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The Journal of Immunology, 2000, 164: 2221-2232.
Copyright © 2000 by The American Association of Immunologists

Production of IFN-{gamma} and IL-10 to Shigella Invasins by Mononuclear Cells from Volunteers Orally Inoculated with a Shiga Toxin-Deleted Shigella dysenteriae Type 1 Strain1

Taraz Samandari*, Karen L. Kotloff*, Genevieve A. Losonsky*, William D. Picking{dagger}, Philippe J. Sansonetti{ddagger}, Myron M. Levine* and Marcelo B. Sztein2,*

* Center for Vaccine Development, Departments of Pediatrics and Medicine, University of Maryland School of Medicine, Baltimore, MD 21201; {dagger} Department of Biology, St. Louis University, St. Louis, MO 63103; and {ddagger} Unité de Pathogénie Microbienne Moléculaire, Unité 389, Institut Nationale de la Santé et de la Recherche Médicale, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Volunteers were orally administered invasive, non-Shiga toxin-producing Shigella dysenteriae 1 to establish a challenge model to assess vaccine efficacy. In stepwise fashion, four separate groups were given 3 x 102, 7 x 103, 5 x 104, or 7 x 105 CFU. Using PBMC, proliferative responses and cytokine production were measured to S. dysenteriae whole-cell preparations and to purified recombinant invasion plasmid Ags (Ipa) C and IpaD. Anti-LPS and anti-Ipa Abs and Ab-secreting cells were also evaluated. Preinoculation PBMC produced considerable quantities of IL-10 and IFN-{gamma}, probably secreted by monocytes and NK cells, respectively, of the innate immune system. Following inoculation, PBMC from 95 and 87% of volunteers exhibited an increased production of IFN-{gamma} and IL-10, respectively, in response to Shigella Ags. These increases included responses to IpaC and IpaD among those volunteers receiving the lowest inoculum. No IL-4 or IL-5 responses were detected. Whereas there were no Ab or Ab-secreting cell responses in volunteers receiving the lowest inoculum, other dose groups had moderate to strong anti-LPS and anti-Ipa responses. These results suggest that in humans, type 1 responses play an important role in mucosal and systemic immunity to S. dysentariae 1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Shigellosis is an important etiologic agent of diarrheal disease and dysentery worldwide, causing an estimated 1.3 million deaths annually, particularly among infants and young children (1). Shigella dysenteriae type 1 is an especially virulent serotype because of its ability to produce devastating epidemics and pandemics of severe disease in countries where sanitation is poor and overcrowding is common.

Information on the human immune response to Shigella infection is limited primarily to humoral immunity, and attempts to correlate specific humoral immunity and protection have been inconsistent. In humans, systemic and mucosal Abs are directed primarily to Shigella LPS, with a lesser response against the invasion plasmid Ags (Ipa)3 A, B, C, and D (2). Some field studies and phase 1 vaccine trials have shown a correlation between protection and Abs against LPS (3, 4), whereas other trials have not (5, 6). For example, in an epidemiological study in Peru, investigators found that in contrast to anti-LPS Ab, anti-Ipa Ab may limit the spread and severity of infection (7).

Because Shigella is an intracellular pathogen, it has been hypothesized that cell-mediated immunity (CMI) may be essential for defense against shigellosis. Evidence is accumulating to support this theory. In a mouse pulmonary model of Shigella infection, IFN-{gamma} was produced by naive and, at even higher levels, by immune mice in response to Shigella infection (8). Using mice deficient in IFN-{gamma} and beige mice (a mouse strain deficient in NK cell activity), one group concluded that NK cell-mediated IFN-{gamma} is essential for resistance following primary Shigella infection (9). In another study, human-derived peripheral blood NK cells were shown to kill Shigella-infected HeLa cells (10). In studies conducted in Bangladeshi patients with acute shigellosis, elevated levels of IFN-{gamma} were detected in rectal biopsies and serum as well as stool, which contained particularly high levels (11, 12). Subsequently, the same investigators found an up-regulation of IFN-{gamma} production and expression of the IFN-{gamma} receptor in the epithelial lining of rectal biopsies from patients convalescing from S. dysenteriae 1 infection (13). Shigella infection has also been found to elicit the appearance of activated T cells in circulation during the course of human disease (14, 15). Despite these observations suggesting an important protective role for CMI, prospective data are not yet available in humans to correlate the generation of CMI responses with protection against Shigella.

Experimental challenge studies in volunteers have become an important step in preliminarily testing the efficacy of candidate vaccines against Shigella sonnei and Shigella flexneri (3, 16, 17, 18). A modification of the model in which wild-type S. flexneri is administered to volunteers with buffer has resulted in a reliable, repeatable model characterized by high attack rates of typical shigellosis. No similar dependable challenge model has yet been developed for S. dysenteriae 1. Among the shigellae, only S. dysenteriae 1 produce the Shiga toxin (Stx). Because Shiga toxin is responsible for the hemolytic uremic syndrome and other complications associated with S. dysenteriae 1 infections, it would have been unacceptable to administer an Stx+ strain in volunteers. Thus, to establish a challenge model to assess the efficacy of new S. dysenteriae 1 vaccines, we used a Shiga toxin subunit A-deficient, {Delta} stxA, S. dysenteriae 1 mutant, SC595, administered with buffer, at several dosage levels. This dose-response trial provided a unique opportunity to characterize the cellular and humoral immune responses to shigellosis in a group of Shigella-naive volunteers. No previous studies have examined the human cellular immune response to purified S. dysenteriae 1 proteins, the dose response for eliciting CMI to S. dysenteriae 1, or the relative time frame in which CMI and Abs develop following S. dysenteriae 1 infection in naive volunteers.

