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Infection and Immunity, March 2003, p. 1083-1090, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1083-1090.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Enteral Immunization with Attenuated Recombinant Listeria monocytogenes as a Live Vaccine Vector: Organ-Dependent Dynamics of CD4 T Lymphocytes Reactive to a Leishmania major Tracer Epitope

Hélène Saklani-Jusforgues,1 Elisabeth Fontan,1 Neirouz Soussi,1 Geneviève Milon,1 and Pierre L. Goossens2*

Unité d'Immunophysiologie et Parasitisme Intracellulaire,1 Unité Toxines et Pathogénie Bactérienne, Institut Pasteur, Paris, France2

Received 8 August 2002/ Returned for modification 23 October 2002/ Accepted 12 December 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Listeria monocytogenes is considered as a potential live bacterial vector, particularly for the induction of CD8 T cells. The CD4 T-cell immune response triggered after enteral immunization of mice has not yet been thoroughly characterized. The dynamics of gamma interferon (IFN-{gamma})- and interleukin-4 (IL-4)-secreting CD4 T cells were analyzed after priming through intragastric delivery of an attenuated {Delta}actA recombinant L. monocytogenes strain expressing the Leishmania major LACK protein; a peptide of this protein, LACK158-173 peptide (pLACK), is a well-characterized CD4 T-cell target in BALB/c mice. Five compartments were monitored: Peyer's patches, mesenteric lymph nodes (MLN), spleen, liver, and blood. A single intragastric inoculation of {Delta}actA-LACK-LM in BALB/c mice led to colonization of the MLN and spleen at a significant level for at least 3 days. Efficient priming of IFN-{gamma}-secreting pLACK-reactive CD4 T cells was observed in all tested compartments. Interestingly, IL-4-secreting pLACK-reactive CD4 T cells were detectable at day 6 or 7 only in blood and liver. The absence of translocation of viable bacteria through the intestinal epithelium after further {Delta}actA-LACK-LM inoculations was concomitant with the absence of an increase in the level of IFN-{gamma} secreted by the MLN, blood, and splenic pLACK-reactive Th1 T cells, although the levels remained significantly above the basal level. No change in this population size was detected in the spleen. However, an increase in the number of intragastric inoculations had a clinical beneficial effect in L. major-infected BALB/c mice. L. monocytogenes thus presents the potential of an efficient vector for induction of CD4 T cells when administered by the enteral route.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Listeria monocytogenes has been considered as a potential live vaccine vector in order to induce in vivo a CD8-dominated immune response (10, 12, 22, 25, 33). The oral route is the natural way of entry of L. monocytogenes in the organism (6). The Listeria translocation (20, 29), the development of immune system-dependent effectors and regulators in secondary lymphoid organs, and their delivery in nonlymphoid tissues (1, 21, 24) have been analyzed after oral and enteral infection of laboratory rodents by using specific epitopes of heterologous antigens expressed by recombinant L. monocytogenes (26, 30) or listerial epitopes (18). However, the CD4 immune response induced by the enteric delivery of L. monocytogenes as an experimental live vector needs more precise characterization (18, 22, 34).

In a previous study we established the efficiency of an attenuated {Delta}actA recombinant L. monocytogenes strain expressing the heterologous LACK protein—a known CD4 T-cell target (14, 19) of the obligate intracellular parasite Leishmania major—to induce a protective Th1 CD4 immune response (34). The intragastric (i.g.) route led to a higher protection level than did the intraperitoneal one. The present study was intended to analyze the local and extraintestinal dynamics of the CD4 T-cell populations primed after enteric delivery of this attenuated recombinant L. monocytogenes. This attenuated mutant lacks the ActA protein, which is necessary for Listeria intracellular motility and subsequent cell-to-cell spread (17), and primes both CD4 and CD8 Listeria-reactive T cells after parenteral inoculation (9). The use of such a mutant should increase the pressure on the gut-associated immune system and enable checking the ability of the locally primed T cells to migrate and express their function(s) in other extraintestinal sites. In the present study, the timing, magnitude and persistence of the LACK-reactive gamma interferon (IFN-{gamma})- and interleukin-4 (IL-4)-secreting CD4 T-cell immune responses generated during enteric immunization with this L. monocytogenes vector were analyzed in the Peyer's patches (PP), mesenteric lymph nodes (MLN), spleen, liver, and blood. Although further i.g. inoculations allow to prevent the translocation of L. monocytogenes to extraintestinal sites, they do not result in further expansion of the size of LACK-reactive CD4 T-lymphocyte population, at least in the MLN, spleen, and blood compartments. We focused on these parameters of the immune response because they reflect the actual effector function(s) that these CD4 T cells are expected to exert in vivo in mice inoculated with L. major.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and bacterial and parasite strains. Female BALB/c mice, aged 8 to 12 weeks, were obtained from Janvier (Le Genest St. Isle, France). The bacteria used was strain PIG41.A1, a recombinant {Delta}actA L. monocytogenes strain expressing the LACK protein of L. major (34), and BUG876, the {Delta}actA parental strain (11). The L. major strain used was National Institutes of Health 173 (MHOM/IR/-/173).

