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*
Department of Immunology, University of Strathclyde, Glasgow, United Kingdom; and
Division of Infection and Immunity and
Wellcome Unit of Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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cpa),
cpb (cpb), or both cpa
and cpb (cpa/cpb) have been generated
by targeted gene disruption. cpa mutants produce a
disease phenotype in BALB/c mice close to that of wild-type L.
mexicana, but cpb mutants are much less
infective, producing very slowly growing small lesions, and
cpa/cpb double mutants do not induce lesion growth.
Immunologic analysis of Ab isotype during infection and splenocyte
IFN-
, IL-2, and IL-4 production following stimulation with
Leishmania Ag or Con A indicates that there was a
significant shift from a predominantly Th2-associated immune response
in mice infected with wild-type L. mexicana to a
Th1-associated response in mice inoculated with cpb
or cpa/cpb. Significantly, cpa
altered the balance of the immunologic response to a lesser extent than
did the other mutants. Similar disease outcomes and switches in the
Th1/Th2 balance were also observed when other L.
mexicana-susceptible mouse strains were infected with the
mutants. BALB/c and C57BL/6 mice vaccinated with
cpa/cpb and CBA/Ca mice vaccinated with
cpb or cpa/cpb were subsequently
more resistant, to varying degrees, than were untreated mice to
infection with wild-type parasites, as measured by development of
lesions and parasite burden. These data implicate leishmanial cysteine
proteinases not only as parasite virulence factors but also in
modulation of the immune response and provide strong encouragement that
cysteine proteinase-deficient L. mexicana mutants are
candidate attenuated live vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which mediates
protection by up-regulating macrophage inducible nitric oxide synthase
expression and nitric oxide production, which is microbicidal for the
parasites (4). Consequently, neutralization of IL-12 or IFN- or
inhibition of nitric oxide production results in disease exacerbation
(2, 5, 6, 7, 8, 9). The immunologic pathways leading to the development of
nonhealing progressive disease are less well characterized and more
contentious. Thus, although a large number of studies have indicated
that susceptibility to L. major (4, 10, 11), L.
mexicana, (12) and L. amazonensis (13) is related to a
developing Th2 response and IL-4 production with down-regulation of
Th1-associated activities, further studies on several
Leishmania species suggest that the inability to mount a Th1
response rather than the presence of a Th2 response may determine
susceptibility (13, 14, 15). Nevertheless, studies using IL-4-deficient
mice from a number of genetic backgrounds have demonstrated an absolute
requirement for this cytokine in determining susceptibility to L.
mexicana, as in the absence of IL-4 lesions develop at the site of
cutaneous infection (12). As most mouse strains develop nonhealing
lesions when infected with L. mexicana (16), this parasite
must be particularly adept at subverting a protective immune response. Of particular interest with regard to the factors that determine the outcome of L. mexicana infection is the idea that cysteine proteinases (CPs)3 might be potentiating Th2 responses by enhancing IL-4 production (17). L. mexicana contains multiple, highly active CPs, many of which are stage regulated (18). Three L. mexicana CP genes are known. These are cpa, a single-copy gene encoding a nonabundant cathepsin L-like CP (19); cpb, a multicopy gene that encodes the major cathepsin L-like CPs of the intracellular mammalian amastigote form of the parasite (20); and cpc, a single-copy gene encoding a cathepsin B-like CP (21). Mutants lacking each of these genes have been generated by targeted gene disruption (21, 22, 23). cpa-deficient mutants were found to resemble wild-type L. mexicana in both in vitro and in vivo phenotypic tests (22). The cpb-deficient mutants proved to be more interesting. It was discovered that although cpb is not essential for growth or differentiation of the parasite in vitro, the null mutant was 5–10-fold less infective to explanted mouse macrophages, indicating that the enzyme is a virulence factor (23). The data suggest that cpb-deficient mutants can only survive in a subpopulation of macrophages, although the parasites that successfully infect these macrophages grow normally. The cpb-deficient mutants were found to infect BALB/c mice but produced only very small, slowly growing lesions (23). Mutants lacking both the cpa and cpb genes were also created, and these were found to have a phenotype similar to that of the cpb-deficient mutant in terms of their ability to infect macrophages in vitro (23), although the infectivity for mice of the cpa/cpb-deficient double mutant had yet to be studied.
