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The Infectious Disease Research Institute, Seattle, WA 98104;
Medical School of Itajuba, Itajuba, Brazil;
Corixa Corporation, Seattle, WA 98104; and
Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15261
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 million people
worldwide every year (1). The currently available vaccine
against TB, bacillus Calmette-Guérin (BCG), is an attenuated
strain derived from Mycobacterium bovis that is highly
variable and has unpredictable efficacy (2, 3). Although
anti-TB combination chemotherapy exists, treatment is long,
expensive, and requires strict compliance to prevent the development of
multidrug-resistant forms of the causative agent, Mycobacterium
tuberculosis. In view of this, there has been a concerted effort
to develop a new and improved vaccine against TB.
Protective immunity to M. tuberculosis is incompletely
understood partly because M. tuberculosis is a facultative
intracellular pathogen that resides in mononuclear cells such as
macrophages. However, it is thought that a coordinated response of the
cellular immune system is fundamental. Consequently, both
CD4+ as well as CD8+ T
lymphocytes are believed to play important roles in immunity to TB, in
part because they secrete IFN-
. In addition, lysis of infected cells
and subsequent killing of M. tuberculosis by cytotoxic cells
including CD4+ T cells,
CD8+ CTL, and NK cells, may be important in
maintaining continuous immune surveillance during quiescent infection
(4, 5). Several studies of murine models of TB have
suggested that both CD4+ and
CD8+ T cells have a protective role in M.
tuberculosis infection (6, 7, 8, 9, 10). Indeed, adoptive
transfer studies of purified T cells have shown that protective immune
responses are induced by Ag-specific IFN--producing
CD4+ T cells, as well as CTL that secrete IFN-
and lyse infected macrophages (11, 12, 13). Therefore,
immunity to TB, and thus the development of a vaccine, relies on the
identification, formulation, and delivery of protective T cell Ags in a
manner that will generate prolonged memory responses of relevant
effector cells.
Thus far, protein subunit TB vaccine candidates have generally proven to have reduced efficacy when compared with BCG, possibly because the likelihood of the component Ags accessing the host cell cytosol and the MHC class I pathway is minor. Given that a critical aspect of protective immunity against TB appears to be presentation of Ags to the immune system via the exogenous and endogenous Ag-processing pathways for both MHC class II and class I recognition by CD4+ and CD8+ T cells, respectively, the identification and delivery of protective Ags in a manner that would stimulate both T cell subsets is optimal. Few examples exist of induction of CTL responses on immunization with Ags delivered in a protein format. However, the application of recombinant DNA technology to the control of M. tuberculosis and other pathogens (14, 15, 16, 17) appears to be a useful mechanism for generating not only CD4+ T cell immunity, but also CD8+ T cell immune responses.
Previous studies have focused on injecting bacterial plasmid DNA
expressing hsp-65, the 36-kDa proline-rich Ag, the 38-kDa
glycolipoprotein, hsp-70, Ag85B, Ag85A, MPT-63, MPT-83, and a
multivalent combination of ESAT-6, MPT-64, MPT-63, and KatG. Many of
these Ags have elicited partial protection when expressed from plasmid
DNA (14, 15, 17, 18, 19, 20, 21). Although Th2 responses with a
predominance of noncytotoxic/CD44low
IL-4-producing cells may be detected in mycobacterial infections,
strong Th1 responses with high levels of IFN- and
CD44high T cells with concomitant down-regulation
of CD45RB are observed during disease. Likewise, DNA vaccination is
associated with the elicitation of both CD8+ and
CD4+ cellular immunity,
CD44high type 1 cytokines such as IFN-, and
humoral responses that can persist for long periods of time (22, 23).
Several lines of evidence suggest that secreted or soluble Ags of
M. tuberculosis are likely targets of protective immune
responses (24, 25, 26, 27). In these experiments, the protective
immune responses were thought to be primarily
CD4+ T cell mediated. In our previous studies,
Ags present in M. tuberculosis culture filtrate (CF) were
identified and evaluated for their ability to stimulate PBMC from
purified protein derivative-positive (PPD+)
healthy donors because these presumably represent TB-protected
individuals (28, 29). Ags that elicited proliferation and
IFN- production from healthy donor PBMC then were tested for their
ability to stimulate protective immune responses in animal models. Mtb
8.4 is one such Ag.