Intragastric inoculations. Bacterial stocks were kept at -80°C in a bacterial culture medium containing 15% glycerol. The conditions of the culture and inoculation were as already described for the acid adaptation of L. monocytogenes (31). The size of the inocula was retrospectively checked by CFU enumeration on Bacto tryptose agar. Some mice were randomly checked for the absence of cultivable L. monocytogenes in the lungs.

Enumeration of L. monocytogenes. At specified time points after inoculation, MLN, spleens, livers, and various parts of the intestinal tract were excised and homogenized in sterile phosphate-buffered saline. Serial dilutions of the homogenates were plated on tryptose agar (Difco, Detroit, Mich.) containing 50 µg of kanamycin (Sigma)/ml. The kanamycin resistance marker present on the hly-lack cassette (34) allowed differentiation of the inoculated L. monocytogenes from the bacteria of the intestinal flora. Results were expressed as the mean log10 CFU per organ ± the standard error of the mean.

Preparation of mouse cell suspensions and ex vivo reactivation of LACK-reactive T lymphocytes. Single-cell suspensions from at least three mice were prepared from spleen, MLN, or PP; the liver lymphoid cells present in the extravascular compartment were isolated as previously described (7). Blood from at least five mice per group was collected in the presence of calciparine (250 IU/ml; Sanofi); mononuclear cells were prepared by centrifugation through a cushion of Lympholyte Mammal (Cedarlane Laboratories) according to the manufacturer's protocol. In some of these experiments, depletion of CD4 T cells was performed by magnetic separation with anti-CD4 antibody-labeled magnetic microbeads on VS+ columns by using the VarioMacs apparatus (Miltenyi Biotec). The depletion efficiency (>99%) was checked by cytofluorimetric analysis by using fluorescein isothiocyanate-conjugated anti-CD4 and phycoerythrin-conjugated anti-CD8 antibody (Becton Dickinson, Le Pont de Claix, France) on a FacScan analyzer (Becton Dickinson).

The presence of LACK-reactive T lymphocytes was assayed by incubating blood, PP, and liver cells (4 x 105 cells per well), MLN (6 x 105 cells per well), or spleen cells (8 x 105 cells per well) in triplicate in a final volume of 200 µl with either the LACK158-173 peptide (pLACK; 7.5 µM) (14), an unrelated antigen (hen egg ovalbumin [OVA], 5 µg/ml), concanavalin A at 5 µg/ml, or the culture medium alone. For blood, MLN, PP, and liver lymphoid cell reactivation, splenocytes from naive BALB/c mice (5 x 105 to 1 x 106 cells/well) irradiated with 10 Gy were added as antigen-presenting cells. Cell culture supernatants were harvested at 48 h for IFN-{gamma} and IL-4 detection and kept at -20°C before cytokine quantification.