These results demonstrate that L. mexicana CPs play an important part in sustaining an infection in the mammalian host. One clear possibility is that they modulate the host immune response to the parasite. The current study was therefore undertaken to determine whether the absence of CPs in the mutant parasites results in a shift in the Th2/Th1 balance and whether such a shift results in protective immunity. If so, such CP-deficient parasites would clearly be vaccine candidates of high potential.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Promastigotes of L. mexicana (MNYC/BZ/62/M379) were grown in HOMEM medium, pH 7.5, containing 10% (v/v) heat-inactivated FCS at 25°C as described elsewhere (19). The following antibiotics were added in combination, as appropriate, for maintenance of drug-selectable markers in the CP-deficient mutants: phleomycin (Cayla, Toulouse, France) at 10 µg/ml, nourseothricin hydrosulfate (Hans-Knoll Institute, Thuringen, Germany) at 25 µg/ml, puromycin (Sigma, Poole, U.K.) at 10 µg/ml, and hygromycin (Boehringer Mannheim, Mannheim, Germany) at 50 µg/ml.
Mice
BALB/c, C57BL/6, CBA/Ca, 129Sv/Ev, and 129Sv/Ev IFN-
receptor-deficient and C57BL/6 recombinant activating gene-deficient
(RAG2-/-) mice were bred and maintained at the Universities of
Glasgow and Strathclyde. In the preliminary study, groups comprised 4
mice each. In later comparative and vaccine studies, however, groups of
20 female, 8–10-wk-old mice were infected s.c. in the shaven rump with
5 x 106 stationary-phase promastigotes of wild-type
or CP-deficient L. mexicana. In each experiment, groups of
normally 5 but no fewer than 4 mice were used from each group per
sample point for immunologic analysis; the data presented are
means ± SEM from the groups of animals. Each experiment was
repeated twice (C57BL/6) or more (BALB/c and CBA/Ca). Lesion size was
measured using a slide gauge micrometer. In the preliminary experiments
undertaken at the University of Glasgow, lesion volume was measured
(23). Thereafter, at the University of Strathclyde, lesion diameters
were measured, and whole parasite burdens from excised disrupted
lesions were assessed using a Neubauer hemocytometer (24).
Detection of Leishmania-specific Abs by ELISA
Peripheral blood was obtained from infected animals by tail bleeding into heparinized capillary tubes. All plasma samples were stored at -20°C before analysis for specific Ab content. Leishmania-specific IgG1 and IgG2a end-point titers were measured by ELISA as previously described (12). Briefly, each well of an Immulon-1 microtiter plate (Dynatech Laboratories, Billingshurst, U.K.) was coated with 1 µg of leishmanial lysate Ag (freeze/thawed wild-type promastigotes in PBS, pH 9.0) by overnight incubation at 4°C. Following incubation of serial dilutions of plasma samples for 1 h at 37°C, bound Abs were detected by incubation with either rat anti-mouse IgG1 horseradish peroxidase conjugate or rat anti-mouse IgG2a horseradish peroxidase conjugate (Southern Biotechnology Associates, Birmingham, AL). Binding of conjugate was visualized with tetramethylbenzidine (0.06 mg/ml) in 0.1 M sodium acetate buffer, pH 5.5, containing 0.03% H2O2. The color reaction was stopped by adding 10% (v/v) sulfuric acid, and the absorbance was measured at 450 nm. Results are expressed as end-point dilutions, for which the end point is defined as the final plasma concentration that yielded an absorbance higher than a negative control plasma sample included in the assay. Comparisons between groups of mice were made with a Mann-Whitney U test.