Herein, we analyze the immunogenicity of recombinant protein and DNA vaccines carrying the gene encoding the Mtb 8.4 Ag of M. tuberculosis (28). In this study, we investigated the immune responses elicited when Mtb 8.4 was presented as either an exogenous (recombinant protein) or endogenous (plasmid DNA) Ag. Our studies demonstrate that immunization with Mtb 8.4 formulated either as a protein mixed with IFA or in a DNA format induces strong Th1 and CTL responses as well as protection against challenge with virulent M. tuberculosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (H-2b) mice aged 6–8 wk old were purchased from Charles River Laboratories (Portage, MI). Animals were maintained in pathogen-free conditions at the Fred Hutchinson Cancer Research Center (Seattle, WA) and were used for experiments beginning between 8 and 10 wk of age. Mice were transferred and maintained in a level 3 physical containment facility before infection with M. tuberculosis H37Rv.
Bacteria and infection
M. tuberculosis H37Rv (a gift from Dr. Sean Skerritt,
Seattle Veterans Affairs Hospital, Seattle, WA) was grown as described
previously (28), frozen in aliquots, and enumerated on
Middlebrook 7H11 agar (Difco, Detroit, MI). Cultures of M.
bovis BCG (Aventis Pasteur, Swiftwater, PA) were grown in 5%
CO2 at 37°C for 1 wk in sterile Middlebrook 7H9
supplemented with 10% ADC enrichment and 0.2% glycerol (Sigma, St.
Louis, MO). Aliquots were frozen at -80°C, and titers were
subsequently determined by complete Middlebrook 7H11 agar. Groups of
five female C57BL/6 mice were infected i.v. via the tail vein with
2 x 105 viable CFU of M.
tuberculosis H37Rv. For challenge experiments, spleens and lungs
were harvested at 3 wk after infection and homogenized in sterile
PBS-0.05% Tween. Serial dilutions of organ homogenates were plated on
Middlebrook 7H11 agar and incubated at 37°C until colonies were
visible 3 wk later. Protective efficacies in each experiment were
expressed as reduced CFU and were compared with the negative control
group (saline) by using the Mann-Whitney test. A p value of
<0.05 was considered statistically significant.
For infection of APCs (macrophages), frozen aliquots of M. tuberculosis H37Rv and M. bovis BCG were used to start cultures at a concentration of 2 x 106/ml in liquid medium (7H9 Middlebrook). Bacteria were grown in 5% CO2 at 37°C for 1 wk, washed in RPMI 1640 (Life Technologies, Grand Island, NY), and sonicated for 10 s in a sonicating water bath (Branson Ultrasonics, Danbury, CT) to disperse clumps. Bacteria were then resuspended in DMEM (Life Technologies) before being used to infect cells.
Expression and purification of rMtb 8.4
As previously reported, Mtb 8.4 (Rv1174C; CAA15851) has an open
reading frame of 330 nt coding for a protein of 110 aa with a consensus
signal peptide of 28 aa (residues 969-1052; Ref. 28). The
predicted molecular mass of the protein in its mature form without the
signal sequence is 8.4 kDa, hence the designation Mtb 8.4. The Mtb 8.4
gene bears a high homology to 2,4-dienoyl-CoA reductase though its
specific function in M. tuberculosis is unknown. As
described previously (28), Mtb 8.4 lacking its signal
peptide sequence was subcloned with six consecutive histidine residues
at its amino-terminal portion (N-terminal HIS-TAG) into the pET-17b
plasmid vector that has the T7 RNA polymerase expression system
(Novagen, Madison, WI). rMtb 8.4 was expressed in BL-21 (DE3) pLysE
cells by isopropyl-
-D-thiogalactoside induction
(Novagen). The purification of the rMtb 8.4 was performed by metal
chelate column chromatography with nickel-nitrilotriacetic acid resin
according to the manufacturer’s recommendations (Qiagen,
Chatsworth, CA).
Plasmids
The gene encoding the mature version of the Mtb 8.4 protein without its signal sequence was subcloned into the pcDNA3.1 expression vector (Invitrogen, San Diego, CA), which is under the control of a CMV promoter, to give the pcDXm8.4 construct. The mature Mtb 8.4 gene also was subcloned into the retroviral expression vector pBIB-X vector, which is under the control of the murine leukemia virus long terminal repeat promoter, to give the pBiXm8.4 construct. The Mtb 8.4 encoding sequence was obtained by PCR amplification with 5' oligonucleotides designed to make PCR products that begin 3' to the secretory sequence. The 3'-oligonucleotide included the stop codon. The 5' primers included sequence for HindIII restriction enzyme sites, and the 3' primers included NotI for directional subcloning into the pcDNA3.1 and pBIB-X expression vectors. The 5' primers also included a KOZAK consensus sequence (GCCGCCACC; Ref. 30) upstream of the initiation codon to ensure efficient translational initiation in both vectors.