Detection of IL-4 and IFN-{gamma}. Enzyme-linked immunosorbent assays (ELISAs) with a pair of monoclonal antibodies specific for IL-4 (11B11 and BVD6-24G2; Pharmingen) and IFN-{gamma} (ATCC HB170 and AN-18.17.24 (27) were designed to quantify the amount of IL-4 and IFN-{gamma} present in the cell culture supernatants according to classical protocols (3). A standard curve for each assay was generated with known concentrations of each cytokine. The IL-4 standard was a supernatant from a transformed cell line derived from X63Ag8-653 (15) kindly provided by F. Melchers (Basel Institute, Basel, Switzerland); murine recombinant IFN-{gamma} was kindly provided by G. R. Adolf (Ernst Boehringer-Institute für Arzneimittelforschung, Vienna, Austria). Standardization and quantification were done with the KC4 software (Bio-Tek Instruments, Inc.). No IL-4 and IFN-{gamma} was detected in the supernatants from pLACK-reactivated cells from nonimmunized and {Delta}actA strain-immunized mice or from OVA-reactivated cells from all animals tested or when pLACK was omitted in the reactivation assays for the LACK-expressing {Delta}actA L. monocytogenes-immunized mice (data not shown).

Enzyme-linked immunospot (ELISPOT) assay. To detect IFN-{gamma}-producing cells in the spleen, 96-well nitrocellulose plates (Millititer, HA; Millipore, Molsheim, France) were coated with anti IFN-{gamma} monoclonal antibody (R4-6A2; Pharmingen). T-cell reactivation was performed in triplicates for a 20-h period at concentrations ranging from 1.25 x 105 to 1 x 106 cells per well in 100 µl in the presence of pLACK (7.5 µM) or with culture medium alone. After revelation with biotinylated anti-IFN-{gamma} monoclonal antibody (XMG1-2; Pharmingen) and avidin-phosphatase (Sigma), spots were developped with the BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium (Sigma) substrate, and the number of spots was determined. The results were expressed as the means ± the standard errors of the mean (SEM) for 106 splenocytes. No spot was detected with cells from nonimmunized and {Delta}actA strain-immunized mice.

Inoculation of BALB/c mice with L. major parasites. L. major promastigotes were obtained after differentiation from amastigotes kept at -80°C as previously described (4). Stationary-phase promastigotes (2 x 105 parasites) were injected subcutaneously into the right hind footpad. The clinical parameters of infection were monitored by measuring the footpad swelling with a dial gauge caliper. The results are expressed as an increase of footpad thickness.

Statistical analysis. The results are expressed as the means ± the SEM. The statistical significance was calculated by using the Student t test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
(i) LACK-reactive CD4 T-cell in PP, MLN, and spleen after multiple {Delta}actA-LACK-LM i.g. inoculations. By using IFN-{gamma} and IL-4 secretion as a readout assay, the timing of the pLACK-reactive CD4 T-cell population priming and persistence was characterized in the PP, MLN, and spleens of BALB/c mice after multiple i.g. inoculations of the attenuated LACK-expressing recombinant {Delta}actA L. monocytogenes ({Delta}actA-LACK-LM). After the first inoculation, IFN-{gamma}-secreting LACK-reactive cells were detected only transiently on day 5 in the PP and MLN (Fig. 1). In spleens, the IFN-{gamma} secretion response peaked between days 5 and 7 before decreasing and remaining at a significantly positive level (~5,000 pg/ml, P < 0.05 versus the naive or {Delta}actA-LM immunized group [data not shown]) for at least 39 days (Fig. 1). After subsequent i.g. inoculations, no significant increase in IFN-{gamma} secretion by pLACK-reactive CD4 T cells could be detected with lymphoid cells from MLN and the spleen (Fig. 1). As expected, since the LACK158-173 peptide binds to IAd, in vitro CD4 depletion before cell reactivation at days 7 and 13 after each inoculation prevented the IFN-{gamma} secretion by the reactivated cells (data not shown). No IL-4 secretion could be detected in supernatants from MLN, PP, and spleens at all time points (data not shown).



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  		FIG. 1. IFN-{gamma} secretion profiles of PP-, MLN-, and spleen-derived pLACK-reactive cells. Cells were collected from BALB/c mice at various time points after 1 to 3 i.g. inoculations of {Delta}actA-LACK-LM (7 x 109 bacteria) at 15-day intervals. IFN-{gamma} secretion levels after ex vivo reactivation in the presence of the LACK158-173 peptide were quantified by ELISA as described in Materials and Methods. No IFN-{gamma} secretion was detected in the supernatants of reactivated cells from the noninoculated and {Delta}actA-LM inoculated mice. The arrows indicate the timing of each vector i.g. inoculation. The ELISPOT assay (bottom right panel) of IFN-{gamma}-secreting pLACK-reactive T cells in the spleen was performed as described in Materials and Methods. No IFN-{gamma}-secreting cells were detected among the spleen cells from uninoculated and {Delta}actA-LM-inoculated mice. The results are expressed as the means of triplicate determinations ± the SEM and represent the combination of at least two separate experiments. ND, not determined.