Splenocyte responses
Spleens were aseptically removed at appropriate times postinfection, as detailed for individual experiments, and cell suspensions were prepared by gently teasing apart the tissue in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.05 mM ß-mercaptoethanol, and 10% (v/v) heat-inactivated FCS (Life Technologies, Paisley, U.K.). Following centrifugation at 200 x g for 10 min at 4°C, cells were resuspended in 3 ml of Boyle’s solution (0.17 M Tris-HCl, pH 7.2, and 0.16 M ammonium chloride) at 37°C for 3 min to deplete red blood cells. Spleen cell suspensions were then centrifuged at 200 x g for 10 min at 4°C, resuspended, washed, and resuspended in 2 ml of complete RPMI 1640 (as above). Viable cells were enumerated by trypan blue exclusion, and the suspensions were adjusted to 5 x 106 cells/ml. Aliquots of the cell suspension (100 µl) were added to 96-well, flat-bottom tissue culture plates (Costar, Cambridge, MA), and 100-µl aliquots of Con A (5 µg/ml) or L. mexicana lysate Ag (5, 10 or 25 µg protein/ml) were added as appropriate. Cultures were then incubated in 5% CO2/95% air for 60 h at 37°C, whereupon cultures were pulsed with 0.25 Ci of [3H]thymidine (sp. act. 2 Ci/mmol) and incubated for a further 12 h. Supernatants were collected from parallel cultures at this time for quantification of cytokine production (see below). Pulsed cells were then harvested onto filter paper using a cell harvester (Skatron, Lier, Norway), and thymidine uptake was determined by liquid scintillation on a beta counter (Pharmacia LKB Biotech, Milton Keynes, U.K.).
IFN-, IL-2, and IL-4 assays
IFN-, IL-2, and IL-4 production by stimulated cells
(stimulated by Leishmania Ag or Con A) and nonstimulated
cells from mice infected with wild-type or CP-deficient parasites were
measured by capture ELISA. Briefly, the wells of Immunol-1 microtiter
plates (Dynatech Laboratories) were coated with capture Ab at 2.0
µg/ml (IFN-, R4-6A2; IL-2, JES6-1A 1.2 (PharMingen, San
Diego, CA); IL-4, 11B11 (Genzyme, Cambridge, U.K.)) in PBS (pH
9.0) or carbonate buffer (0.05 M, pH 9.5) by overnight incubation at
4°C. Wells were then washed three times with PBS, pH 7.4/0.05%
Tween-20 and blocked by incubation with 10% (v/v) FCS for 1 h at
37°C. The culture supernatants and appropriate recombinant standards
(rIFN- and rIL-2, PharMingen; rIL-4, Genzyme) were then added to
individual wells. For standard curves, rIFN- (0–10 ng/ml), IL-2
(0–1350 pg/ml), and rIL-4 (0–1000 pg/ml) were used. Following
incubations at 37°C for 2 h, the wells were washed three times
with PBS, pH 7.4/0.05% Tween-20, and then biotinylated rat
anti-mouse IFN- (XMG1.2, PharMingen; 1 µg/ml),
biotinylated rat anti-mouse IL-2 (JES6–5H4, PharMingen;
1 µg/ml), or biotinylated goat polyclonal anti-IL-4 (Genzyme; 1
µg/ml) was added and incubated for 1 h at 37°C. For the
detection of bound biotinylated rat Ab, 100 µl of
streptavidin-alkaline phosphatase conjugate (diluted 1/1000,
PharMingen) was added to each well for 45 min at 37°C, and following
further washing, binding was visualized with substrate consisting of
p-nitrophenyl phosphate (1 mg/ml, Sigma) in glycine buffer
(0.1 M, pH 10.4). The absorbance was subsequently measured at 405 nm on
a Titertek Multiscan Plate Reader. For detection of bound biotinylated
goat Ab, 100 µl of streptavidin-horseradish peroxidase conjugate
(diluted 1/500, Genzyme) was added to each well for 30 min at 37°C
and following further washing was incubated with tetramethylbenzidine
as described above. Cytokine concentrations in the cell cultures were
determined from the standard curve (regression coefficient,
r = 0.990 or better). All assays were conducted in
triplicate. Comparisons between groups were made using the Student’s
t test. P values of <0.05 were considered
significant.
Vaccination studies
Two mouse strains (BALB/c and CBA/Ca) that develop nonhealing
lesions when infected with L. mexicana and have been used
previously for vaccine studies were examined (25), as well as C57BL/6
mice, which normally develop nonhealing lesions when infected with this
parasite. The protocols for each mouse strain were modified to reflect
their response to CP mutants. At 2 and 4 mo after vaccination by
inoculation s.c. in the flank with 5 x 106
stationary-phase promastigotes of cpa/cpb, BALB/c mice
were infected s.c. in the shaven rump with 5 x 106
wild-type parasites, and disease progression was compared with that of
nonvaccinated mice. CBA/Ca and C57BL/6 mice were vaccinated with
cpb or cpa/cpb (s.c. inoculation of 5
x 106 stationary-phase promastigotes) and challenged 8 wk
later with 106 stationary-phase promastigotes of wild-type
L. mexicana. Lesion growth was monitored up to 20 wk and
compared with that of nonvaccinated control animals.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice inoculated s.c. with 5 x 106
stationary-phase promastigotes of wild-type L. mexicana
developed rapidly growing, nonhealing lesions (Fig. 1
). While lesion growth following s.c.
