCG. In addition, mortality was used as a biologic endpoint. To
address
these apparent inconsistencies, we tested the efficacy
of BCG with and
without IL-12 following an infectious challenge
by both aerosol and
systemic routes. As shown in Fig.
4A,
consistent
with the data shown above, mice vaccinated with BCG plus
IL-12
had a fivefold reduction (
P < 0.01) in
mycobacterial burden compared
with mice vaccinated with BCG alone
following an aerosol challenge.
In contrast, there was essentially no
difference in the mycobacterial
burden in the spleens of these mice
compared with those of mice
receiving BCG alone. Similarly, there was a
fivefold reduction
(
P < 0.01) in the mycobacterial
burden in the lungs but not the
spleens of mice vaccinated with BCG
plus IL-12 compared with BCG
alone following i.v. challenge (Fig.
4B).
Taken together, these
data show that a single dose of IL-12 with BCG is
effective at
reducing the mycobacterial load in the lungs following a
systemic
or aerosol challenge. In contrast, IL-12 does not affect the
mycobacterial
load in spleens following a systemic or aerosol
challenge.
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FIG. 4.
Mice vaccinated with BCG plus IL-12 have reduced
mycobacterial loads in the lungs but not in the spleen following an
aerosol or i.v. challenge. BALB/c mice were initially vaccinated
(n = 6 to 10/group) with 106 BCG bacteria
(s.c.) with or without a single i.p. injection of IL-12 (500 ng). Six
weeks later, mice were challenged with 500 virulent M. tuberculosis bacteria via the aerosol route (A) or 105
virulent M. tuberculosis bacteria i.v. (B). Six weeks
postinfection, spleens or lungs of individual mice (n = 6) were harvested and numbers of CFU per organ were quantitated.
*, P < 0.01 compared with BCG alone.
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Mice vaccinated with BCG plus CpG ODN have reduced pulmonary
mycobacterial burdens following aerosol challenge with virulent
M. tuberculosis.
In a separate experiment, the efficacy of
CpG ODN as a vaccine adjuvant with BCG was also assessed for the
ability to alter the mycobacterial burden in the lungs following
infection (Fig. 5). Similar to the
results obtained as described above using IL-12 protein, mice
vaccinated with BCG plus CpG ODN had a two- to fivefold bacterial load
reduction compared with mice receiving BCG alone. This effect was
similar whether the CpG ODN was given in the same s.c. injection as the
BCG or given separately i.p. (Fig. 5A). As it may be necessary to
direct immune cells to the site of infection for successful vaccination
against M. tuberculosis, we also determined whether i.n.
delivery of BCG with and without CpG ODN affected the biologic efficacy
of BCG following infectious challenge. Mice receiving i.n. BCG alone
showed a 1- to 2-log bacterial load reduction compared with
unvaccinated mice. The bacterial loads of mice vaccinated with BCG plus
CpG ODN in the same i.n. preparation were further reduced by nearly 1 log compared with those of mice vaccinated i.n. with BCG (Fig. 5B);
however, vaccination with BCG i.n. plus CpG ODN at a separate site i.p.
did not improve the efficacy of vaccination. Thus, CpG ODN can enhance
the efficacy of BCG vaccination, provided that it is given at the same
general site as BCG.
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FIG. 5.
Mice vaccinated with BCG plus CpG ODN have decreased
mycobacterial loads following infectious challenge. BALB/c mice were
initially vaccinated (n = 6 to 10/group) with BCG
(106 bacteria) s.c. with or without CpG ODN given either in
the same shot or at a different site (i.p.) (*, P < 0.05 compared with BCG alone) (A) or i.n. with or without CpG ODN
given either in the same preparation i.n. with BCG or at a different
site (i.p.) (*, P < 0.01 compared with BCG alone)
(B). Shown are numbers of CFU per lung calculated from lungs of six
individual mice harvested 6 weeks after an aerosol challenge with a
virulent strain of M. tuberculosis.
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Pathologic effect of BCG vaccination.