 
To further analyze the kinetics of the LACK-reactive CD4 T-cell population primed during multiple enteric L. monocytogenes infections, quantification of the IFN-{gamma}-secreting LACK-reactive T cells was carried out in the spleen by using an ELISPOT assay after reactivation with the LACK158-173 peptide (Fig. 1). A >50-fold increase in the frequency of IFN-{gamma}-secreting LACK-reactive T cells was detected after the first i.g. inoculation; a plateau was reached by day 7 and lasted for at least 39 days. The frequency of LACK-reactive T cells was estimated at ~50 LACK-reactive T cells for 106 splenic cells, amounting to ~1.5 x 104 IFN-{gamma}-secreting LACK-reactive T cells per spleen. The second and third inoculations did not significantly increase this frequency. The IFN-{gamma}-secreting LACK-reactive cells detected in the ELISPOT assay were of the CD4 phenotype, as observed after in vitro depletion (data not shown).

(ii) LACK-reactive CD4 T-cells in blood and liver after multiple or unique {Delta}actA-LACK-LM i.g. inoculation(s). In order to be recruitable and/or recruited in the sites where their ligands (MHC II-pLACK) are displayed on antigen-presenting cells, these activated T cells are expected to recirculate. Analysis of the recirculating pool of LACK-reactive CD4 T cells was carried out in the blood compartment after multiple i.g. inoculations of {Delta}actA-LACK-LM. After the first inoculation, the presence of IFN-{gamma}-secreting LACK-reactive cells peaked on day 7 before decreasing and remaining at a significant level (~4,000 pg/ml, P < 0.05 versus the naive or {Delta}actA-LM immunized group [data not shown]) for at least 39 days (Fig. 2). Interestingly, a peak of IL-4 secretion was transiently detected after LACK158-173 peptide restimulation of blood lymphoid cells on day 7 after the primo-inoculation (Fig. 2); later on, the IL-4 secretion level was either below detection or slightly above basal level. Ex vivo CD4 depletion before cell reactivation at day 7 prevented the IFN-{gamma} and IL-4 secretions (data not shown). The second and third reinoculations did not significantly modify these patterns of IFN-{gamma} and IL-4 secretion (Fig. 2).



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  		FIG. 2. IFN-{gamma} and IL-4 secretion profiles of pLACK-reactive mononuclear cells isolated from the blood and the liver extravascular compartment components. The inoculation protocol is as described in Fig. 1. IFN-{gamma} and IL-4 secretion levels in the supernatants were quantified by ELISA as described in Materials and Methods. No IFN-{gamma} or IL-4 secretion was detected in the supernatants of cells from the unimmunized or {Delta}actA-LM-inoculated mice. The arrows indicate the timing of each vector i.g. inoculation. The results are expressed as the means of triplicate determinations ± the SEM and represent the combination of at least two separate experiments. ND, not determined.

 
The liver is one of the main targets of intravenously delivered L. monocytogenes (8). Furthermore, due to its position on the blood circulation, the liver functions as a filter for microorganisms, whether they are alive or dead (particulate or microbial molecules). Analysis of the recirculating pool of LACK-reactive CD4 T cells was thus carried out in the extravascular space of this organ after single i.g. inoculations of {Delta}actA-LACK-LM. A peak of IFN-{gamma} and IL-4 secretion was transiently detected after LACK158-173 peptide restimulation on days 6 and 7 after the primo-inoculation (Fig. 2).

(iii) Bacterial intestinal transit and translocation after multiple {Delta}actA-LACK-LM i.g. inoculations. To correlate the temporal variations in the tissue distribution of these LACK-reactive CD4 populations with the fate of live L. monocytogenes in the tissues, the timing of L. monocytogenes intestinal transit, translocation, and subsequent entry and growth in target organs such as MLN, spleen, and liver were analyzed after each inoculation of {Delta}actA-LACK-LM. Transit was similar after one, two, or three inoculations (Fig. 3A) and was characterized by a transient passage through the small intestine and a more prolonged residence at a high level in the cecum and colon compartment components.



