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Infect Immun, June 1998, p. 2951-2959, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Evaluation of New Vaccines in the Mouse and Guinea
Pig Model of Tuberculosis
Susan L.
Baldwin,1
Celine
D'Souza,1
Alan D.
Roberts,1
Brian P.
Kelly,1
Anthony A.
Frank,2
Margaret A.
Lui,3
Jeffrey B.
Ulmer,3
Kris
Huygen,4
David M.
McMurray,5 and
Ian M.
Orme1,*
Mycobacteria Research Laboratories,
Department of Microbiology,1 and
Department of Pathology,2 Colorado
State University, Fort Collins, Colorado 80523;
Department of
Virus and Cell Biology, Merck Research Laboratories, West Point,
Pennsylvania 194863;
Department of
Virology, Pasteur Institute, B-1180 Brussels,
Belgium4; and
Department of Medical
Microbiology and Immunology, Texas A&M University, College Station,
Texas 778435
Received 20 October 1997/Returned for modification 8 January
1998/Accepted 12 March 1998
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ABSTRACT |
The results of this study provide the first evidence that two
completely separate vaccine approaches, one based on a subunit vaccine
consisting of a mild adjuvant admixed with purified culture filtrate
proteins and enhanced by the cytokine interleukin-2 and the second
based on immunization with DNA encoding the Ag85A protein secreted by
Mycobacterium tuberculosis, could both prevent the onset of
caseating disease, which is the hallmark of the guinea pig aerogenic
infection model. In both cases, however, the survival of vaccinated
guinea pigs was shorter than that conferred by Mycobacterium bovis BCG, with observed mortality of these animals probably due to consolidation of lung tissues by lymphocytic granulomas. An additional characteristic of these approaches was that neither induced
skin test reactivity to commercial tuberculin. These data thus provide
optimism that development of nonliving vaccines which can generate
long-lived immunity approaching that conferred by the BCG vaccine is a
feasible goal.
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INTRODUCTION |
Tuberculosis is the leading
worldwide cause of death from infectious disease, with a recent report
on the global epidemiology of tuberculosis predicting that without
worldwide control measures it could be responsible for 30 million
deaths between the years 1990 and 2000 (21).
Humans exhibit a wide range of responses to tuberculosis infection. The
majority are resistant, dealing with exposure to the bacillus by
killing it via innate mechanisms of immunity or by generating a strong
state of acquired cellular immunity that usually leads to control of
the infection. In these latter individuals, the only visible symptom of
exposure is conversion to a positive state of skin test reactivity to
tuberculin purified protein derivative (PPD). In a small percentage of
this latter group of people, however, the initial infection is not
contained but instead gives rise to progressive pulmonary infection.
These two ends of the spectrum of disease in humans can be modeled in
mice, which are resistant to tuberculosis, and in guinea pigs, which
are susceptible to the disease. Mice generate a strong cellular immune
response against tuberculosis, which controls bacterial growth and
limits damage to the lungs. Guinea pigs also initially develop strong
immunity, but this is eventually associated with considerable tissue
damage, leading to extensive caseation and tissue necrosis that
eventually kills the animal. As such, this is a useful model of events
in infected humans, which follow a similar pattern.
The attenuated BCG strain of Mycobacterium bovis has been
extensively used as a vaccine against tuberculosis for the past several
decades. The vaccine has several virtues, including the fact that it
can be safely given to young children, is cheap to produce, and gives
rise to a long-lived state of host resistance (4). It has
been comprehensively evaluated in a relatively large number of
controlled vaccine trials, and in various populations and geographic
regions the calculated protective efficacy of the vaccine has
unfortunately varied between 0 and 80% (24, 29). This
extreme variability has prompted new research into replacing BCG with a
more effective vaccine against tuberculosis (18, 23).