For PCR, standard reactions were conducted in a Peltier Thermal Cycler (DNA Engine PTC-200; MJ Research, Watertown, MA) with Pfu polymerase (Stratagene, La Jolla, CA). The resulting PCR fragments were purified and ligated into appropriately digested plasmid vectors with T4 DNA ligase. Sequencing confirmed that there were no mutations generated by the PCR amplification and subcloning. Plasmid DNA for transfections and immunizations were generated using an endotoxin free DNA purification kit (Qiagen). DNA concentrations were determined by OD at 260 nm.
Generation of Mtb 8.4 CTL targets
To obtain cells stably expressing Mtb 8.4, a retroviral expression system was used. The transductant EXm8.4 was generated by transducing EL4, a chemically induced thymoma of C57BL/6 origin, with viral supernatants taken from a Phoenix-Ampho retroviral packaging line. To obtain viral supernatants for these transductions, the retroviral expression construct pBiXm8.4 was transfected into the Phoenix-Ampho line by using a modified version of the calcium phosphate precipitation protocol described elsewhere (31). To enhance transduction efficiency, transductions were performed in Retronectin (10 µg/ml; Takara Biomedicals, Shiga, Japan)-treated V-bottom tissue culture plates (Costar, Cambridge, MA). The viral supernatants generated with pBiXm8.4, as well as control viral supernatants (50 µl of each), were added to 50 µl/well of EL4 cells at 0.25 x 106/ml. Plates were centrifuged at 1200 rpm for 20 min and then placed at 32°C overnight. After 24 h the plates were incubated at 37°C for 96 h. Transduction efficiency was measured by flow cytometric analysis with EL4 cells transduced with pBIB-EGFP (enhanced green-fluorescent protein) viral supernatants as a positive control. All transductants were selected with blastocidin-S (Calbiochem, San Diego, CA) at a concentration of 10 µg/ml. Expression of Mtb 8.4 by transduced cells was confirmed by using lysates from transduced cells to stimulate Mtb 8.4-specific T cells (data not shown).
Immunization procedures
C57BL/6 mice were immunized three times with 50 µg of pcDXm8.4 1 mo apart in each tibialis muscle. Another group of C57BL/6 mice was immunized s.c. three times 2 wk apart with 15 µg of rMtb 8.4 formulated in IFA. Control animals were injected with saline, control plasmid (pcDNA3.1), IFA alone, or once s.c. with 5 x 104 CFU of viable M. bovis BCG (Aventis Pasteur) at the time of the first DNA or protein immunization. Serum was collected 2 wk after the last injection. Three weeks after the last DNA or protein injection, mice were challenged i.v. (2 x 105 CFU) with virulent M. tuberculosis H37Rv, or were sacrificed to conduct the analysis of immune responses including proliferation, cytokine production, and CTL induction.
ELISA for anti-Mtb 8.4 IgG
Serum samples were taken from all animals, and Ag-specific ELISAs were performed for the identification of specific anti-Mtb 8.4 IgG1 and IgG2a. Briefly, 96-well microtiter plates (Costar) were coated with 100 µl/well of rMtb 8.4 at 2 µg/ml in PBS for 4 h at 37°C. Plates were washed and blocked overnight at 4°C with 200 µl/well 10% FCS in PBS-Tween. Serum samples were diluted to 1:100 with PBS-Tween/10% FCS and applied to plates in 2-fold serial dilutions. Plates were incubated at 37°C for 4 h. Plates were washed, and HRP-conjugated goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates, Birmingham, AL) was added at a 1:2000 dilution for 2 h at 37°C. Plates were detected with tetramethylbenzidine substrate (3,3',5,5'-tetramethybenzidine; Kirkegaard and Perry, Gaithersburg, MD). The OD was determined at 450 nm with 570 nm as a reference wavelength.