In this report we describe the results of studies on Ab production as well as Ab-secreting cell (ASC) and cytokine and proliferative responses to crude S. dysenteriae preparations and to purified recombinant Shigella proteins IpaC and IpaD using pre- and postinoculation PBMC from volunteers infected with the {Delta}stxA S. dysenteriae 1 strain, SC595.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Challenge strain

The deletion mutant SC595 was derived from S. dysenteriae 1 (strain 7/87) (19) and was constructed in the following stages. Briefly, the tox operon was cloned from S. dysenteriae 1 7/87, and the toxA gene encoding the catalytic subunit of Shiga toxin was deleted. The P1 promoter from pBR322 was inserted upstream of the gene encoding the Shiga toxin B subunit to provide the {Delta}stxA mutant with the capacity to express the B subunit and elicit anti-toxin immunity. The construct was subcloned into the suicide vector pC527K followed by insertion of a mercury resistance gene upstream of the P1-toxB construct. The HgR-P1-toxB sequence was exchanged with the StxA/B operon in S. dysenteriae 1 7/87. Therefore, SC595 is {Delta}stxA, HgR, StxB+.

Subjects and clinical protocols

Healthy adult volunteers, 18–40 years of age, from the Baltimore-Washington community participated in these studies. Procedures for medical screening and enrollment were the same as those for previous trials (17). No volunteer had been previously involved in any other Shigella trial. Protocols were approved by the institutional review board of the University of Maryland (BB-IND 6521). In a stepwise fashion, groups of volunteers were challenged orally with SC595 in a bicarbonate solution as described in earlier trials involving S. flexneri 2a (20). The inoculum contained 3 x 102, 7 x 103, 5 x 104, or 7 x 105 CFU of SC595. The volunteers were observed for 12–14 days on the Research Isolation Ward of the Center for Vaccine Development: 2 days before and 10–12 days after ingestion of the challenge strain. Volunteers were carefully monitored for any evidence of illness, which was defined as oral temperature >=100oF, dysentery (gross blood in a liquid stool), or diarrhea (>=3 liquid stools/48 h totaling 200 ml or a single liquid stool >=300 ml). Either 5 days (volunteers receiving 3 x 102 or 7 x 103 CFU inocula) or 7 days (those receiving the larger inocula) postchallenge, the volunteers were started on a 5-day course of ciprofloxacin (500 mg every 12 h). Blood samples were drawn before challenge and on days 7, 14, 21, and 28 after challenge to measure serologic responses. Blood samples were drawn on days 7, 10, and 14 after challenge to measure the ASC response. Blood was drawn before inoculation and 28 days after challenge to measure cell-mediated responses.

Isolation of PBMC and volunteer identification

PBMC were isolated and cryopreserved in 10% DMSO in liquid nitrogen as previously described (21). Frozen PBMC were quickly thawed, except for PBMC from patients receiving 7 x 105 CFU, which were used fresh. No differences were observed in the proliferative responses of freshly obtained or cryopreserved PBMC in response to either tetanus toxoid (TT) or PHA stimulation. Volunteers numbered 1–4 in this study were given the 7 x 103 CFU dose, volunteers 5–10 were given the 5 x 104 CFU dose, volunteers 11–15 were given the 3 x 102 CFU dose, and volunteers 16–21 were given the 7 x 105 CFU dose. PBMC isolated on the day of inoculation are referred to as day 0 PBMC or preinoculation, and those drawn 4 wk later were called day 28 PBMC or postinoculation.

Reagents

Complete RPMI consists of RPMI 1640 medium (Biofluids, Rockville, MD) supplemented with 10% FCS (HyClone, Logan, UT), 2 mM glutamine (Biofluids), 50 µg/ml gentamicin (Life Technologies, Grand Island, NY), and 10 mM HEPES (Biofluids). AIM-V serum-free lymphocyte medium was purchased from Life Technologies, and TT was obtained from Connaught Laboratories (Toronto, Canada). PHA, BSA, and DMSO were obtained from Sigma (St. Louis, MO); [3H]thymidine was purchased from NEN-DuPont (Boston, MA); and ß plate Scint scintillation mixture was purchased from Fisons Chemical (Loughborough, U.K.). Anti-cytokine (capture) and biotinylated (detection) mAbs against human IL-2, IL-4, IL-5, IL-10, IL-12, IL-15, and IFN-{gamma} were obtained from PharMingen (San Diego, CA); anti-human TGF-ß (capture) and biotinylated (detection) Abs were obtained from R&D Systems (Minneapolis, MN).

Preparation of bacterial Ags

Whole cell and homogenated bacterial Ags used in the cytokine and proliferation assays were derived from a {Delta}guaBA,{Delta}stxA S. dysenteriae 1 strain, CVD 1251, and a Bacillus subtilis strain (Ehrenberg) Cohn from the American Type Culture Collection (no. 7067; Manassas, VA) that was used as a control in these studies. An overnight culture of bacteria was used to inoculate 500 ml of appropriate broth (either Luria-Bertoni broth or Luria-Bertoni broth supplemented with 0.005% guanine in the case of the Shigella auxotroph, CVD1251). The bacteria were centrifuged and resuspended in 15 ml of sterile PBS. A French press (SLM Instruments, Rochester, NY) with a 1-in. diameter piston was used at 1260 psi to prepare a homogenate of the bacteria, which were then centrifuged at 3000 rpm for 10 min and filter-sterilized with a 0.45-µm pore size filter (Acrodisc, Gelman Sciences, Ann Arbor, MI) and stored at -85°C. The protein concentration in the homogenate was quantified using the BCA protein assay system from Pierce (Rockford, IL). The particulate preparation was made by heating the bacteria at 60°C for 1 h and then resuspending the preparation in phenol at a final concentration 0.5% (v/v). Before heating, a measured volume of bacterial culture was serially diluted and counted after overnight growth on agar plates. The particulate preparation was kept at 4°C in the PBS/phenol solution. Before use in the stimulation assays, the phenol was removed from the particulate preparations by washing three times with sterile PBS at 10,000 rpm for 3 min in a microfuge. LPS Ag for coating ELISA plates and ASC analysis was prepared from S. dysenteriae 1 by the hot aqueous phenol extraction method of Westphal and Jann (22) and was further purified by the procedure of Thomashow and Rittenberg (23). The IPA preparation for coating ELISA plates was made according to the procedure described by Oaks et al. (24). It should be noted that the IPA preparation used for the measurement of Ab levels and ASC determinations is a crude preparation containing a mixture of Ipa proteins that must be distinguished from the highly purified recombinant IpaC and IpaD protein preparations used in the proliferation and cytokine assays.