infection with the cpa-deficient mutants (cpa)
was slower than that recorded with wild-type infections, lesion growth
resulting from infection with cpb-deficient mutants
(cpb) was considerably slower, and only very small
lesions were formed (Fig. 1). In contrast, no lesions were detected
even 9 mo postinfection in any mice inoculated with
cpa/cpb-deficient double mutants (cpa/cpb).
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Plasma levels of Leishmania-specific IgG1 and IgG2a
were determined 6 mo postinfection (Fig. 2). Animals infected with wild-type
L. mexicana had pronounced Leishmania-specific Ab
levels, primarily of the IgG1 subclass. Mice infected with
cpa also had large Ab titers, primarily of the IgG1
subclass but with significantly higher IgG2a titers than those
following infection with wild-type L. mexicana
(p < 0.01). Mice infected with
cpb had significantly less IgG1 Ab than animals infected
with wild-type L. mexicana (p <
0.01), and there was a distinct and significant increase in the
IgG2a/IgG1 ratio. Mice infected with cpa/cpb had very
little detectable Leishmania-specific Ab.
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Both wild-type and CP-deficient mice were able to mount
Ag-specific and Con A-induced proliferative responses at all time
points examined (2, 3, 4, 6, and 9 mo) in all experiments. From 2 mo
onward the Ag-specific and from 6 mo onward the Con A-induced
proliferative responses were significantly greater in mice inoculated
with cpa/cpb (p < 0.05 for Con A
and Ag) and mice inoculated with cpb
(p < 0.05 for Con A and Ag) than in those mice
inoculated with wild-type parasites (Fig. 3).
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production by stimulated splenocytes from infected mice
IFN- production from splenocytes isolated from BALB/c mice 9 mo
postinfection and stimulated with parasite Ag (5 µg protein/ml) or
Con A are shown in Fig. 4, a
and b. Similar results were found for different amounts of
Ag used and at earlier time points postinfection (not shown). Ag
stimulation resulted in significantly increased IFN- production in
comparison with background levels (p < 0.05)
by all splenocyte cultures (Fig. 4a). However, production
was significantly greater by Ag-stimulated splenocytes from animals
infected with CP-deficient mutants than with wild-type L.
mexicana (cpa and cpb,
p < 0.05; cpa/cpb, p <
0.005). Moreover, Ag-induced IFN- production was significantly
greater from splenocytes isolated from mice infected with
cpb (p < 0.05) and
cpa/cpb (p < 0.01) than from
splenocytes isolated from cpa-infected mice. Con A
stimulation increased IFN- production to significantly greater
levels than background in all splenocyte cultures. Under these
conditions, splenocytes derived from mice infected with
cpa (p < 0.05) and
cpb (p < 0.02) but not
cpa/cpb produced significantly more IFN- than
splenocytes derived from mice with wild-type infections (Fig. 4b).
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In follow-up studies, we concentrated on comparing primarily the
developing immune response in mice inoculated with
cpa/cpb and wild-type parasites. In addition to
confirming differences in splenocyte IFN- production, we measured
IL-2, IL-4, IL-5, IL-10, and IL-12 production. While no differences in
splenocyte IL-5, IL-10, and IL-12 production were observed between mice
infected with cpa/cpb and those infected with wild-type
parasite, profound differences in IL-2 and IL-4 production were
observed, depending on the infectious agent, at 2 and particularly 4
and 6 mo postinfection (Fig. 5,
a and b). While splenocytes from animals infected
with wild-type parasites failed to produce a significant Ag-induced
increase in IL-2 production, those infected with cpa/cpb
produced, following stimulation with Ag, IL-2 significantly over
background (p < 0.01). Ag-stimulated
splenocyte IL-4 production was, however, significant over background
for both animals infected with cpa/cpb
(p < 0.001) and those infected with wild-type
parasites (p < 0.001). However, the increase
in splenocyte IL-4 production was significantly greater in wild-type
parasite-infected animals than in animals inoculated with
cpa/cpb (p < 0.001).