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  		FIG. 3. {Delta}actA-LACK-LM transit and translocation in BALB/c mice after one, two, or three i.g. inoculations at a 15-day interval (7.2 x 109 bacteria for the first inoculation, 6.9 x 109 bacteria for the second and third inoculations). The arrows indicate the timing of each vector i.g. inoculation. The dotted lines indicate the threshold of the CFU assays; for the MLN and spleen from days 1 to 6 the whole homogenates were plated. (A) Transit. At various time points after each inoculation, the L. monocytogenes load in the entire small intestine and the cecum and colon were determined. (B) Translocation. At the indicated time points, the bacterial loads were determined in the MLN, spleen, and liver. The results are expressed as the mean log10 CFU per gut compartments or per organ ± the SEM (four mice per time point). ND, not determined.

 
Translocation through the intestinal epithelium was efficient upon the first inoculation (Fig. 3B), with a 3- to 4-log10 plateau of viable bacteria in the MLN and spleen from days 1 to 3; the presence of the attenuated {Delta}actA-LACK-LM in liver was only transient (24 h) and at a lower level than in the MLN and spleen. Interestingly, L. monocytogenes reached the liver and spleen as soon as 5 h after the inoculation but could not be detected in the MLN at this time point. The translocation of viable, i.e., cultivable, bacteria in the MLN and spleen was prevented after the second and third inoculations (Fig. 3B).

(iv) Effect of {Delta}actA-LACK-LM multiple i.g. inoculations on L. major cutaneous challenge. Since the LACK antigen coinjected with IL-12 is an essential target of a protective Th1-orientated immune response in mice (23), the correlation between the level of LACK-targeted immune response and the extent of protection against a subcutaneous high-dose L. major challenge (2 x 105 stationary-phase promastigotes) was characterized in susceptible BALB/c mice (Fig. 4). Intragastric inoculations of the recombinant listeriae induced a delay in the onset of clinically detectable lesions in a significant proportion of the mice, i.e., 30, 60, and 50% after one, two, and three inoculations, respectively. In these mice, after one or two {Delta}actA-LACK-LM i.g. immunizations, lesion progression was slower than in the control groups, leading to more chronic process without ulceration (Fig. 4). In the group receiving three i.g. immunizations, 37% of the mice showed efficient control of footpad swelling (<1 mm).



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  		FIG. 4. Effect of multiple {Delta}actA-LACK-LM i.g. (IG-1 to -3) immunizations on L. major lesion development in BALB/c mice. {Delta}actA-LACK-LM (6 x 109 bacteria) was inoculated by the i.g. route once (10 mice), twice (10 mice), or thrice (8 mice) at a 15-day interval. At 14 days after the last immunization, 2 x 105 NIH173 L. major stationary-phase promastigotes were inoculated subcutaneously into the right hind footpad, and the resulting footpad swelling was monitored. Controls were unimmunized mice (n = 10) and {Delta}actA-LM-inoculated mice (IG-1, n = 9; IG-2, n = 8; IG-3, n = 9). The results are expressed as the means ± the SEM. {Delta}actA-LACK-LM-immunized mice were divided in two or three subgroups, depending on the degree of lesion containment (i.e., the number of mice in each subgroup in parentheses).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A model of i.g. immunization with the {Delta}actA L. monocytogenes vector was developed to generate protective Th1 CD4 T lymphocytes by using a LACK peptide as a tracer epitope. The vector translocation across the intestinal barrier was efficient since {Delta}actA-LACK L. monocytogenes reached the liver and spleen as soon as 5 h after i.g. delivery. The bacteria were detected in the MLN later; this observation suggests that, in addition to spreading through the lymphatic system, early dissemination to deep organs could occur by hematogenic route. Spreading to the MLN seemed slower, but rapid inactivation of the viable L. monocytogenes reaching the MLN could not be excluded, considering that MLN are lymph nodes continuously exposed to viable bacteria and bacterial molecules from the indigenous intestinal flora. In contrast, Pron et al. (29) observed an equivalent seeding in the liver, spleen, and MLN 3 h after inoculation of wild-type or actA-deficient L. monocytogenes. These differences are very likely due to the variety of experimental procedures: instead of the delivery of a high inoculum in a ligatured ileal intestinal loop, we used i.g. delivery with an acid-adapted L. monocytogenes strain that survived the low gastric pH and spread physiologically along the whole gut (31). Variation in the spreading route may also depend on the gut compartment, i.e., small intestine versus the cecum and colon. Lymphatic draining is richer in the small intestine (chyliferes) and, in our model, L. monocytogenes went rapidly through the small intestine and persisted at a higher level and for a greater period of time in the cecum and colon.