In this regard, we have hypothesized that proteins produced and
secreted by the metabolizing M. tuberculosis bacilli (which are present in culture filtrate), rather than constitutive or stress
proteins, are the key antigens recognized by the protective immune
response (16, 17). This hypothesis has received increasing support in the field, and reports from four separate laboratories have
shown variable degrees of success in vaccinating mice or guinea pigs
against experimental tuberculosis by using culture filtrate proteins
(CFP) (2, 10, 20, 22). To date, however, no information has
been presented about the ability of these vaccines to prevent the
development of lethal pathologic changes, such as pulmonary caseous
necrosis, over a sustained period. This information would be important,
given that BCG vaccination limits the production of caseation within
the lungs of guinea pigs. Naive guinea pigs infected via the aerosol
route show evidence of caseation and necrosis a few weeks after
exposure. This gradually leads to caseous necrosis within lesions,
which can either mineralize or cavitate. Outward signs of these events
are shallow breathing, sudden significant weight loss, and death
occurring 8 to 20 weeks postinfection.
For a new vaccine to have any credence as a potential replacement for
BCG, it is imperative to demonstrate that (i) it can prevent this
caseating disease and instead induce a cellular response in the lungs
similar to that induced by BCG, and (ii) it can protect the animal over
the long term, at least to the extent provided by BCG (classical
studies [13] show that BCG can protect guinea pigs for
about 200 to 400 days postinfection).
In the present study these questions were investigated in the guinea
pig model after a series of comprehensive pilot studies in the mouse.
The results indicate that two vaccine types, one based upon a mixture
of monophosphoryl lipid A (MPL) adjuvant, CFP, and the cytokine
interleukin-2 (IL-2) and the other based upon a DNA plasmid vaccine
encoding a major culture filtrate protein from M. tuberculosis (Ag85A), could prolong the survival of infected guinea pigs and prevent them from developing caseating disease. In
these animals, a lymphocytic granulomatous response ensued, similar to
that seen in animals vaccinated with BCG.
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MATERIALS AND METHODS |
Animals.
Specific-pathogen-free female C57BL/6 mice and
female outbred Hartley guinea pigs were purchased from Charles River
Laboratories (North Wilmington, Mass.) and held under barrier
conditions in an ABL-3 biohazard laboratory at Colorado State
University. Mice were 6 to 8 weeks old at the beginning of the
experiments and were housed four to a cage. The mice were sacrificed 30 days after infection with M. tuberculosis Erdman. Guinea
pigs weighed approximately 500 to 600 g at the beginning of the
experiment and were housed two to a cage. Guinea pigs in the first
study were sacrificed 30 days after aerogenic infection with M. tuberculosis H37Rv. A second set of vaccinated guinea pigs was
monitored for several months after aerogenic infection with M. tuberculosis H37Rv. Because of expense, we were limited to four
animals per group for these studies. The animals were weighed every few
days, and those showing evidence of sudden significant weight loss were
euthanized. Animals were allowed free access to water and standard
mouse or guinea pig chow, respectively.
Bacterial infections.
M. tuberculosis Erdman and H37Rv
and M. bovis BCG Pasteur were previously grown to early
mid-log phase in Proskauer Beck medium containing 0.05% Tween 80. Cultures were aliquoted into 1-ml tubes and stored at 
70°C until
used. Thawed aliquots were diluted in double-distilled sterile water to
the desired inoculum concentrations. An aerosol generation device
(Glas-Col, Terre Haute, Ind.) was used to expose animals to an aerosol
of M. tuberculosis and was calibrated to deliver
approximately 20 to 50 bacilli into each guinea pig lung. Mice were
aerogenically infected with approximately 100 bacilli.
CFP.
Purified CFP from M. tuberculosis were
kindly provided by John Belisle, Colorado State University.
Cytokines.
Recombinant murine IL-12 (rIL-12) was kindly
provided by Genetics Institute, Cambridge, Mass. Polyethylene glycol
recombinant human IL-2 was kindly provided by Chiron Corp., Emeryville,
Calif.
Adjuvant.
Adjuvant formulations based upon MPL were kindly
provided by Ribi ImmunoChem Research, Inc., Hamilton, Mont. MPL is a
nontoxic derivative of the lipid A from Salmonella
minnesota. MPL was solubilized in triethanolamine (TeoA) by
sonication; stock solutions contained 0.02% TeoA and 0.4% dextrose.