Construction and purification of recombinant IpaC and IpaD

The coding sequence of ipaC was amplified from the virulence plasmid of S. flexneri 2a strain 2457T using PCR and inserted into the plasmid vector pET15b (Novagen, Madison, WI) to give pWPC15 for expression in Escherichia coli BL21(DE3) as described previously (25, 26). The coding sequence of ipaD, also from S. flexneri, was cloned into the pET15b vector to give pWPD10 (27). Synthesis of IpaC or IpaD was induced in 400 ml mid-log phase cultures with 1 mM isopropylthio-ß-D-galactoside. Recombinant IpaC and IpaD were purified by virtue of a short (20-aa) leader sequence that contained six tandem histidine residues (His-Tag) using nickel chelation affinity chromatography as previously described (25, 27). Briefly, after induction of protein synthesis, the cells were harvested by centrifugation, and the pellets were resuspended in 20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, and 5 mM imidazole (binding buffer). The cells were then frozen, quickly thawed, and sonicated, and the solution was clarified by centrifugation at 39,000 x g for 20 min. The supernatant fraction was passed over HisBind resin that had been charged with NiSO4 and equilibrated with binding buffer. The column was then washed with the same buffer containing 60 mM imidazole. The recombinant protein was eluted using 20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, and 1 M imidazole (elution buffer). Following elution of the purified protein from the column, the sample was dialyzed against 20 mM phosphate (pH 7.2) with 0.15 M NaCl (PBS). Analysis of the protein products by SDS-PAGE and immunoblot analysis using convalescent serum from monkeys that had been experimentally infected with S. flexneri (provided by Dr. Edwin V. Oaks, Department of Enteric Infections, Walter Reed Army Institute of Research, Washington, D.C.) was used to monitor the purity of the product, and final protein concentrations were determined using the BCA assay (Sigma, St. Louis, MO) according to the manufacturer’s instructions. An SDS-PAGE of IpaC and IpaD is shown in Fig. 1Go. The IpaC and IpaD derived from S. dysenteriae 1 (GenBank accession no. X60777) are 98 and 94% identical to the respective proteins derived from S. flexneri 2a (GenBank accession no. J04117). There were <70 endotoxin units (EU)/ml of LPS in the IpaC or IpaD samples as measured by the Kinetic QCL Chromogenic LAL test (BioWhittaker, Walkersville, MD). We determined that this amount of LPS was unable to induce significant levels of IFN-{gamma} or IL-10 production by PBMC isolated from healthy volunteers (data not shown).



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  		FIGURE 1. Coomassie blue-stained gel electrophoresis of recombinant IpaC and IpaD proteins. Following affinity purification, IpaC and IpaD were separated by SDS-PAGE using a 12% gel. The first lane shows m.w. markers. The second lane shows ~0.5 µg of purified IpaC, which is >90% pure (indicated by the top arrow at the right). The third lane shows 4 µg of purified IpaD, which is >90% pure (indicated by the bottom arrow at the right).

 
Incubation of PBMC with Ags

PBMC were quickly thawed and immediately resuspended in complete RPMI. They were then washed, counted, and resuspended in AIM-V medium at a density of 1.5 x 106 cells/ml. The Ags were prepared in AIM-V medium and then added to the PBMC at the following final concentrations: S. dysenteriae homogenate at 1, 10 or 25 µg/ml; S. dysenteriae particulate at 5 x 104, 2 x 105 or 8 x 105 particles/well (1 particle = 1 CFU of heat-phenolized whole-cell bacteria); IpaC at 6 or 12 µg/ml; IpaD at 6 or 12 µg/ml; B. subtilis homogenate at 1, 10, or 25 µg/ml; B. subtilis particulate at 5 x 104, 2 x 105 or 8 x 105 particles/well; TT at 2 µg/ml; PHA at 1.8 µg/ml; and BSA at 25 µg/ml. PBMC (7.5 x 105 and 1.5 x 105/well) were plated in 24- and 96-well plates (Corning, Corning, NY), for cytokine and proliferation assays, respectively. Plates were incubated at 37°C with 5% CO2 in humidified chambers for the indicated times.

Proliferation assays

PBMC were incubated in triplicate wells with appropriate Ags in 96-well plates for 6 days before being pulsed with 1 µCi/ml [3H]thymidine diluted in AIM-V media. Cells were harvested the next day using a Tomtec cell harvester (Orange, CT) onto filter mats (Wallac, Turku, Finland). PHA-stimulated PBMC were pulsed 2 days after incubation and harvested on the third day. Scintillation mixture was added to the filter mats, and the samples were read in a Microbeta liquid scintillation counter (Wallac). Proliferation assays were performed on PBMC for 20 of the 21 volunteers. Data are presented as net counts per minute and were calculated by subtracting the average of the medium from the average of the Ag being evaluated for each individual volunteer on each day (i.e., preinoculation or postinoculation day).

Cytokine analysis

PBMC were incubated in 24-well plates with the various Ags at the concentrations indicated above. Supernatants were collected after 3 days of incubation with Ags and either tested immediately or maintained at -70°C until analyzed. Chemiluminescence ELISAs were performed on each sample (28). Briefly, 100 µl of each sample was placed in duplicate in opaque ELISA plates. Due to the variable number of PBMC collected from the volunteers, it was not possible to test the cytokine responses from all volunteers for all Ags. Overall, from the 21 volunteers who participated in all the groups described in this report, PBMC from the following numbers of individuals were tested for production of each cytokine as follows: 21 for IL-2, 11 for IL-4, 11 for IL-5, 21 for IFN-{gamma}, 15 for IL-10, 7 for IL-12, 7 for IL-15, and 8 for TGF-ß. ELISAs were read in a Microbeta luminescence counter (Wallac, Turku, Finland) immediately after addition of chemiluminescence working solution.