Our results showed that enteric immunization with the attenuated {Delta}actA-LACK-LM induced a transient peak of IFN-{gamma}-secreting pLACK-reactive CD4 T lymphocytes in PP and MLN by days 5 to 7 and resulted in the presence, within extraintestinal lymphoid (spleen) and nonlymphoid (blood and liver) tissues, of IFN-{gamma}-secreting LACK-reactive CD4 T cells that persisted for at least 39 days. The induction of this immune response was correlated with the ability of the attenuated {Delta}actA-LACK-LM to colonize MLN and spleen at a significant level for at least 3 days after a single i.g. inoculation. The rapid elimination of the vector and its antigens between the third and sixth day may explain the transient nature of the peak of the CD4 response. The dynamics of the CD4 T lymphocytes in our model of immunization differs from the T CD8 response described after an i.g. immunization with OVA-expressing L. monocytogenes (26); the induced OVA-reactive CD8 T lymphocyte population peaked in the gut, liver, and spleen on day 9 after immunization; expansion of gut and liver OVA-reactive CD8 populations was shown to depend on CD4 T-lymphocyte participation through CD40L-CD40 costimulation. Our results are thus in keeping with this temporal relationship since, in our model system, the peak of IFN-{gamma}-secreting LACK-reactive CD4 T lymphocytes occurred on day 5 in PP, MLN, spleen, liver, and blood. In addition to IFN-{gamma}-secreting pLACK-reactive CD4 T cells, IL-4-secreting pLACK-reactive cells could be transiently detected in the liver and blood but not in the MLN and spleen. These recirculating T cells could originate from the cross-priming with the bacteria from the intestinal microflora as previously suggested (13). This observation, together with the reported abundance of IL-4-secreting NKT cells in the liver (16), stresses the originality of this organ in the development of the immune response.

Further inoculations of the attenuated {Delta}actA-LACK-LM did not significantly modify the immune parameters analyzed in the MLN, spleen, and blood; moreover, comparison of the ELISPOT and reactivation assays showed that the sustained number of splenic IFN-{gamma}-secreting LACK-reactive CD4 T lymphocytes was correlated with a lower IFN-{gamma} secretion capacity per cell, at least in our ex vivo reactivation conditions. This absence of in vitro recall response was concomitant with the efficient inhibition of viable L. monocytogenes translocation in MLN and spleen. Tolerance was not induced since, under the same i.g. inoculation conditions, we previously showed that IFN-{gamma}-secreting LACK-reactive CD4 T cells were recruited in the lymph nodes draining the site of an L. major challenge (34). A similar absence of in vitro recall response was also reported after i.g. delivery of OVA-expressing wild-type L. monocytogenes since no increase of the OVA-reactive CD8 T-lymphocyte population was detected after a second i.g. inoculation of the recombinant vector (26). In that study—as well as in the present study—the lamina propria was not monitored for the presence and level of CD4 and CD8 T lymphocytes reactive to the "tracer epitopes." However, two recent studies have shown that, by using either an OVA-derived or a listeriolysin O-derived peptide as a tracer peptide, a CD4 recall response was detected in the lamina propria after a second i.g. inoculation (18, 21). Even if no translocated bacteria could be detected in a viable form, killed bacteria and their derived immunogenic peptides (in our study, the LACK peptide) may nevertheless lead to restimulation of the memory effector CD4 T populations in this particular compartment and explain the delay of onset and attenuation of L. major lesions observed in the present study after the second and third i.g. inoculations. Similarly, after oral inoculation of a human immunodeficiency virus (HIV)-Gag-expressing L. monocytogenes strain, even though the HIV-Gag reactive cytotoxic-T-lymphocyte activity was no longer detected, protection against HIV-Gag vaccinia virus was still achieved after mucosal challenge (30).