Vaccinations.
For mice, vaccines containing a total of 100 µg of M. tuberculosis CFP were emulsified in MPL-TeoA
adjuvant. In addition, some preparations contained rIL-12 (500 ng per
mouse) or rIL-2 (100 µg per mouse). Vaccines were given twice,
subcutaneously, 3 weeks apart. In mice which received both cytokines,
rIL-12 was included in the first immunization and rIL-2 was included in
the second. BCG at 106 bacilli was injected subcutaneously
and was given once at the same time as the second immunizations.
Animals were aerogenically challenged with approximately 100 M. tuberculosis bacilli 30 days after vaccination.
Guinea pigs were immunized with 150 µg of CFP in MPL-TeoA. In
addition, some vaccines contained rIL-12 (1 µg) and/or rIL-2 (20 µg). Vaccines were injected subcutaneously three times, at 3-week
intervals. The vaccine containing CFP and IL-2 was given only twice due
to a slight hypersensitivity reaction in some animals. BCG
(103 bacilli/guinea pig) was injected intradermally (i.d.)
once at the same time as the third immunizations. The animals were
aerogenically challenged with approximately 50 M. tuberculosis bacilli 6 weeks later.
DNA vaccines, consisting of the control plasmid vector V1Jns
(DNA-vector) and V1Jns containing the genes encoding the secreted and
nonsecreted forms of M. tuberculosis Ag85A protein
(DNA-Ag85A), were kindly provided by the Merck Research Laboratories
(West Point, Pa.). Vaccines were given intramuscularly three times at 3-week intervals. Each guinea pig was given 200 µg of plasmid DNA in
saline per quadricep muscle (400 µg total per immunization). The
animals were then infected aerogenically as above.
Delayed-type hypersensitivity (DTH) measurements.
Tuberculin
PPD (lot CT68) was purchased from the Connaught Laboratories (Toronto,
Canada). Mice were injected in the left hind footpad with 5 µg of PPD
in 50 µl of sterile saline via a 30-gauge needle. Phosphate-buffered
saline (PBS) alone was injected into the contralateral footpad as a
negative control. Footpad thickness was measured 48 h later with
calipers capable of measuring 0.05-mm increments in thickness.
Guinea pigs were shaved on the back, and 1 µg of each skin test
reagent suspended in 50 µl of sterile saline was injected
i.d. into
different sites via a 30-gauge needle. The skin test
reagents consisted
of saline and bovine serum albumin, as negative
controls, PPD,
M. tuberculosis H37Rv lipoarabinomannan-free purified
CFP, and
purified 19-kDa, 45-kDa, and Ag85A proteins from
M. tuberculosis.
Induration was measured 48 h after injection by
using a dial gauge
caliper.
Histological analysis.
Tissues were fixed in 10% neutral
buffered formalin for routine microscopic processing. All tissues were
stained with hematoxylin and eosin. In each case, the left lower lobe
was sagittally sectioned through the middle of the lobe. The tissues
were coded and evaluated by a board-certified pathologist without
knowledge of the treatment groups.
The following parameters were subjectively assessed in tissue sections:
severity (degree of parenchymal involvement), size
of typical
granulomas, amount of caseous necrosis, relative number
of neutrophils
and lymphocytes, degree to which lymphocytes were
organized in the
granuloma, and extent to which the granulomas
were organized (sharp
demarcation from surrounding tissue, often
with lymphocytes and/or
fibrosis at the periphery). Sections were
evaluated at least twice
without knowledge of treatment or previous
grading, and the results
were reproducible. Although there was
some lesion variability within
vaccination groups, presumably
due to the use of outbred animals, this
variability was much less
pronounced than was lesion variability
between vaccine groups.
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RESULTS |
Protection studies with mice.