Culture of spleen and lung cells
Proliferative and cytokine responses were measured by preparing
single-cell suspensions of spleens with Lympholyte-M density gradient
centrifugation (Cedar Lane Laboratories, Hornby, Ontario). Splenic
mononuclear cells were resuspended in complete medium (RPMI 1640
supplemented with 10% FCS, 50 µg/ml gentamicin, 2 mM
L-glutamine, and 5 x 10-5 M
2-ME), and plated at 2.5 x 106/well in
24-well flat-bottom plates (Costar) for cytokine assays and at 5
x 105/well in 96-well flat-bottom plates for
proliferation assays. The spleen mononuclear cells were cultured in the
presence of anti-IL-4R Ab (Immunex, Seattle, WA) at 1 µg/ml to
block the use of secreted IL-4 by activated T cells, thereby increasing
the accuracy of quantitating IL-4 without affecting proliferation or
IFN- production. The spleen cells were stimulated in vitro at 37°C
in 5% CO2 with rMtb 8.4 (10 µg/ml), CF (10
µg/ml), or medium alone. Anti-CD3 at 10 µg/ml was coated in some
wells 24 h before executing these assays to serve as an additional
control. Supernatants were taken after 72 h of culture and tested
by ELISA for the presence of IL-4 and IFN- as described previously
(28). Alternatively, after 5 days, the cultures were
pulsed with 1 µCi of [3H]thymidine for
18 h, harvested, and counted in a gas scintillation counter.
For analysis of Mtb 8.4-specific CTL, splenic mononuclear cells prepared as described above were cultured in 24-well plates at a density of 5 x 106 per well and stimulated with 2.5 x 105 per well of irradiated (10,000 rad) EXm8.4 transductants in complete medium supplemented with MEM nonessential amino acids and MEM-sodium pyruvate solution (Life Technologies). The cultures were restimulated once weekly for 2 wk by culturing 4 x 105 T cells per well with 2 x 105 stimulator cells, 4 x 106 irradiated syngeneic splenocytes, and 20 U/ml of rIL-2 (Genzyme, Cambridge, MA). After 3 wk of in vitro culture, the splenocyte population was characterized by flow cytometry with fluorescently labeled anti-mouse CD4 (L3T4; GK1.5) and anti-mouse CD8a (Ly-2; 53-6.7), both obtained from BD PharMingen (San Diego, CA).
Single-cell suspensions also were obtained from lungs of mice that were infected for various periods of time. RBC were lysed with NH4Cl-Tris solution, and cells were washed twice. Cells were plated in 96-well U-bottom plates (Corning, Corning, NY) in DMEM supplemented with 10% certified FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES (Life Technologies), 50 µM 2-ME (Sigma), 30 µg/ml gentamicin (Life Technologies), 15–20 U/ml murine rIL-2 (Boehringer Mannheim, Indianapolis, IN), and 1 mM aminoguanidine (Sigma) at 2 x 105 cells/well. Irradiated EXm8.4 cells were added at 6.5–7x103 cells/well. After 2–3 days of culture, 100 µl of media were removed from each well and replaced with fresh media containing IL-2. Cells were cultured for an additional 3–4 days and restimulated twice before FACS analysis and CTL assays.
Cytotoxicity assays
Lung and spleen lymphocytes harvested from two weekly
restimulation cultures were tested in 4- to 6-h
51Cr release assays. To prepare targets, EXm8.4,
EL4-EGFP, or parental cell line were harvested and labeled with 100
µl of Na51CrO4 (Amersham,
Arlington Heights, IL) in teflon jars (Savillex, Minnetonka, MN) for
1 h at 37°C. Cells were washed three times with DMEM and were
plated at 4x103 cells/well in 96-well U-bottom
plates for assays involving lung lymphocytes, and at 5 x
103/well in V-bottom plates for assays involving
spleen lymphocytes. The targets were allowed to settle for 20 min
before addition of T cells. For lung cytotoxicity assays, various E:T
ratios were cultured in a total volume of 100 µl of DMEM supplemented
with 10% certified FBS, 1 mM sodium pyruvate, 2 mM
L-glutamine, 25 mM HEPES, and 50 µM 2-ME. After 4 h,
85 µl of supernatant was removed from each well without disturbing
the cells and counted in a counter. For spleen lymphocyte
cytotoxicity assays, serial dilutions of 5 x
105 to 1.5 x 104 per
well of effector spleen T cells in a total volume of 200 µl were
added to the labeled target cells. After 6 h incubation at 37°C,
the supernatants were harvested with macrowell tube strips (Skatron,
Sterling, VA) and counted in a Cobra II counter (Packard, Downer’s
Grove, IL). Spontaneous release was determined by culturing target
cells in medium alone, and maximum release was determined by adding
0.1% Triton X-100 to target cells. Specific lysis =
[(experimental release - spontaneous release)/(maximum
release - spontaneous release)] x 100%.