The sensitivities of the ELISAs were as follows: IL-2, 13 pg/ml; IL-4, 23 pg/ml; IL-5, 80 pg/ml; IFN-{gamma}, 18 pg/ml; IL-10, 10 pg/ml; IL-12, 44 pg/ml; IL-15, 45 pg/ml; and TGF-ß, 11 pg/ml. Data are presented as net cytokine production and were calculated by subtracting the cytokine levels (in picograms per milliliter) of the medium alone control wells from cytokine levels of Ag-stimulated wells in each day (day 0 or 28).

Measurement of serum Abs

Levels of Abs to S. dysenteriae 1 LPS and Ipa IgA, IgM, and IgG were measured by ELISA. Briefly, polystyrene U-bottom 96-well microtiter plates (Dynex Technologies, Chantilly, VA) were coated overnight with either S. dysenteriae 1 LPS or Ipa Ags at the optimal coating concentrations of 10 and 2 µg/ml, respectively, and then blocked with PBS containing 5% FBS. Serial 2-fold dilutions of sera were added after washing the plates. After incubating at 37°C for 1 h, the plates were washed, and alkaline phosphatase-conjugated goat anti-human IgA, IgG, or IgM (Kierkegaard & Perry, Gaithersburg, MD) was added. After incubation at 37°C for 1 h, the plates were washed, and the phosphatase substrate p-nitrophenyl phosphate (Kierkegaard & Perry) was added. The OD at 405 nm (A405) in each well was then recorded by an automated TiterTek ELISA reader (Huntsville, AL). End-point titers were defined for each assay at the following absorbances: 0.3 for S. dysenteriae 1 LPS IgA and IgG, 0.35 for IgM, 0.15 for Ipa IgA, 0.4 for IgG, and 0.25 for IgM. Cut-off absorbances were determined using a negative control population. A 4-fold rise in titer from pre- to postinoculation samples was reported as positive. For Ab titers against Ipa, the background was removed by performing an ELISA with T55 Ag (an Ipa preparation from a S. dysenteriae 1 strain missing the invasiveness plasmid) and subtracting the response of each individual from the ELISA using Ipa Ag.

Measurement of ASC

ASC assays were performed as previously described (29) except for being modified for S. dysenteriae 1 LPS or Ipa Ags using optimal coating concentrations of 10 and 2 µg/ml, respectively. Results are recorded as the number of ASC per 106 PBMC. For each Ag and Ab class, a positive response was defined as one that exceeded the preinoculation geometric mean cell number + 2 SDs given a minimum of four cells.

Statistical analysis

For lymphocyte proliferative responses, the net counts per minute in triplicate of day 28 PBMC were compared with the net counts per minute in triplicate of day 0 PBMC incubated with the same concentration of the same Ag by paired two-tailed t tests (null hypothesis: counts per minute after inoculation is equal to the day 0 value). Subjects for whom the null hypothesis was rejected were considered responders. For cytokine responses, the duplicate chemiluminescence units (CU; in relative light units per second) after inoculation following PBMC exposure to the same concentration of the same Ag were compared with CU on day 0 for each subject by paired two-tailed t tests (null hypothesis: CUs after inoculation are equal to the day 0 CUs by Ag and dilution). Statistical tests with resulting probabilities <=5% were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical response to challenge with SC595

A total of 21 volunteers ingested SC595. None of the five volunteers given a 3 x 102 CFU dose of SC595 developed symptoms. One (no. 2) of the four individuals challenged with 7 x 103 CFU had fever, diarrhea, and dysentery. One (no. 5) of the six volunteers given 5 x 104 CFU had low grade fever (100.7oF) and diarrhea. Among those who ingested 7 x 105 CFU, one (no. 17) of the six volunteers had low grade fever (100.4oF), and two (no. 19 and 20) had diarrhea.

Cytokine production and proliferative responses by PBMC of volunteers inoculated with 7 x 103 CFU of the SC595 S. dysenteriae 1

PBMC from the four volunteers inoculated with 7 x 103 CFU of the modified Shigella strain were incubated with homogenate and particulate preparations of S. dysenteriae and the purified recombinant proteins IpaC and IpaD. Culture supernatants were then evaluated for proliferative responses and production of IFN-{gamma}, IL-10, IL-2, and TGF-ß by chemiluminescence ELISA. Fig. 2Go shows peak IFN-{gamma} production in response to S. dysenteriae 1 homogenate and particulate preparations. In three of four individuals, there was a significant increase in IFN-{gamma} production in response to S. dysenteriae 1 homogenate by day 28 PBMC (p < 0.05). Moreover, three of the four volunteers exhibited specific increases in IFN-{gamma} production in response to IpaC, and 1 of 4 had an increase in response to IpaD (Fig. 3Go). Taken together, these data show that all four volunteers had specific increases in IFN-{gamma} production (p < 0.05) in response to at least one of the Shigella Ags. Of note, we observed that the homogenate and particulate Shigella preparations and, to a lesser extent, purified IpaC and IpaD were able to induce IFN-{gamma} production by PBMC isolated before exposure to SC595, albeit at generally lower levels than those observed following immunization. It is important to emphasize that a significant proportion of the IFN-{gamma} induced by homogenate and particulate preparations was observed following incubation with IpaC and IpaD, suggesting that these molecules contribute significantly to the overall responses observed against Shigella Ags. Incubation of PBMC from days 0 and 28 with BSA at 25 µg/ml stimulated no more than 2 pg/ml of IFN-{gamma} cytokine in all groups (data not shown).