Because of the expense of the
guinea pig model, a comprehensive series of pilot experiments was
conducted with mice. Two inoculations were given, 3 weeks apart,
followed by an aerosol challenge with M. tuberculosis Erdman
4 weeks later, and bacterial numbers in the lungs were determined 30 days postchallenge. It was decided to avoid strong adjuvants in these
studies because (i) it would be unlikely that such preparations could
be used in humans, and (ii) they also induce very strong DTH reactions.
As a result, studies were performed with the adjuvant MPL-TeoA, which
is known to enhance both cell-mediated and humoral responses. This
adjuvant, which is currently undergoing human clinical safety trials
(8, 12), has many of the immunomodulatory properties of
lipopolysaccharide without the toxicity typically associated with
endotoxins. MPL induces macrophages to secrete several cytokines
including IL-1, IL-6, IL-8, granulocyte-macrophage colony-stimulating
factor, and tumor necrosis factor (14) and enhance antigen
uptake, processing, and presentation (25).
In initial studies, CFP given in alum or in MPL was not effective (data
not shown). As a result, several further experiments
were conducted in
which the vaccine formulation was supplemented
with IL-12, given the
property of this cytokine to enhance the
secretion of the protective
cytokine gamma interferon by TH
1-type
CD4 T cells in
several infectious-disease models (
1,
5-7,
15,
26,
27), and
with IL-2 in an attempt to expand the number
of useful clones of
mycobacterium-specific T cells. (A long-lived
form of IL-2 conjugated
to polyethylene glycol was used; this
material has been reported to
exhibit a 15-fold decrease in plasma
IL-2 clearance compared with that
exhibited by unmodified IL-2
[
28].)
The results of one of several representative experiments are shown in
Table
1. The BCG control-vaccinated mice
had a 1.34-log
reduction in bacterial numbers in the lungs, compared to
PBS controls.
None of the vaccine formulations containing various
combinations
of MPL, CFP, and cytokines had any protective effect,
except when
both IL-2 and IL-12 were administered, resulting in a
statistically
significant 0.91-log reduction in bacterial load.
Combinations
of adjuvant and cytokines had no effect alone (data not
shown).
Interestingly, despite this protective effect, these animals
showed
no evidence of DTH reactivity to PPD injection.
Short-term protection responses in guinea pigs.
Experiments
were then performed with guinea pigs to determine if similar conditions
of cytokine enhancement were necessary for protection in this
susceptible animal model. Vaccines were given three times, 3 weeks
apart, to groups of outbred guinea pigs, which were then challenged via
aerosol infection with approximately 50 viable M. tuberculosis H37Rv bacilli six weeks following the last injection.
The results of this study (Table
2)
showed a 1.35-log reduction in the lungs of guinea pigs that had
received intradermal
(i.d.) BCG vaccination. A marginal but
statistically significant
(
P = 0.048) reduction in
bacterial counts within the lungs was
also seen in guinea pigs given
the MPL-CFP vaccine which had been
supplemented with IL-12 and IL-2.
None of the other vaccine groups
exhibited any statistically
significant reduction in lung bacterial
counts.
All the guinea pigs used in these studies were tested for DTH against a
panel of mycobacterial antigens just before aerosol
challenge. Animals
were injected i.d. on the back, and induration
was measured 48 h later.
As shown in Fig.
1, animals vaccinated
with MPL formulations gave weak responses to purified CFP but
not to
PPD. Only BCG-vaccinated guinea pigs gave strong responses
to most of
the antigen panel.
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FIG. 1.
Development of DTH reactions (induration) 48 h
after i.d. injection with a panel of mycobacterial antigens in guinea
pigs previously immunized with the indicated vaccines. Data are
presented as the mean diameter of induration ± standard deviation
(n = 4).
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Representative histological appearances of the lung tissues of each
group of infected guinea pigs 30 days after aerosol infection
are shown
in Fig.
2.
Cellular details of the granulomatous lesions
are depicted in Fig.
3. In the animals injected with PBS, the
lung lesions tended to be moderately large (approximately 50%
of the
pulmonary parenchyma was involved [Fig.