While IL-12 and CpG ODN
diminished the infectious burden following challenge, the enhanced
production of proinflammatory cytokines (i.e., IFN-) induced by
these adjuvants raised the concern that this could be associated with
greater lung pathology. To address this point, lungs from mice
vaccinated and challenged with M. tuberculosis were
harvested and processed to examine the histopathologic effects
(cellular infiltrate, size and appearance of granulomas) induced by the
various vaccine protocols. The numbers of granulomas were similar in
all groups, but granulomas in BCG-vaccinated mice were consistently
about one-half of the volume of those in unvaccinated mice (Fig.
6). Granulomas from vaccinated mice
contained 20 to 40% lymphocytes, compared with approximately 15% in
unvaccinated mice. The addition of IL-12 or CpG ODN to BCG vaccination
did not further alter the size or appearance of granulomas, and no pulmonary damage was seen aside from the granulomas. Virtually all of
the cells in the granulomas were lymphocytes or activated macrophages,
and no necrosis was observed.
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FIG. 6.
Addition of IL-12 or CpG ODN to BCG vaccination does not
affect pulmonary granuloma formation. Mice were vaccinated, challenged
with a virulent strain of M. tuberculosis, and sacrificed 6 weeks following infectious challenge. (A) Unvaccinated mouse; the
granuloma is larger than that in vaccinated mice and is composed almost
entirely of macrophages. (B) BCG-vaccinated mouse; the granuloma is
smaller than that in the unvaccinated mouse, and small lymphocytes
constitute a substantial portion of the cells. (C and D) Mice
vaccinated with BCG plus IL-12 and BCG plus CpG ODN, respectively; the
granulomas are similar to that shown in the BCG-vaccinated mouse.
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| |
DISCUSSION |
Over the past 60 years, BCG has been widely used to control
tuberculosis infection worldwide. BCG is given systemically and can
confer protection against systemic or meningeal disease when exposure
to M. tuberculosis occurs shortly after immunization. BCG is
also inexpensive for use in underdeveloped countries. For these
reasons, BCG remains useful and elimination of BCG vaccination is not
feasible at this time. Of greater concern is why BCG is not universally
effective at preventing adult pulmonary disease.
The increasing attenuation of BCG has been discussed in several
reports. The diversity and variable efficacy of BCG were first noted by
showing that clinical outcome appeared to correlate with specific
strains of BCG rather than with purified protein derivative reactivity
(M. A. Behr and P. M. Small, Letter, Nature
389:133-134, 1997). More recent publications suggest that
gene deletions have allowed the evolution of BCG over time, so that a
myriad of BCG strains are now used in different regions of the world
(1). This attenuation of BCG through laboratory passaging
may have limited its clinical efficacy, making it necessary to optimize this vaccine. To this end, numerous groups attempted to either enhance
the effectiveness of BCG or explore other vaccine strategies, such as
plasmid DNA vaccination (11, 14, 15, 22). With regard to DNA
vaccination, while initial reports are promising, this approach has not
proven to be more effective than BCG vaccination itself. This prompted
us to evaluate whether BCG vaccination could be optimized.
The use of cytokines to enhance the efficacy of BCG vaccination in the
mouse model has been previously studied. Murray et al. constructed
recombinant BCG strains that were able to secrete cytokines such as
IL-2, granulocyte-macrophage colony-stimulating factor, and IFN-
(17). While this study was an elegant proof of principle, it
was not demonstrated whether these strains could improve protection
following challenge with M. tuberculosis. The study by Flynn
et al. (7) was similar in concept to our study in using
IL-12 as an adjuvant with BCG. In that study, IL-12 provided no
increase in protection compared with BCG alone. The differences highlighted above (endpoint of study, systemic versus pulmonary challenge, IL-12 dosage) may help explain the contrasting results outlined in this report. Furthermore, with regard to IL-12 dosage, in
the study by Flynn et al., IL-12 was given continuously with BCG
(7) rather than in a single injection as reported here. It
is possible that the continued use of IL-12 with BCG led to more rapid
clearance of BCG, thus limiting its immunogenicity and therefore its effectiveness.