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cpa/cpb parasites failed to induce lesion growth in
C57BL/6, CBA/Ca, 129Sv/Ev, IFNR-/- 129SvEv, and RAG2-/- C57BL/6
mice up to 6 mo postinfection (results not shown). Small lesions were
induced in C57BL/6 and RAG2-/- mice, but not in any of the other
strains used, by inoculation with cpb (results not
shown). All animals infected with wild-type parasites developed large,
nonhealing lesions. The immunologic responses generated by wild-type
parasites or CP-deficient mutants in the wild-type mouse strains were
examined and were similar to those observed in BALB/c mice. In C57BL/6
mice, Ag-induced splenocyte cytokine production in animals inoculated 6
mo previously with cpa/cpb consisted entirely of IFN-
(p < 0.01) and IL-2 (p
< 0.05), with virtually no IL-4 produced above background levels (Fig. 6, a through c).
However, Ag-stimulated splenocytes from animals inoculated with
wild-type parasites produced not only significant levels of IFN-
(p < 0.05) but also IL-4
(p < 0.01), with little or no IL-2 above
background levels. Although IFN- levels following Ag stimulation
were similar in wild-type and cpa/cpb infected
mice groups, the increase in splenocyte IFN- production in
cpa/cpb-inoculated animals over background (5.8 ±
1.5 and 0.11 ± 0.05 pg/ml, respectively) was 10-fold greater than
that produced over background by splenocytes from wild-type
parasite-infected mice (6.0 ± 3.2 and 0.99 ± 0.43 pg/ml,
respectively). Con A-induced splenocyte IFN- production was also
significantly greater (p < 0.05) in mice
inoculated with cpa/cpb than in mice inoculated with
wild-type parasites.
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BALB/c mice vaccinated 2 or 4 mo before infection with wild-type
parasites produced more slowly growing lesions (Fig. 7, a and b), which
contained significantly fewer parasites, than similarly infected
nonvaccinated mice. At wk 8 postinfection with wild-type L.
mexicana, the wild-type parasite burdens in mice vaccinated 2 and
4 mo previously with cpa/cpb were significantly less than
those of nonvaccinated mice (for vaccinated mice: 2 mo, 2.7 x
106 ± 5.4 x 105; 4 mo, 1.6 x
106 ± 3.2 x 105; for nonvaccinated
mice: 2 mo, 6.2 x 107 ± 2.3 x
107; 4 mo, 8.4 x 107 ± 2.4 x
107; p < 0.002 and p <
0.001, respectively). Whereas all nonvaccinated and the vast majority
of vaccinated mice went on to develop nonhealing lesions, 2 of the 10
mice infected 4 mo after vaccination failed to develop lesions up to 12
wk postchallenge. C57BL/6 mice vaccinated with cpa/cpb
also developed significantly smaller lesions following challenge
infection with wild-type parasites than nonvaccinated mice (Fig. 8, p < 0.005 wk 14
postchallenge). The lesions in the vaccinated C57BL/6 mice but not the
control mice began to decrease in size after this period.
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cpb. Nine of 10 nonvaccinated mice developed
nonhealing cutaneous lesions by wk 14 following infection with
wild-type L. mexicana promastigotes. However, only 1 of 10
mice vaccinated with cpb and 1 of 10 mice vaccinated with
cpa/cpb had developed lesions over the experimental
period following the same challenge with wild-type parasites (Table I). Analysis of splenocyte Ag-specific
IFN- production at 8 and 14 wk post-challenge infection demonstrated
the significantly enhanced ability of cpa/cpb-vaccinated
mice to produce this cytokine over control nonvaccinated animals
infected with wild-type parasites (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cpa to BALB/c mice is similar to that of wild-type
L. mexicana (Ref. 22 and Fig. 1
), whereas deletion of the
multicopy cpb genes results in mutants that form only very
slowly growing small lesions (Ref. 23 and Fig. 1). The finding that
cpa/cpb-deficient double mutants do not form lesions in
BALB/c mice suggests that deletion of both genes has a synergistic
effect in reducing infectivity. Furthermore, the finding that
cpa/cpb mutants infect macrophages at a similar rate as
do cpb mutants (23) confirms that the in vivo situation
is more complex than that in vitro and implicates host factors, in
addition to the susceptibility of macrophages, as playing parts in
determining whether the parasites cause lesions. The difference between
the ability of the CP-deficient mutants to infect mice was also
observed in C57BL/6 mice. Two other strains (CBA/Ca and 129Sv/Ev) did
not form lesions when inoculated with either cpb or
cpa/cpb, whereas nonhealing lesions resulted from
inoculation with wild-type L. mexicana. An examination of
the humoral and cellular immune responses of BALB/c mice following
infection with the CP-deficient or wild-type L. mexicana
suggests that the CPs of the parasite may not only be important
virulence factors but may also play a crucial role in modulating the
host immune response.