2A]), with scattered
infiltrates of lymphocytes admixed with epithelioid macrophages
(Fig.
3A) and only modest organization. In contrast, granulomas
in the lungs
of BCG-vaccinated animals affected a smaller portion
of the pulmonary
parenchyma (approximately 25 to 33%), were small
and compact with
sharp lines of demarcation to the surrounding
parenchyma (Fig.
2B), and
had increased numbers of lymphocytes
throughout the lesions (Fig.
3B).
No caseation was observed in
the BCG group, and caseation was minimal
in the saline control
group. In animals inoculated with MPL alone,
MPL-CFP, or MPL-CFP-IL-12,
large portions of the lungs were affected
(
50%), with individual
granulomas characterized by large size,
modest demarcation from
the surrounding parenchyma (Fig.
2C, D, and F),
occasional small
areas of central caseation (Fig.
3D), and generally
scant, unorganized
lymphocyte infiltrations (Fig.
3C). Animals
vaccinated with MPL-CFP-IL-2
(Fig.
2E) and MPL-CFP-IL-12-IL-2
(Fig.
2G) developed granulomas
that were small and of limited extent,
similar to the BCG group.
Lesion size and extent were intermediate in
the lungs of DNA-Ag85A-vaccinated
animals (Fig.
2H). The lesions in the
last three groups contained
lymphocytic infiltrates varying from
scattered to heavier infiltrates
(Fig.
3E and F). Lesion demarcation
was sharp, and caseation was
not observed.
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FIG. 2.
Representative photomicrographs of lung tissue sections
harvested from vaccinated guinea pigs 30 days after an aerosol
infection with M. tuberculosis H37Rv. (A) PBS control.
Approximately 50% of the parenchyma is replaced by multiple,
moderately sized granulomas. (B) BCG. The limited area of affected
parenchyma contains small, focal granulomas. (C) MPL control. There is
extensive parenchymal destruction by a large, poorly demarcated
granuloma. (D) MPL-CFP. This lesion is similar to that in the MPL
control (C), except that this extensive granuloma has central caseation
(see Fig. 3D). (E) MPL-CFP-IL-2. A small, sharply demarcated
granuloma affects a minimal amount of parenchyma. (F) MPL-CFP-IL-12.
This lesion is essentially identical to that in the MPL-CFP animal (D).
(G) MPL-CFP-IL-12-IL-2. The limited parenchymal involvement is
characterized by small granulomas similar to those found with BCG
treatment (B) and MPL-CFP-IL-2 treatment (E). (H) DNA-Ag85A. An
intermediate amount of parenchyma is affected by moderately sized,
well-demarcated granulomas. Magnification, ×20.
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FIG. 3.
Higher-magnification photomicrographs of some of the
lesions depicted in Fig. 2, demonstrating cytological details. (A) PBS
control. Scattered lymphocytes are admixed with epithelioid
macrophages. (B) BCG. Numerous lymphocytes are present throughout the
section. There is no caseation. (C) MPL control. Note the relative
paucity of lymphocytes amid numerous foamy macrophages. (D) MPL-CFP.
This section demonstrates an area of central caseation, the same lesion
seen in the lungs of MPL-CFP-IL-12-vaccinated animals (higher
magnification not included). (E) MPL-CFP-IL-2. Note the similarity to
the lesion in the BCG control (B). This is essentially the same lesion
observed in MPL-CFP-IL-2-IL-12-vaccinated animals (higher
magnification not included). (F) DNA-Ag85A. Relatively numerous
lymphocytes are admixed with fields of epithelioid macrophages.
Hematoxylin and eosin stain; magnifications, ×156.
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Long-term responses in guinea pigs.
The results of the study
of long-term responses in guinea pigs are shown in Fig.
4. Placebo control animals (PBS or MPL
alone) began to lose weight about 2 weeks after aerosol challenge,
after which time their weight plateaued for several weeks. Then, 8 to 18 weeks postchallenge, these animals died. One animal in the MPL group
began to lose weight at 15 weeks, but not quite at a rate at which
euthanasia was deemed appropriate.