The experiments reported here were designed to enhance the
effectiveness of BCG vaccination in the mouse pulmonary model of M. tuberculosis infection. In many mouse models of
intracellular infection, the magnitude of the Th1 response often
correlates with the subsequent control of infection. These studies
focused on the use of immune adjuvants such as IL-12 or CpG ODN with
BCG as a means to further increase the quantitative aspect of the Th1
response induced by BCG vaccination. While both IL-12 and CpG ODN are
potent inducers of Th1 responses, CpG ODN may offer some additional
advantages over IL-12. For example, CpG ODN directly induce the
production of IL-12, as well as other inflammatory cytokines (tumor
necrosis factor alpha, IL-6) (13, 19), and have also been
shown to directly enhance T-cell activation (2). The
potential advantage of CpG ODN compared to IL-12 was shown in vivo
using the mouse model of Leishmania major infection. In these studies, treatment of mice with CpG ODN several days following infectious challenge enabled mice to control the infection (23, 24). This contrasted with previous reports showing that IL-12 treatment must be initiated at the time of infection for it to confer
protective immunity (9, 20).
While there did not appear to be any advantage in the use of CpG ODN
over that of IL-12 protein as an immune adjuvant with BCG at 6 weeks
postchallenge, preliminary data suggest that CpG ODN may be more
effective at later time points (12 weeks postchallenge; data not
shown). Furthermore, with regard to production of IFN- following
vaccination, mice vaccinated with BCG plus IL-12 or CpG ODN had
enhanced IFN- production and mRNA expression in the spleen prior to
infection compared with mice vaccinated with BCG alone (Fig. 1 and 2).
In contrast, we could not detect any mRNA for IFN- in lungs of mice
vaccinated with BCG s.c. with or without IL-12 or CpG ODN (data not
shown). These data are consistent with our finding that, following s.c.
vaccination with BCG, BCG was detectable in the spleens but not in the
lungs by quantitative culture (data not shown). It is notable that
despite the increase in IFN- production in the spleens of mice
vaccinated with BCG plus IL-12- or CPG ODN, the mycobacterial load
reductions in the spleens following infection were comparable among all
groups of vaccinated mice. These data are compatible with the notion
that while the general types of immunity required for protection
against tuberculosis (i.e., Th1) are similar between systemic disease and pulmonary disease, there are likely differences in the qualitative and quantitative aspects of the immune response between the spleen and
the lungs. In this regard, there is a critical threshold of Th1
cytokine production for protection in a variety of intracellular infection models. Thus, due to the greater number of immune cells (i.e., T cells) in the spleen compared with the lungs, the threshold of
IFN- required for a significant mycobacterial load reduction in the
spleen is reached by BCG alone. By contrast, due to the relative
paucity of T cells in the lungs compared with the spleen, an increase
in the number of IFN--producing cells induced by BCG and IL-12 or
CpG ODN would have a greater effect in reducing mycobacterial loads in
the lungs following an infectious challenge. We postulate that
following s.c. vaccination with BCG plus IL-12 or CpG ODN, an increase
in the number of tuberculosis-specific IFN--producing cells is
generated in the spleen. These cells then migrate to the lungs
following an infectious challenge, causing a reduction in mycobacterial growth.
This report shows that the addition of immune adjuvants can increase
the magnitude of the Th1 response induced by BCG vaccination and reduce
the pulmonary bacterial load after a challenge with M. tuberculosis. Because BCG remains the standard for protection in
the mouse model, we believe that any consistent improvement over BCG
alone represents a potentially significant finding. The real issue is
whether this approach will offer increased protection against disease
in humans. In addition, we are investigating whether the effects of
addition of IL-12 or CpG ODN to BCG remain durable over a period of
time. Our experiments have focused on the use of combinations of BCG,
immune adjuvants, and plasmid DNA encoding antigen not present in BCG
as a way to further optimize vaccination against M. tuberculosis.
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ACKNOWLEDGMENTS |
We thank Brenda Rae Marshall for editorial assistance.
L.S. and G.B.M. are HHMI-NIH research scholars.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: LCI, NIAID, 10 Center Dr., Room 10/11C215, NIH, Bethesda, MD 20892. Phone: (301)
402-4816. Fax: (301) 480-1936. E-mail:
rseder{at}niaid.nih.gov.
Editor:
W. A. Petri Jr.
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Abstract
Introduction