The resistance of BALB/c mice to cutaneous lesion growth following
infection with CP-deficient mutants was closely related to the ability
of their splenocytes to produce IFN- following specific Ag
stimulation. However, as the CP-deficient mutants failed to induce
lesions in IFN-R-/- mice, raised IFN- is perhaps a beneficial
consequence of infection with these parasites but not the reason for
their low virulence. Nevertheless, as they cause a significant
elevation of parasite-specific IFN- production, these CP mutants may
be excellent vaccine candidates. Previous reports for the L.
mexicana complex (12, 13, 15) have shown that there is a strong
correlation between IFN- production and resistance to infection,
sometimes irrespective of the presence of IL-4 (13, 15). In addition,
CPs have previously been reported to induce IL-4 production (17), and
throughout the course of infection in this study, splenocytes from mice
infected with wild-type parasites produced significantly more IL-4,
following stimulation, than those infected with cpa/cpb
mutants. Indeed, Ag-induced splenocyte IL-4 production was barely
detectable in the majority of C57BL/6 mice infected with
cpa/cpb. These results are consistent with the finding
that lesions failed to develop following L. mexicana
infection in susceptible mice totally deficient in IL-4 (12).
Furthermore, although lesions developed in IL-4-/-
splenocyte-reconstituted SCID mice following infection with L.
mexicana, these healed, unlike IL-4+/+ splenocyte-reconstituted
SCID mice, which continued on to develop fatal infection (26). Thus,
while healing of L. mexicana lesions and resistance to
lesion growth may take place in the presence of IL-4 and is associated
with IFN- production, a nonhealing response is probably dependent on
the presence of lymphocyte-derived IL-4. Taken together, our results
further emphasize the finding originally described by Heinzel et
al. (10), and now well documented, of the counterregulatory activities
of IFN- and IL-4 in determining the outcome of cutaneous
leishmaniasis.
While Ag-induced splenocyte IL-4 production was reduced or absent in
cpa/cpb-infected mice, IL-2 production was significantly
enhanced. No increased IL-2 production was generally observed in
similarly treated wild-type parasite-infected mice. While several
reports have suggested a potential disease-exacerbating role for IL-2
in cutaneous leishmaniasis (24, 27), this has been related to the
ability of IL-2 to promote IL-4 production (27). In the present study
this is clearly not the situation, as the cytokines appear to be
counterregulatory. What may be more important in this study on L.
mexicana, which is known to disseminate and visceralize under
controls similar to those described for L. donovani (16), is
that IFN- and IL-2 have been shown to synergize to protect against
L. donovani (28). Indeed, we have found that although local
treatment of L. mexicana lesions with rIL-2 did not reduce
local parasite burdens (24), it did prevent disease dissemination
(unpublished observation).
Concurrent with enhanced Ag-induced IFN- production by splenocytes
and lymphocyte proliferation responses from mice infected with
cpb and cpa/cpb, there was a significant
reduction in parasite-specific Ab levels. This is reminiscent of the
immunologic status of humans at the resistant end of the disease
spectrum (reviewed in 29 . Similarly, high Ab levels and low
IFN- production displayed by animals infected with wild-type
L. mexicana are generally reminiscent of individuals with
nonhealing disease (29). Although producing high specific Ab levels,
splenocytes derived from cpa-infected mice did produce
more IFN- than their wild-type counterparts upon Ag stimulation and
consequently significantly more IFN--dependent IgG2a Ab (30).