In this respect, interrelation between the cutaneous and mucosal immune responses has been recently reported. First, dendritic leukocytes derived from a cutaneous site can stimulate mucosal immunity (5). Second, transforming growth factor ß, known to act in the gut-associated lymphoid tissues (GALT) as a signal that confers unique tissue-homing properties to primed T lymphocytes, can induce the cutaneous lymphocyte antigen or P-selectin ligand (2). Third, in sites of chronic inflammation (35), mucosal addressin-cell adhesion molecule 1 (MadCAM-1) is detected on postcapillary venules of secondary lymphoid organs outside the GALT area. The expression of its ligand, the {alpha}4ß7 integrin (a signature of the GALT-derived CD4 T lymphocytes), and of cutaneous lymphocyte antigen could then contribute to the extravasation of the GALT-primed CD4 T cells in the L. major-infected cutaneous sites. Clearly, analysis of the quality and dynamics of the intraepithelial and lamina propria-residing CD4 T lymphocytes in relation to the number of immunizing oral inoculations will help us to understand the potential implications of a mucosa-induced response in the control of a cutaneous infection.

These primed LACK-reactive Th1 CD4 T lymphocytes were able to migrate into a L. major-infected cutaneous site (34) and be protective; i.g. immunization with the recombinant Listeriae induced a delay in the lesion onset induced by a high dose of L. major and 37% of the mice showed efficient control of footpad swelling; it is noteworthy that, in the same experimental conditions (i.e., three i.g. inoculations of the attenuated {Delta}actA-LACK-LM) (34), 80% protection was achieved against a low-dose L. major infection (3 x 104 to 5 x 104 parasites), closer to the dose naturally delivered by the sandfly (32). Interestingly, although the attenuated {Delta}actA-LACK-LM reached a 1- to 2-log10-lower extraintestinal bacterial load compared to a wild-type L. monocytogenes strain (data not shown), the efficiency of {Delta}actA-LACK-LM immunization on complete control of a high-inoculum L. major lesion (37% [the present study] and 31% [34]) was similar to the one observed with a wild-type derivative (wt-LACK-LM, 29% [34]). This is in keeping with the observation (28) that transport of L. monocytogenes by dendritic cells from PP to draining MLNs and subsequent spreading to mononuclear phagocytes occurred through actA-independent mechanisms; thus, the similarity of the early intestinal invasion process of {Delta}actA-LACK-LM or wt-LACK-LM could explain the equivalent level of protection observed.

In conclusion, our study showed that enteric administration of the attenuated {Delta}actA-LACK L. monocytogenes as a live immunization vector was efficient in promoting a local and systemic functional Th1 CD4 immune response able to prevent Listeria translocation after further inoculations and to counterbalance the naturally driven L. major Th2-orientated immune response. Even if no recall response was observed at the systemic level after i.g. reinoculations, both the delay in the onset of the lesion and the attenuation of clinical signs in the L. major-infected cutaneous site suggested the existence of GALT-located processes that extend the functions of these GALT-primed CD4 T lymphocytes to the skin-draining lymph node complex.


   ACKNOWLEDGMENTS
 
We thank Pascale Cossart, Christine Kocks, and Edith Gouin (Unité Interactions Bactéries Cellules, Institut Pasteur, Paris, France) for kindly providing the {Delta}actA mutant; Micheline Lagranderie for information on the use of the ELISPOT assay; Karim Sebastien for expertise with mice; and Christine Maillet for preparation of the many liquid and solid media used in this study.

Neirouz Soussi was a recipient of a grant from the Fondation Marcel Mérieux. This work was supported by Institut Pasteur, as well as by grants from "Le Programme Microbiologie du Ministère de l'Education Nationale, de la Recherche et de la Technologie (France)" and from "Le Programme Puces à ADN du CNRS (Appel d'Offres 2000)."


   FOOTNOTES
 
* Corresponding author. Mailing address: Unité Toxines et Pathogénie Bactérienne, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: 33-1-45-68-82-49. Fax: 33-1-45-68-89-54. E-mail: pierre.goossens{at}pasteur.fr. Back

Editor: J. D. Clements


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Infection and Immunity, March 2003, p. 1083-1090, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1083-1090.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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