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  		FIGURE 2. Peak PBMC IFN-{gamma} responses to particulate and homogenate Ags in volunteers administered 7 x 103 CFU of modified S. dysenteriae type 1. PBMC obtained from volunteers before and after inoculation were incubated in vitro with Shigella Ags as described in Materials and Methods. The Ags used include S. dysenteriae 1 homogenate (Sd-h), B. subtilis homogenate (Bs-h), S. dysenteriae 1 particulate (Sd-p), and B. subtilis particulate (Bs-p). Vol., volunteer number. Results represent the mean of duplicate ELISA wells of supernatants exhibiting the peak cytokine response from the various Ag concentrations evaluated as described in Materials and Methods. SDs are shown as error bars. An asterisk indicates where a day 28 response is significantly greater than a day 0 response with p < 0.05. The p value of the IFN-{gamma} response of PBMC from volunteer 3 to Sd-p is 0.14, and that of volunteer 4 to Sd-h is 0.09.

 


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  		FIGURE 3. Peak PBMC IFN-{gamma} responses to purified protein Ags in volunteers administered 7 x 103 CFU of modified S. dysenteriae 1. PBMC obtained from volunteers before and after inoculation were incubated in vitro with Shigella Ags as described in Materials and Methods. The Ags used include medium, B. subtilis homogenate (Bs-h), IpaC, and IpaD. See Fig. 2Go for details. The p values of the IFN-{gamma} responses of PBMC from volunteer 1 to IpaC and IpaD are 0.15 and 0.26, respectively.

 
Figs. 4Go and 5 show peak IL-10 production by PBMC from these same four volunteers. There was a specific increase in IL-10 production by day 28 in all four volunteers in response to homogenate or particulate preparations of S. dysenteriae or to either IpaC or IpaD (p < 0.05). Upon in vitro exposure to all Shigella Ags, significant production of IL-10 was observed in PBMC derived from all four volunteers before inoculation with SC595. Small increases in IL-10 production were also noted in three of four volunteers incubated with the control (B. subtilis) particulate preparation, but not with the B. subtilis homogenate (Fig. 4Go).



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  		FIGURE 4. Peak PBMC IL-10 responses to particulate and homogenate Ags in volunteers administered 7 x 103 CFU of modified S. dysenteriae 1. PBMC obtained from volunteers before and after inoculation were incubated in vitro with Shigella Ags as described in Materials and Methods. Ags used include S. dysenteriae 1 homogenate (Sd-h), B. subtilis homogenate (Bs-h), S. dysenteriae 1 particulate (Sd-p), and B. subtilis particulate (Bs-p). Vol., volunteer number. The p value of the IL-10 response to Sd-h by PBMC from volunteer 4 is 0.14.

 
No significant increases in IL-2 production by PBMC of any of these volunteers were observed following exposure to Shigella Ags, and three of four had weak (10–75 pg/ml) to moderate (76–150 pg/ml) increases in TGF-ß production by day 28 PBMC (data not shown). There was essentially no proliferation by PBMC of any individual in response to Shigella or B. subtilis Ags in this clinical trial.

Cytokine production by PBMC of volunteers inoculated with 5 x 104 CFU of the S. dysenteriae 1 challenge strain

In this dose group, a 1-log higher inoculum of the challenge strain and a larger panel of cytokines were investigated. Table IGo displays the complete panel of responses by examining increases in cytokine production by day 28 PBMC over the day 0 PBMC against a given Ag preparation. There was virtually no production of IL-2, IL-4, IL-5, IL-12, or IL-15 in any volunteer. All six volunteers had some IFN-{gamma} production increase by PBMC on day 28 when exposed to at least one Shigella Ag, and three of these were strong increases. Five of the volunteers had a significant increase in day 28 IL-10 production in response to one or more of the Shigella antigenic preparations, with two of these volunteers having very strong responses (Table IGo).


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  		Table I. Cytokine production in response to various Shigella Ags before and after inoculation in volunteers administered 5 x 104 cfu of modified S. dysenteriae type 1 strain, SC5951

 
Results from proliferative responses in this clinical trial are shown in Table IIGo. There were relatively minor, albeit significant, increases in proliferation (p < 0.05) by day 28 PBMC against the S. dysenteriae 1 particulate preparation for three of the six volunteers; however, overall the data indicate poor proliferative responses to Shigella Ags.


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  		Table II. Peak proliferative responses to ShigellaAgs in volunteers administered 5 x 104 cfu of modified S. dysenteriae type 11

 
Induction of IFN-{gamma} and IL-10 production as a function of the size of bacterial inoculum

The abilities of varying doses of the SC595 S. dysenteriae 1 strain to induce proliferative and cytokine responses were evaluated in two additional groups of subjects who were given oral inoculation with 3 x 102 CFU and 7 x 105 CFU. Fig. 6Go summarizes the peak IFN-{gamma} production by PBMC in response to the various Shigella Ags tested at all four dosage levels involving a total of 21 volunteers. Twenty of the 21 (95%) volunteers showed significant increases in IFN-{gamma} production from day 0 in response to at least one Shigella Ag. Fourteen of these individuals had strong increases, and six had weak to moderate increases. Statistical analysis of the mean IFN-{gamma} production by day 28 PBMC compared with day 0 PBMC within each dose group indicated a significant increase in mean IFN-{gamma} production only in the 3 x 102 CFU and 7 x 103 CFU groups in response to the homogenate preparation and in the 5 x 104 CFU group in response to the particulate preparation (p < 0.05). The absolute amount of IFN-{gamma} produced was higher using both the homogenate and the particulate preparation relative to IpaC or IpaD. Almost half (10 of 21) of the volunteers had IFN-{gamma} production following stimulation of day 0 PBMC.



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  		FIGURE 6. Peak IFN-{gamma} responses to Shigella Ags in the entire series. Results represent the average of a duplicate ELISA of the supernatant of PBMC from a given volunteer taken from the highest response to a range of Ag concentrations. Different symbols represent specific individuals in each trial. Each trial had unique participants. The mean IFN-{gamma} production of controls in all these groups is as follows: B. subtilis homogenate: day 0, 9 pg/ml; day 28, 23 pg/ml; B. subtilis particulate: day 0, 20 pg/ml; day 28, 39 pg/ml; BSA, <2 pg/ml. IFN-{gamma} production in response to IpaC was not evaluated in the 7 x 105 CFU trial. The horizontal line represents the mean of the data points displayed for that given day.