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FIG. 4.
Body weights of individual guinea pigs, given subunit
vaccines (A) or DNA vaccines (B), after aerogenic infection with
M. tuberculosis. Arrows indicate the last weight measurement
before death. All remaining animals were euthanized at 31 weeks
postinfection.
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Animals given MPL-CFP increased in weight past 10 weeks and appeared
relatively healthy, but then three of the four died around
week 16. One
guinea pig with significantly lower lung bacterial
counts (see below)
survived to the end of the experiment (212
days).
A very surprising result was seen in the MPL-CFP-IL-12-IL-2 group,
which was the only group to show significant protection
at day 30 after
aerosol challenge. Three of the four animals did
not thrive and died
between 11 and 16 weeks. The fourth animal
gained weight but also died
20 weeks post challenge. A similar
result was seen in animals given
MPL-CFP-IL-12.
Somewhat better results were seen when only IL-2 was added to the
MPL-CFP vaccine. One guinea pig began to lose weight 17
weeks
postinfection and died shortly thereafter, and another guinea
pig in
this group died after 27 weeks. The remaining two guinea
pigs, however,
continued to thrive and still appeared healthy
when the study was
terminated.
In the BCG-vaccinated group, three of four animals appeared healthy
throughout, while one animal began to lose weight and
was found to have
higher bacterial counts in the lungs than the
others did (Table
3).
In a parallel experiment testing the vaccine efficacy of DNA-Ag85A, one
animal died after 11 weeks and another died at 26
weeks (this animal
had signs of lymphadenopathy, which may have
contributed to its death).
Two others, however, appeared healthy
at the end of the study (although
one was starting to show evidence
of weight loss). Vector control
guinea pigs died between 6 and
23 weeks.
Histological examination of survivors and animals that died during the
experiment revealed dramatic differences (representative
examples are
shown in Fig.
5, with cytological details
given in
Fig.
6). Lungs from placebo
control groups (PBS, MPL, DNA-vector)
as well as from the CFP-MPL group
exhibited extensive (>80%) granulomatous
pneumonia throughout the
vast majority of their tissue (Fig.
5A
to C), with dystrophic
calcification observed within the centers
of caseation (Fig.
6A).
Granulomas within the lungs of BCG and
surviving MPL-CFP-IL-2
vaccinated animals were smaller and more
compact, involving less than
50% of the lung tissue (Fig.
5D and
E), whereas granulomatous
pneumonia involving about 75% of the
lung parenchyma was observed in
animals vaccinated with the DNA-Ag85A
vaccine (Fig.
5F). Granulomas in
each of the surviving animals,
primarily those from the BCG,
MPL-CFP-IL-2, and DNA-Ag85A groups,
were highly lymphocytic (Fig.
6C
to F), whereas only scattered
lymphocytes were seen in groups of
animals dying of infection
(Fig.
6A and B). Significantly, even animals
from the DNA-Ag85A
group, with extensive granulomatous pneumonia,
lacked pulmonary
necrosis and caseation and had a high percentage of
lymphocytes
within the lesions (Fig.
5F and
6E and F).
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FIG. 5.
Representative photomicrographs of lungs from vaccinated
guinea pigs infected via the aerosol route with M. tuberculosis H37Rv at least 15 weeks previously. (A) PBS control
euthanized due to rapid weight loss after 15 weeks. Granulomatous
pneumonia replaces the vast majority of the parenchyma. (B)
MPL-CFP-vaccinated animal that died 18 weeks postinfection. The lesions
are similar to those seen in the PBS control (A). (C) DNA-vector
control animal that died 18 weeks postinfection. The lesions are
similar to those seen in the PBS control (A). (D) BCG-vaccinated animal
euthanized at 31 weeks postinfection (study termination). Smaller,
discrete granulomas affect approximately 40 to 50% of the pulmonary
parenchyma. (E) MPL-CFP-IL-2-vaccinated animal euthanized at 31 weeks
(study termination). The extent of the lesions is very similar to that
in the BCG-vaccinated animal (D). (F) DNA-Ag85A-vaccinated animal
euthanized at 31 weeks (study termination). The lesions are extensive,
involving 75 to 80% of the parenchyma. Hematoxylin and eosin stain;
magnifications, ×3.5.