Clearly, however, the increase in the ability to produce IFN- was
not sufficient to shift the balance toward a protective Th1 response as
the cpa-deficient mutants formed nonhealing lesions, albeit
somewhat more slowly growing than those caused by wild-type L.
mexicana. While it has been suggested from studies on BALB/c mice
infected with L. major (31) that simply reducing the
infectious parasite dose can have immunomodulatory effects similar to
those observed here, we have found no healing responses following
infection with low doses of wild-type L. mexicana in the
mouse strains used in this study.
Our observations therefore support the theory that CPs have a
Th2-potentiating role (17). This previous study provided preliminary
evidence that the CP papain was a potent allergen that, following
inoculation into the footpad, induced a 10–30-fold increase in IL-4
mRNA expression in mice within 1 h. Other CPs are also allergens,
such as those derived from house dust mite feces (Der P1) (32) and two
Ags from schistosomal ova that induce a strong Th2 response (33, 34).
Therefore, it is particularly significant that Ag from wild-type
L. mexicana promastigotes enhances IL-4 production over
background levels in nonprimed splenocytes while down-regulating
IFN- production (unpublished observation). Interestingly, not only
has L. major been shown to induce rapid IL-4 production from
CD4+ NK1.1- cells in vivo (35), but the
ability of a single Leishmania Ag LACK
(Leishmania homologue of receptors for activated kinase) to
promote IL-4 production in susceptible mice has also been observed for
these parasites (36, 37). Thus, L. major-susceptible mice
made tolerant to LACK respond with a Th1 rather than a Th2 response
when challenged with wild-type parasites (37). The discovery of how
L. mexicana CPs mediate their effect awaits further study.
Interestingly, however, leishmanial CPs have been implicated in the
inhibition of macrophage Ag presentation (38, 39) by degrading MHC
class II molecules in the parasitophorous vacuole (39). Limiting the
ability of macrophages to present Ag has previously been shown to
potentiate Th2 responses by favoring Ag presentation by accessory cells
of other lineages (40).
Targeted gene deletion of an essential metabolic gene, dihydrofolate
reductase-thymidylate synthase, has been used to create L.
major auxotrophic mutants (41). These parasites failed to
replicate in macrophages but persisted in BALB/c and nu/nu BALB/c mice
for 2 mo without causing overt disease (42). By contrast,
cpa/cpb multiplied in a small number of macrophages in
vitro (23) and persisted in mice for up to 6 mo postinfection although
without lesion development. While persistence of parasites without
clinical infection is well documented (43) and may be necessary to
maintain an anamnestic response (44), there is the risk of subsequent
disease reactivation in immunodepressed individuals. It was extremely
encouraging, therefore, that these CP-deficient mutants not only failed
to induce overt disease in a number of L.
mexicana-susceptible mouse strains such as 129Sv/Ev, but also
failed to induce lesion growth in IFN-R-/- 129Sv/Ev mice and in T
and B cell-deficient RAG2-/- mice on a C57BL/6 background.
The majority of mouse strains are susceptible to L. mexicana
(16, 45) and develop nonhealing lesions that may metastasize to the
extremities or the viscera. Thus, this parasite in mice offers an
excellent model system for putative vaccine studies against
disseminating disease in a variety of susceptible genotypes. It is
therefore extremely encouraging that cpa/cpb successfully
limited lesion and parasite growth in BALB/c mice and C57BL/6 mice and
that both cpb and cpa/cpb virtually ablated
the incidence of lesion development in CBA/Ca mice challenged with
wild-type parasites following s.c. vaccination. This is particularly
promising, as vaccination by this route in the absence of a cytokine or
suitable adjuvant generally results in exacerbated disease (3, 4). The
positive findings clearly suggest that the CP-deficient L.
mexicana are vaccine candidates of high potential.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. G. H. Coombs, Division of Infection and Immunity, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ, U.K. E-mail address:
3 Abbreviations used in this paper: CP, cysteine proteinase; cpa, gene encoding a L. mexicana CP; cpa, L. mexicana cell line with the cpa gene deleted; cpb, gene encoding a multicopy L. mexicana CP; cpb, L. mexicana cell line with all the cpb genes deleted; cpa/cpb, L. mexicana cell line with both cpa and cpb genes deleted.
Received for publication May 21, 1998. Accepted for publication August 25, 1998.
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and TNF-
mRNA expression in DBA/2 mice infected with Leishmania mexicana. Immunology 84:1.[Medline]
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), or
). The lesions resulting from infection with
) or vaccinated with 