 
Surprisingly, inoculation with as few as 3 x 102 S. dysenteriae 1 bacteria induced strong IFN-{gamma} production by PBMC in response to Shigella Ags (Fig. 6Go). However, no dose-response differences were noted in IFN-{gamma} production with increasing inoculum size (Fig. 6Go). All six volunteers who received 5 x 104 CFU had significant postinoculation increases in IFN-{gamma} production following exposure to Shigella Ags despite the lower levels of IFN-{gamma} production by PBMC isolated from these volunteers compared with those who received different inocula (Table IGo). The fact that similar proliferative responses were induced by TT and PHA stimuli in PBMC isolated from volunteers in the 5 x 104 CFU group as well as those from volunteers inoculated with the other doses, and the fact that similar IL-10 responses were observed among all volunteers (Fig. 7Go), indicate that the PBMC from the volunteers vaccinated with 5 x 104 CFU were not compromised.



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  		FIGURE 7. Peak IL-10 responses to Shigella Ags in the entire series. Results represent the average of a duplicate ELISA of the supernatant of PBMC from a given volunteer taken from the highest response to a range of Ag concentrations. Different symbols represent specific individuals in each trial. Each trial had unique participants. The mean IL-10 production of controls in all these groups is as follows: B. subtilis homogenate: day 0, 5 pg/ml; day 28, 15 pg/ml; B. subtilis particulate: day 0, 18 pg/ml; day 28, 40 pg/ml; BSA, <2 pg/ml. IL-10 production was not evaluated in the PBMC from the 7 x 105 CFU trial.

 
IL-10 production in all groups is summarized in Fig. 7Go. Overall, 13 of 15 (87%) of the volunteers tested had a significant increase in IL-10 production on day 28 when exposed to one or more of the Shigella Ags. Three of these were strong increases, five were moderate increases, and five were weak increases. Although there was a trend toward increased IL-10 production against each Shigella Ag in all of the groups, in these small samples a statistically significant increase was observed only for the 3 x 102 CFU series against the homogenate preparation and in the 5 x 104 CFU series against the particulate preparation (p < 0.05). Of note, 10 of 13 (77%) of the volunteers had significant IL-10 production by day 0 PBMC. No dose-response patterns in terms of IL-10 production were observed with the different Shigella inocula (Fig. 7Go).

Induction of IL-2, IL-4, IL-5, IL-12, IL-15, and TGF-ß production and proliferative responses in volunteers inoculated with the SC595 S. dysenteriae 1 strain

Overall, the following significant increases in cytokine production were observed in volunteers following inoculation: 3 of 21 volunteers tested had weak to moderate increases by day 28 PBMC over day 0 PBMC in IL-2 production, 2 of 11 had weak to moderate IL-4 production, 1 of 7 had a weak IL-12 response; 3 of 8 produced weak to moderate TGF-ß responses, and 0 of 11 and 0 of 7 volunteers produced IL-5 and IL-15, respectively. In the entire series, there were minimal proliferative responses, with only 5 of 20 proliferating weakly in response to Shigella Ags.

Kinetics of IL-10, IL-12, IL-15, and IFN-{gamma} production

It is well established that IL-12 and IL-15, produced by monocytes/macrophages (30, 31), and IL-10, produced by macrophages and lymphocytes (32), are released soon after macrophage activation. It was therefore possible that we failed to detect measurable levels of IL-12 and IL-15 during the studies described above at least in part due to the fact that we measured these cytokines in supernatants collected 3 days after exposure to the Ags rather than at earlier times. Given the importance of IL-12 in inducing type 1 (T1) responses, characterized by IFN-{gamma} production (33, 34), and of IL-15 in promoting lymphocyte proliferation, it was of importance to explore whether these cytokines were produced at earlier time points. To this end, we measured these cytokines in supernatants collected at 24, 48, and 72 h. IFN-{gamma} was also measured in these experiments as a positive control to confirm that this cytokine followed the kinetics of production of T cell-derived cytokines, i.e., a gradual rise, reaching maximum levels in the supernatant 3 days following Ag stimulation. Results are shown in Fig. 8Go, A and B. As expected for lymphocyte-derived cytokines, a steady increase was noted over the 3 days in the production of IFN-{gamma} for both volunteers in response to S. dysenteriae Ags. In contrast, one individual (no. 3) produced significant and steady levels of IL-10 on all 3 days against the particulate preparation of Shigella as well as to IpaC and IpaD, while the other volunteer (no. 16) did not release IL-10 at any time point. No IL-12 or IL-15 production was observed at any time. These results support the contention that the IL-10, IL-12, and IL-15 responses observed in 3-day culture supernatants are representative of the levels of these cytokines observed at earlier time points.



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  		FIGURE 8. Kinetics of cytokine production by PBMC from two volunteers in different dose groups. Values represent the mean of results of duplicate chemiluminescence ELISAs with the SDs. All values (in picograms per milliliter) are peak postinoculation (day 28) minus preinoculation (day 0) PBMC responses for the same Ag at the same concentration. Sd homog, S. dysenteriae 1 homogenate; Sd partic, S. dysenteriae 1 particulate; Bs homog, B. subtilis homogenate. A, Volunteer 3 (7 x 103 CFU recipient). Day 2 production of IFN-{gamma} on PBMC from this volunteer (no. 3) drawn preinoculation were not available; preinoculation IFN-{gamma} production from day 3 was subtracted instead. B, Volunteer 16 (7 x 105 CFU recipient).