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FIG. 6.
Higher-magnification photomicrographs of some of the
lesions depicted in Fig. 5, demonstrating cytological detail. (A) PBS
control (same animal as in Fig. 5A). Areas of fibrosis (arrow) surround
a zone of necrosis, characterized by cytolysis (indistinct cells and
cell margins) and karyolysis, with a central core of dystrophic
calcification (deeply basophilic deposits [arrowhead]).
Magnification, ×103.5. (B) Higher magnification of the specimen in
panel A. Note the vast predominance of epithelioid macrophages and a
multinucleated giant cell. Magnification, ×172.5. (C) BCG-vaccinated
animal (same animal as in Fig. 5D). Note the decreased fibrosis,
increased number of lymphocytes, lack of necrosis, and excellent
granuloma organization compared to PBS controls (A). Magnification,
×103.5. (D) Higher magnification of the specimen in panel C. A typical
section with abundant lymphocytes and numerous macrophages is shown.
Magnification, ×172.5. (E) DNA-Ag85A-vaccinated animal (same animal as
in Fig. 5F). Note the extensive granulomatous pneumonia, similar in
cytological makeup to that in the BCG-vaccinated animal (C) and the
MPL-CFP-IL-2-vaccinated animal (higher magnification not shown).
Magnification, ×121. (F) Higher magnification of the specimen in panel
E. A typical section similar to that for the BCG-vaccinated animal is
shown. Magnification, ×224. All panels stained with hematoxylin and
eosin.
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In general, there was good correlation between mortality of individual
animals and bacterial counts (Table
3). Given that
the bacterial load
30 days after aerosol inoculation was in the
5.5- to 6.0-log range,
some surviving animals showed evidence
of reduction (<5 log) whereas
mortality was invariably associated
with counts in the 7- to 8-log
range.
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DISCUSSION |
The results of this study provide the first evidence that two
nonliving vaccine formulations, one based on the CFP of M. tuberculosis and the other consisting of a DNA vaccine encoding a
major immunogenic antigen of CFP (Ag85; mycolyl transferase
[3]), can confer in the definitive guinea pig model
increased survival compared to appropriate controls and prevention of
caseating disease that gradually develops 10 to 20 weeks after aerosol
infection. Instead, infected animals immunized with both vaccines
developed lung disease similar to that seen in BCG-vaccinated control
animals, characterized by a lymphocytic form of granulomatous response.
Hence, while these new vaccines did not prevent mortality in a manner
comparable to that conferred by BCG, these results generate optimism
that a new generation of nonliving vaccines can be developed in the near future.
It was noticeable that with one exception other than BCG, bacterial
counts in the lungs of both animal models were not reduced below those
of placebo controls 30 days after aerosol challenge (the "gold
standard" in most studies published to date). However, at least two
vaccines showed evidence of causing prolonged survival compared to
controls. This may be providing an important lesson, namely, that
short-term reduction in bacterial counts may not, in fact, be the most
important criterion and that survival/pathology data in the guinea pig
model may in fact give a better picture of the long-term effectiveness
of a vaccine. We believe our results suggest that the survival data in
this model is influenced by the type of lesion produced. While lesion
severity is undoubtedly a critical component of survival, the
cytological character of the lesion within the DNA-Ag85A group,
particularly the degree of lymphocytic infiltration, may have a better
correlation with survival in this model.
This concept was best illustrated by the results obtained with
formulations containing IL-12. When IL-12 was given with IL-2, day 30 protection was seen in both models. However, 8 to 10 weeks after
aerosol challenge of guinea pigs, these animals all began to die. The
reason for this is not known, but our speculation at present is that
IL-12 enhances an initial gamma interferon response by T cells but in
the process drives these antigen-reactive cells into a short-lived
mode. As a result, these cells are absent when the infection begins to
progress or reactivate a few weeks later.