Infection of macrophages
Macrophages were obtained from bone marrow precursor cells eluted from the femurs of C57BL/6 mice. Bone marrow cells were washed once in HBSS and plated at 2 x 105/ml in 6-well plates (Costar) in DMEM containing 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, and 10% L929 cell-conditioned medium as a source of macrophage CSF (complete medium) or 10 ng/ml rGM-CSF (Immunex). The medium was changed on days 2 and 4 of culture. On day 5 of culture, adherent cells (macrophages) were washed twice with cold PBS, harvested on ice, and infected with M. tuberculosis H37Rv or M. bovis BCG.
Macrophages were infected in a level 3 physical containment facility for 15–20 h at a multiplicity of infection of 2–4. At the end of infection, noninternalized bacteria were separated from macrophages by washing with warm HBSS. Macrophages were cultured in fresh medium for an additional 36–48 h before being used in T cell stimulation assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protective immunity against M. tuberculosis is believed
to be associated with the induction of Th1 cellular immunity. To
monitor Ag-specific immune responses primed by Mtb 8.4 immunization,
humoral and cellular responses were examined in mice immunized with Mtb
8.4. The Ab titers against Mtb 8.4 were determined in sera harvested
from immunized mice 3 wk after the third injection with DNA encoding
the mature version of Mtb 8.4, or with rMtb 8.4 formulated with IFA.
Serum from animals after three inoculations of the Mtb 8.4 plasmid
contained very low levels of Mtb 8.4-specific IgG1 and IgG2a (Fig. 1
). Anti-Mtb 8.4 IgG1 Abs could only be
detected at a 1:100 dilution in the pcDXm8.4-immunized mice (Fig. 1A). In contrast, immunization with rMtb 8.4 formulated in
IFA elicited Ag-specific IgG Abs that were elevated in both IgG1 (OD
2.70 ± 0.09 at a 1:100 dilution to 1.55 ± 0.06 at a 1:12,
800 dilution) and IgG2a (OD 1.20 ± 0.13 at a 1:100 dilution to
0.1 ± 0.04 at a 1:12, 800 dilution). Serum from animals receiving
BCG, saline, IFA, and the control pcDNA3.1 vector were essentially
negative for Mtb 8.4-specific IgG1 and IgG2a.
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is produced. This corresponds to the finding that
splenocytes and lymph node cells harvested from mice vaccinated with
pcDXm8.4 or rMtb 8.4 plus IFA proliferated when stimulated in vitro
with M. tuberculosis H37Rv CF (10 µg/ml) or rMtb 8.4 (10
µg/ml; Fig. 2A), and
produced IFN- (Fig. 2B). The elicitation of
IFN- occurred in a dose-dependent manner after in vitro stimulation
with rMtb 8.4 that was titrated from 10 µg/ml (19.9 ± 0.3
ng/ml) to 0.1 µg/ml (10 ± 0.1 ng/ml; data not shown). Although
the elicitation of IgG1 is associated with a Th2 phenotype in which
IL-4 might be secreted, splenocytes from mice immunized with rMtb 8.4
formulated in IFA produced almost undetectable levels of IL-4 when
stimulated in vitro with rMtb8.4 (Fig. 2 C).
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DNA immunization has been shown to be effective in inducing class
I-restricted CTL effector cells. Therefore, the induction of Mtb
8.4-specific CTL was examined in Mtb 8.4 DNA-immunized mice and
compared with the responses in mice immunized with rMtb 8.4 formulated
with IFA. Splenocytes from DNA-vaccinated and recombinant
protein-immunized mice were harvested, and CTL responses were assessed
after two in vitro stimulations with EXm8.4 stimulator cells. T cells
from mice receiving rMtb 8.4 formulated with IFA and Mtb 8.4 DNA
demonstrated the ability to specifically lyse Mtb 8.4-expressing EL4
cells after only one round of in vitro stimulation (data not shown).