 
Ab and ASC responses in all volunteers

Peak ASC and Ab responses of all volunteers in each dose group are shown in Tables III and IV. There were no anti-LPS or anti-Ipa ASC responses in subjects who received 3 x 102 CFU. The ASC responses to LPS in the 7 x 103, 5 x 104, and 7 x 105 CFU groups were of similar magnitude. Anti-Ipa ASC responses were generally of considerably lower magnitude than anti-LPS ASC responses. There was a dose-response trend in the mean postinoculation anti-LPS IgA, IgG, and IgM Ab titers and in the anti-Ipa IgG and IgM titers that did not reach statistical significance given the wide distribution of responses among the volunteers in each dose group. Overall, 71% (15 of 21) volunteers had IgM, IgA, and/or IgG responses to LPS, and 38% (8 of 21) of subjects mounted an IgA or IgG Ab response to Ipa, although these responses were generally of moderate magnitude.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We provide new insights into the human host immune response to S. dysenteriae 1 by examining in detail the induction of serum Ab, ASCs, proliferative responses, and cytokine release patterns of PBMC from 21 healthy Maryland volunteers inoculated orally with varying doses of a virulent, non-Shiga toxin-producing strain of S. dysenteriae 1 (SC595). The salient findings of this work include the novel observations that 1) in most volunteers, inoculation with SC595 induces IFN-{gamma} and IL-10 production by PBMC to Shigella homogenate and particulate preparations; 2) inoculation with SC595 induces robust IFN-{gamma} and IL-10 production by PBMC in response to purified recombinant Shigella invasins, IpaC and IpaD; 3) IFN-{gamma} and IL-10 responses of similar magnitude were observed at all doses of bacterial inocula, including volunteers inoculated with the lowest dose (3 x 102 CFU); 4) when exposed to Shigella Ags, IL-10 and IFN-{gamma} are produced by PBMC obtained before inoculation in many volunteers, although at lower levels than in day 28 PBMC; and 5) strong CMI responses can be detected as early as 28 days after exposure to Shigella.

The observations that preinoculation PBMC from 10 of 21 volunteers produced IFN-{gamma} in response to Shigella Ags and that 20 of 21volunteers inoculated with the virulent S. dysenteriae 1 strain developed a specific IFN-{gamma} response after inoculation lead us to conclude that we observed both nonspecific (innate) and specific IFN-{gamma} responses. Because IFN-{gamma} is produced by T lymphocytes and NK cells, both of which are present in PBMC isolates (35), it is reasonable to hypothesize that NK cells may be contributing the nonspecific (innate) preinoculation IFN-{gamma} response and that T cells produce the greatly increased levels of IFN-{gamma} present in postinoculation samples. In support of the idea that there is a nonspecific NK cell IFN-{gamma} response, several groups have observed NK cell activity against shigellosis in both human and animal models (9, 14, 36). For example, in a mouse pulmonary model a sizable IFN-{gamma} response was detected in naive mice, and a greater and brisker IFN-{gamma} response occurred in previously immunized mice (8). The possibility that there may have been an increase in NK cell numbers in our volunteers is unlikely because of the observation that NK cell numbers are not increased in natural S. dysenteriae infections (14). Our kinetic studies (Fig. 8Go) that showed a steady increase over the 3 days of culture are consistent with IFN-{gamma} being produced mainly by activated lymphocytes. The increase in IFN-{gamma} production observed in PBMC collected postinoculation suggests that T1 lymphocyte activation in shigellosis may stimulate macrophages leading to the elimination of phagocytosed Shigella organisms. In addition to promoting T1 responses, IFN-{gamma} serves as an effector molecule by preventing Shigella invasion of eukaryotic cells (37). The negligible IL-4 and IL-5 responses, two key type 2 (T2) cytokines (33, 34), further support the idea of a predominantly T1 response in shigellosis. Interestingly, we observed that while as few as 3 x 102 CFU of SC595 induced a predominantly type 1 response, characterized by strong IFN-{gamma} production by PBMC in the absence of Ab or ASC, at higher doses (7 x 103 CFU and above) both humoral and IFN-{gamma} responses were evoked. This type 1 dominance suggests that IFN-{gamma} may play a key role in the host’s immune response to Shigella.

IL-10 is produced by T cells, B cells, and activated monocytes, all of which are found in PBMC isolates (38, 39, 40). Once again what appears to be both a nonspecific (innate) and an Ag-specific IL-10 response is observed in pre- and postinoculation PBMC. Studies in Bangladeshi patients also showed evidence of IL-10 production in the acute phase of shigellosis (41). In the kinetics experiment (Fig. 8Go), it was observed that while IFN-{gamma} steadily increased over a 3-day period, IL-10 increased in day 28 PBMC by the first day and remained at a stable level for the entire period. This pattern is consistent with a predominant monocyte response. However, the observations that higher levels of IL-10 were induced in the supernatants of PBMC from volunteers following inoculation points to the presence of an Ag-specific component in addition to the innate response.

Given the marked anti-inflammatory and the Ab-promoting properties of IL-10, this molecule might have a dual role in shigellosis. By inhibiting T1 responses (42), NK responses (43), and the strong induction of IL-1 observed in shigellosis (44), IL-10 may limit the sequellae of the inflammatory response. Furthermore, IL-10 may stimulate B lymphocyte proliferation (45) and Ab secretion in synergy with TGF-ß (46).

Our observation that IFN-{gamma} and IL-10 are produced in response to recombinant IpaC and IpaD Shigella proteins provides the first evidence of CMI directed specifically against these purified proteins. These immune responses were specific, since neither TT nor BSA induced IFN-{gamma} or IL-10 production. The observation that cytokine responses to the Ipa proteins are of considerable magnitude, yet less than those observed against the homogenate or particulate preparations, suggests that the response to the Ipa proteins accounts for a substantial fraction of the response to the whole-cell preparations. In contrast to anti-LPS Ab, the fact that Abs to IpaC and IpaD are elicited (47, 48) implies T cell help, supporting our contention that specific T cells induced after inoculation may also act as promoters of B cell proliferation and Ab secretion.