Much better survival and less severe lung disease (characterized by
increased lymphocyte infiltration) were seen if IL-2 alone was added to
the MPL-CFP vaccine. These data suggest the hypothesis, therefore, that
IL-2 was sufficient to drive a long-lived memory T-cell response to
this vaccine formulation. This is certainly an approach worthy of
further investigation, especially considering the almost identical lung
disease seen in these animals and BCG controls (Fig. 5D and E).
Similar disease, although with more lung consolidation, was seen in
animals given the DNA vaccine. In contrast to earlier studies in the
mouse in which some degree of protection was observed (11),
the DNA-Ag85A did not reduce bacterial numbers at day 30 in the guinea
pig; nevertheless, there was long-term lymphocytic granulomatous
disease similar to (but more extensive than in) the BCG controls.
Whether this observation reflects strong generation of memory immunity
or prolonged stimulation of the immune system through expression of
Ag85 antigen by host muscle cells is not currently known.
There was no apparent association between an increase in bacterial
numbers in the surviving groups and lung consolidation; if anything,
bacterial numbers were reduced. This implies, therefore, that it was
the continuing host granulomatous response to the infection, rather
than the infection per se, that was responsible for the progressive
infiltration of lung tissue and the eventual death of the animals, a
process which would probably eventually kill the BCG-vaccinated group.
This in turn implies, therefore, that dampening the inflammatory
response in the lungs (without reactivating the remaining bacteria)
should further lengthen survival times.
To the very limited extent to which they can be compared, the findings
of this study are similar to those of Horwitz et al. (9),
who showed that various components of CFP delivered in a different
(Syntex) adjuvant resulted in small reductions in bacterial load in the
lungs and protected these animals for 10 weeks after aerosol challenge.
An appropriate BCG control was not included in that study, but even so
we would argue that the experiments were curtailed long before any
pulmonary caseation had fully developed, hence preventing demonstration
of this central tenet of the guinea pig model.
Our different tactic, to use a mild adjuvant rather than a potent one
and then enhance it by the use of IL-2, not only was successful in the
guinea pig model but had the additional benefit of not inducing a skin
test reaction to PPD. As a result, this vaccine should not disable the
clinical diagnosis test. (We should emphasize, however, that these
animals still gave small reactions to purified CFP, indicating that
they are not actually anergic.)
Turning to practical matters, how could these new vaccines be used in
the clinical setting? The majority of the worldwide population has been
given BCG or is otherwise sensitized by exposure to environmental
mycobacteria. As we have discussed elsewhere (18), perhaps a
more realistic use of these new vaccines (given the fact that their
capacity to confer survival was less than that conferred by BCG) would
be to boost individuals already previously vaccinated or those who may
be at risk of reactivation disease due to latent tuberculosis or
drug-resistant tuberculosis that is refractory to treatment. To address
these questions, we are conducting experiments with guinea pigs
vaccinated in early life with BCG to see if boosting with the new
vaccines in midlife will prolong survival over that obtained with BCG
alone, as well as experiments with inbred strains of mice prone to
reactivation tuberculosis to see if this event can be prevented or
delayed.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grant AI-75320, by a grant from
the Colorado Institute for Biotechnology, and by generous contributions from Merck, Chiron Corp., and Ribi Immunochem.
We thank Elisa French for performing necropsies on the
long-term-infected guinea pigs and the staff at the CSU Laboratory of
Animal Resources for their excellent care and monitoring of the mice
and guinea pigs. We are very grateful to J. Terry Ulrich, and Marty
Giedlin for their invaluable advice and to Mike Jessen for technical
assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Mycobacteria
Research Laboratories, Department of Microbiology, Colorado State
University, Fort Collins, CO 80523. Phone: (970) 491-5777. Fax: (970)
491-5125. E-mail: iorme{at}lamar.colostate.edu.
Editor: R. N. Moore
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Abstract
Introduction