After a second round of in vitro stimulation, the specific lysis of
EXm8.4 at an E:T ratio of 100:1 increased from 43 ± 3 to 76
± 8% for pcDXm8.4-vaccinated mice; at an E:T ratio of 3:1, the
specific lysis of EXm8.4 was 16 ± 10% (Fig. 3). The splenocytes isolated from mice
injected with rMtb 8.4 formulated in IFA also demonstrated strong
specific CTL activity (49 ± 7% at an E:T ratio of 100:1) after
in vitro stimulation with EXm8.4 transductants (Fig. 3). Thus, our data
demonstrates that T cells isolated from mice immunized with either rMtb
8.4 protein formulated with IFA or pcDXm8.4 have the ability to lyse
homologous transfected target cells. The lysis of untransduced EL4
cells by splenocytes isolated from these vaccinated mice varied from
10% to 0 (pcDXm8.4) and 11% to 0 (rMtb 8.4 + IFA). This observed
background lysis with EL4 cells was subtracted from the CTL results
shown in Fig. 3. T cells from these mice also were unable to lyse EL4
transduced with EGFP and other mycobacterial genes (data not shown). In
addition, T cells from mice immunized with control plasmids did not
lyse EXm8.4 (Fig. 3). By flow cytometric analysis, the phenotype of the
Mtb 8.4 CTL lines generated from both Mtb 8.4-DNA or rMtb 8.4
protein-immunized mice was found to be >99%
CD8+ (data not shown).
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C57BL/6 mice are known to control infection with M.
tuberculosis or M. bovis BCG and are relatively
resistant to TB. As such, they provide a good model for defining the
necessary immune responses and components associated with those exposed
to the bacillus but who do not develop disease. To determine whether
mice infected with M. tuberculosis or M. bovis
BCG could mount a CTL response to Mtb 8.4, spleen or lung cells were
removed from these groups of mice after 4 wk and restimulated in vitro
for two weekly cycles with the EL4 cells expressing Mtb 8.4. These
studies clearly demonstrate that Mtb 8.4-specific CTL activity could be
measured in spleen (53 ± 8% at an E:T ratio of 50:1; Fig. 4A) or lung (23 ± 2% at
an E:T ratio of 50:1; Fig. 4B) T cell cultures derived from
mice previously infected i.v. with M. tuberculosis (2
x 105 CFU). Mtb 8.4-specific CTL activity also
could be measured in mice receiving 2 x 105
CFU M. bovis BCG i.v. (31 ± 2% at an E:T ratio of
50:1) or 5 x 104 CFU M. bovis
BCG s.c. (22 ± 4% at an E:T ratio of 50:1; Fig. 4C).
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production by Mtb 8.4-specific CTL after stimulation with
M. tuberculosis-infected macrophages
To examine whether Mtb 8.4 CTL might contribute to protective
immunity against M. tuberculosis by IFN- production, the
elicitation of IFN- from Mtb 8.4 CTL stimulated with M.
tuberculosis-infected macrophages was measured. Mtb 8.4-specific
CTL lines derived from mice that were infected with M.
tuberculosis for 6 wk or from mice that were immunized with Mtb
8.4 DNA were stimulated for 72 h with bone marrow-derived
macrophages that were uninfected or infected with either live M.
bovis BCG or M. tuberculosis H37Rv for 36–48 h. High
levels of IFN- were produced by both Mtb 8.4 CTL lines on in vitro
stimulation with M. tuberculosis-infected macrophages (Fig. 5). The elicitation of IFN- was
stronger in the CTL isolated from Mtb 8.4 DNA-immunized mice (37221
pg/ml; Fig. 5B) than from the mice previously infected with
M. tuberculosis (11354 pg/ml; Fig. 5A). On
stimulation with M. bovis BCG-infected macrophages, lower
levels of IFN- were elicited from the CTL isolated from the mice
previously infected with M. tuberculosis (1205 pg/ml).
IFN- was not elicited from Mtb 8.4 CTL when stimulated with
uninfected APCs in vitro.
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To examine whether immunization with Mtb 8.4 DNA or rMtb 8.4
formulated with IFA would confer protection against challenge with
M. tuberculosis, groups of mice were challenged by i.v.
administration of virulent M. tuberculosis H37Rv 3 wk after
their second vaccination boost. Four weeks after challenge with
M. tuberculosis, spleens and lungs were harvested from these
mice and the bacterial loads were quantified by plating serial 5-fold
dilutions of organ homogenates onto 7H11 agar plates. The mean splenic
and lung CFU values are shown in Fig. 6.