To rule out the possibility that the minute quantities of LPS (14–70 EU/ml) detected in the purified IpaC and IpaD preparations activated monocytes/macrophages and subsequently produced IFN-{gamma} and/or IL-10, we exposed PBMC from two volunteers to varying concentrations of LPS for 72 h. We observed that at LPS concentrations of <=95 EU/ml there was no detectable production of IL-10, and production of IFN-{gamma} by PBMC was <50 pg/ml. In contrast, these PBMC produced up to 180 pg/ml of TNF-{alpha} in response to the same LPS concentrations (data not shown). These results make it highly unlikely that the traces of LPS present in purified recombinant IpaC and IpaD preparations contributed significantly to the observed cytokine responses induced by Shigella invasins.

Although an unexpectedly poor proliferative response to all Shigella Ags was observed in the entire series, this observation is consistent with the absence of two key cytokines that promote proliferation, i.e., IL-2 and IL-15. It is possible that the strong IL-10 production observed in our studies leads to an inhibition of Th1-derived IL-2 (42) and monocyte-derived IL-15 (49), resulting in limited proliferative responses. Moreover, we cannot rule out that IFN-{gamma} is exerting anti-proliferative effects, because it has also been shown to suppress the proliferation of normal and tumor cells (35). We also observed some TGF-ß production by day 28 PBMC against Shigella-specific Ags in three of eight volunteers (data not shown). One of the most potent activities of TGF-ß on lymphocytes is an antiproliferative effect (50). Therefore, the various cytokines that have been measured at high levels in our system may act in concert to inhibit lymphoproliferative responses. Finally, a growing body of evidence indicates that many bacterial species can inhibit the proliferative response by a variety of independent mechanisms (51, 52). For example, we have recently reported that killed whole-cell S. flexneri inhibits human lymphocyte proliferation in response to a heterologous Ag (53). Furthermore, some investigators have suggested that Shigella may subvert the host’s defenses by inhibiting macrophage presentation of Shigella Ags and possibly even by inducing apoptosis of macrophages (54, 55, 56).

Taken together, all the above observations suggest that a T1 response prevails during shigellosis, consistent with the fact that Shigella is an intracellular pathogen. We hypothesize that IFN-{gamma} is the predominant cytokine produced by T cells following Ag exposure and by NK cells during both the primary and secondary responses. The IFN-{gamma} production may be modulated by IL-10 release, possibly aided by TGF-ß1, which also limits the IL-1-promoted inflammatory response characteristic of shigellosis.

Results from some field trials suggest that serum Ab responses are associated with protection, and it is possible that systemic Ab levels are a surrogate marker for local immunoprotective responses (4). Other investigators surmise that local (secretory IgA), rather than systemic (IgG), immunity holds the key to protection against Shigella infection, because shigellosis is a disease restricted to the gastrointestinal tract (5). In the studies described in this manuscript, although secretory IgA was not measured, we show the induction of circulating ASC that produce IgM, IgG, and IgA to Shigella Ags. Because it is well accepted that following oral immunization ASC represent transiently detected Ab-secreting B cells that originate in the afferent gut mucosal immune system and after processing in regional lymph nodes re-enter the circulation to seed other mucosal sites (57, 58), we conclude that mucosal immunization with Shiga toxin-deficient S. dysenteriae 1 elicits both serum Ab and mucosal immune responses.

To summarize, we provide evidence from individuals living in an area nonendemic for S. dysenteriae 1 that a specific, acquired, predominantly T1 (e.g., IFN-{gamma} production in the absence of IL-4 and IL-5) immunity is produced with as few as 3 x 102 micro-organisms. A significant IL-10 component of the acquired immune response was also discovered, which suggests a role for IL-10 as a stimulant of Ab production and as an anti-inflammatory agent. Future studies of cells isolated from systemic and mucosal sites and the analysis of clonal cell populations from these tissues should enhance our understanding of the CMI elicited by oral inoculation with attenuated strains of Shigella. Our observations of CMI directed against IpaC and IpaD strengthen the importance of these proteins as potentially important targets in the human host immune response to shigellosis. This work underscores the importance of evaluating the role of CMI in protection against Shigella and encourages future investigations of new attenuated Shigella vaccine candidate strains as well as exploration of this micro-organism as a vector for Ag or DNA vaccines.



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  		FIGURE 5. Peak PBMC IL-10 responses to purified protein Ags in volunteers administered 7 x 103 CFU of modified S. dysenteriae 1. PBMC obtained from volunteers before and after inoculation were incubated in vitro with Shigella Ags as described in Materials and Methods. Ags used include medium, B. subtilis homogenate (Bs-h), IpaC, and IpaD. Vol., volunteer number. The p values of the IL-10 responses by PBMC from volunteer 2 to IpaC and volunteer 4 to IpaD are 0.15 and 0.21, respectively.

 

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  		Table III. Peak ASC responses following inoculation by individual1

 

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  		Table IV. Peak Ab responses following inoculation by individual1

 

   Acknowledgments
 
We express our appreciation to Annick Fontaine-Thompson for her contribution to the construction of the Shigella strain, SC595.


   Footnotes
 
1 This work was supported in part by National Institute of Allergy and Infectious Disease Grants K08-AI01507 (to T.S.), R21-AI42802 (to M.B.S.), N01-AI45251 (to M.M.L.), and R29-AI34428 (to W.D.P.). Back

2 Address correspondence and reprint requests to Dr. Marcelo B. Sztein, Center for Vaccine Development, Department of Pediatrics, University of Maryland, 685 West Baltimore Street, Room 480, Baltimore, MD 21201. E-mail address: Back

3 Abbreviations used in this paper: Ipa, invasion plasmid Ags; ASC, Ab-secreting cell; CMI, cell-mediated immunity; Stx, Shiga toxin; TT, tetanus toxoid; EU, endotoxin units; CU, chemiluminescence units; T1, type 1; T2, type 2. Back

Received for publication September 8, 1999. Accepted for publication December 3, 1999.


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