Viable bacterial counts in the spleens and lungs of mice receiving DNA
encoding mature Mtb 8.4 (pcDXm8.4) or rMtb 8.4 formulated with IFA were
reduced postinfection when compared with mice injected with saline or
control plasmid DNA (p < 0.05). The levels of
protection induced by the Mtb 8.4 DNA vaccine measured as a reduction
in viable bacilli were comparable to that induced by BCG vaccination
(1.0 and 1.2 log CFU, in the spleen and lung, respectively; Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our initial strategy for identifying candidate Ags for development of a
subunit vaccine for TB involved screening for Ags that would stimulate
T cells from human PPD+ healthy donors that are
presumably controlling their M. tuberculosis infection. With
this approach, we previously identified and characterized an Ag in
M. tuberculosis CF, Mtb 8.4 (28), and showed
that it elicited from human and murine cells Th 1-type cellular
responses that are considered to be essential for inducing protective
immune responses against M. tuberculosis (34, 35). In the current study, we have extended our observations to
show that immunization with rMtb 8.4 formulated with IFA elicited
strong humoral responses of a mixed IgG isotype (IgG1 and IgG2a),
whereas, i.m. immunization of mice with plasmid DNA encoding the
mycobacterial Mtb 8.4 Ag elicited little, if any, IgG Abs. In addition,
our studies demonstrate that immunization with either rMtb 8.4 in IFA
or Mtb 8.4 DNA evoked strong Mtb 8.4-specific Type 1 T cell immune
responses characterized by IFN- secretion and CTL activity.
Moreover, vaccination with either rMtb 8.4 formulated with IFA or Mtb
8.4 DNA decreased the bacterial load or induced a degree of protection
on challenge with virulent M. tuberculosis. Thus, these data
clearly illustrate that Ab production to Mtb 8.4 is not essential for
protective immunity against M. tuberculosis in this instance
because mice receiving Mtb 8.4 DNA had reduced mycobacterial loads when
compared with saline and vector alone controls.
Mtb 8.4-specific CTL were detected not only from Mtb 8.4 immunized mice but also from cells isolated from both the spleens and lungs of mice infected with M. tuberculosis. This is an important finding given that other studies have reported that mice infected with M. tuberculosis do not mount CTL responses to other mycobacterial candidate vaccine Ags such as the Ag85A (14). Thus, although Ag85A DNA induces a CTL response in mice, these CTL are unable to recognize M. tuberculosis-infected cells and the protective effect of Ag85A DNA appears to be independent of CD8+ T cells (36). As far as we are aware, CTL in mice infected with M. tuberculosis have been found to few other mycobacterial candidate vaccine Ags, including the 38-kDa Ag and hsp-65 (22, 32, 37). Mtb 8.4-specific cytolytic activity also was observed with T cells isolated from mice that were infected i.v. or intradermally with M. bovis BCG. Thus, our studies demonstrate that in vivo, Mtb 8.4 CTL can be primed and activated during infection with either M. tuberculosis or M. bovis BCG. Given these results, it will be of interest to test Mtb 8.4 in prime-boost strategies with Mtb 8.4 DNA or protein as the prime with M. bovis BCG or a recombinant virus as a live vector boost. This approach has been shown to elicit exceptionally potent CTL responses (38).
Another important finding of this study was the elicitation of IFN-
from Mtb 8.4 CTL when stimulated with macrophages infected with
M. tuberculosis. In murine models, the production of IFN-
is essential to activate macrophages to kill or limit the replication
of M. tuberculosis organisms. Given that Mtb 8.4 CTL are
primed for IFN- production when stimulated with M.
tuberculosis-infected APCs vs noninfected APCs suggests that Mtb
8.4 is a relevant Ag that is processed and presented in the context of
class I MHC by infected APCs during M. tuberculosis
infection. This finding also argues against the hypothesis that our
findings are a consequence of cross-priming of CTL to Ags not expressed
by M. tuberculosis-infected macrophages or of priming via
apoptotic cell fragments in vivo. The reason that Mtb 8.4-specific CTL
strongly recognized M. tuberculosis-infected macrophages but
only showed weak recognition of BCG-infected macrophages is not
immediately apparent. However, it has been suggested that BCG is
inefficient at delivery of Ags into the MHC class I processing pathway
compared with M. tuberculosis (39, 40). Indeed,
studies in 2-microglobulin-deficient mice have
shown that although these mice are quite susceptible to M.
tuberculosis infection, they are capable of controlling infection
with BCG. In line with these data, we found that infection with BCG was
found to be less efficient at priming a Mtb 8.4-specific CTL response
in mice than infection with M. tuberculosis (Fig. 4
).
M. tuberculosis bacilli reside primarily within phagosomes that are normally inaccessible to the MHC class I Ag-processing pathway. At present, the mechanism by which mycobacterial Ags such as Mtb 8.4 might gain access to the cytosol, and thus the MHC class Ia pathway for CD8+ T cell activation, is not understood. Nonetheless, recent studies have proposed different mechanisms by which mycobacterial proteins and exogenous Ags might gain access to MHC class I molecules, including bacilli escape from the phagosome to the cytosol, macropinocytosis, the involvement of pore-forming molecules such as a hemolysin which may permit phagosome to cytosol traffic of mycobacterial Ags, and the formulation of "exogenous Ags" with complex detergent-type adjuvants (14, 17, 40, 41, 42, 43, 44, 45).
Previous studies with DNA vaccines encoding mycobacterial Ags such as MPT-63, MPT-83, or ESAT-6 have demonstrated reduced M. tuberculosis bacterial lung burdens or elicited relative protective responses of 0.41–0.61 when compared with control groups of mice (21, 46). Our studies have shown that vaccines containing Mtb 8.4 have the ability to elicit levels of protection that have only been reported previously with a vaccine containing a combination of four M. tuberculosis DNA constructs (21). Moreover, the levels of protection induced by both rMtb 8.4 and Mtb 8.4 DNA, measured as a reduction in bacilli counts, were comparable to that induced by BCG vaccination (1.0–1.2 log).
DNA vaccination has many inherent attractions as a mode of vaccination: intrinsic adjuvanticity, stability, extreme versatility for different Ags, diverse epitopes, targeting different Ag presentation pathways, ease of production that avoids the problem of denaturing proteins during purification, and DNA-encoded cytokines can easily be incorporated to enhance protective responses. Given that degrees of protective immunity and prevention of caseating disease have been demonstrated with various components of M. tuberculosis (25, 47) and with DNA vaccination (17, 32) the view that only a live attenuated organism such as BCG could confer immunity to TB can be repudiated. The prospect of clinically evaluating Mtb 8.4 DNA vaccination against M. tuberculosis infection is also feasible given that safety and Ag-specific immunogenicity have been demonstrated by using this mode of vaccination in HIV-infected (48) and Plasmodium species-infected individuals. DNA vaccines have many advantages, but disadvantages have surfaced when DNA vaccination has been extrapolated to larger animals. Thus, it may prove easier to vaccinate against M. tuberculosis with a vaccine format comprising protein Ags with potent adjuvants. Therefore, it is significant that rMtb 8.4 formulated with IFA elicited not only CTL responses, but also protective immunity. Previously reported vaccine studies have not noted IFA as a potent adjuvant for CTL induction. Thus, it is more likely that our results might be better explained by the physicochemical properties of Mtb 8.4, which are currently being investigated.
At this point, it is not possible to definitively state which are the best Ags to include in a subunit vaccine against TB. The number of potential Ags is large (25, 29, 47, 49, 50, 51), and more continue to be described after the sequencing of the M. tuberculosis genome. However, candidate Ags are those that are released or processed by the intracellular mycobacteria, contain many promiscuous epitopes that are presented on MHC class I and MHC class II, and elicit strong Ag-specific type 1 immune responses. Mtb 8.4 is an Ag that meets many of these criteria. The generation of Mtb 8.4-specific CD4+ and CD8+ T cell responses through immunization is important for vaccine development against TB, as various studies have demonstrated the importance of these lymphocytes in protective immunity against M. tuberculosis (9, 52, 53, 54). Previous studies have shown that Mtb 8.4-reactive CD4+ T cells are present in healthy PPD+ donors (28), and we are currently assessing whether Mtb 8.4-specific CD8+ CTL also are present in healthy PPD+ individuals protected from active disease with M. tuberculosis. In addition to testing the longevity of Mtb 8.4-induced protective immunity in C57BL/6 mice, Mtb 8.4 vaccines also are being tested in other strains of mice and the more vigorous guinea pig aerosol TB challenge model.
Most probably the level of Th1-type immune responses with IFN-
production, CTL activity, and protective immunity could be improved if
rMtb 8.4 were formulated with a more potent adjuvant than IFA, and
importantly, an adjuvant that is suitable for use in humans. In
addition, the level of protection provided against M.
tuberculosis infection as a result of Mtb 8.4 immunization may be
enhanced through the inclusion in a subunit vaccine, of other
mycobacterial Ags that also stimulate strong Th 1-type responses with
relevant cytokine production, as well as by optimization of the
delivery of the vaccine in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Drs. Rhea N. Coler and Antonio Campos-Neto, Infectious Disease Research Institute, 1124 Columbia Street, Suite 600, Seattle, Washington 98104.
3 Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Guérin; CF, culture filtrate; PPD, purified protein derivative; EGFP, enhanced green-fluorescent protein.
Received for publication October 31, 2000. Accepted for publication March 2, 2001.
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