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Concomitant Induction of CD4⁺ and CD8⁺ T Cell Responses in Volunteers Immunized with Salmonella enterica Serovar Typhi Strain CVD 908-htrA ¹

Rosângela Salerno-Gonçalves^*, Timothy L. Wyant^*, Marcela F. Pasetti^*, Marcelo Fernandez-Viña, Carol O. Tacket^*, Myron M. Levine^* and Marcelo B. Sztein^2,*

^* Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201; and Bill Young DoD Marrow Donor Program, Naval Medical Research Center, Georgetown University, Kensington, MD 20895

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Type 1 cell-mediated immunity might play an important role in protection from typhoid fever. We evaluated whether immunization with Salmonella enterica serovar Typhi (S. Typhi) strain CVD 908-htrA (a aroC aroD htrA mutant), a leading live oral typhoid vaccine candidate, elicits specific CD4⁺ and CD8⁺ S. Typhi immune responses. Potent CTL responses and IFN- secretion by CD8⁺ T cells were detected following immunization with CVD 908-htrA in high (4.5 x 10⁸ CFU) and low (5 x 10⁷ CFU) dosages. S. Typhi-specific CTL were observed in six of eight vaccinees (four high and two low dose) after immunization. Mean increases in the frequency of IFN- spot-forming cells (SFC) in the presence of S. Typhi-infected targets were 221 ± 41 SFC/10⁶ PBMC and 233 ± 87 SFC/10⁶ PBMC, in the high and low dose groups, respectively. Strong CD4⁺ T cell responses were also observed. Increases in the IFN- production to soluble S. Typhi flagella (STF) occurred in 82 and 38% of the volunteers who received the high and low doses, respectively. Robust correlations were observed between volunteers that responded with IFN- SFC to stimulation with S. Typhi-infected cells and IFN- released in response to stimulation with STF Ags (r = 0.822, p < 0.001) and between CTL and IFN- production to STF (r = 0.818, p = 0.013). These data demonstrating the concomitant induction of both CD4- and CD8-mediated CMI are consistent with a significant role for type 1 immunity in controlling typhoid infection and support the continuing evaluation of CVD 908-htrA as a typhoid vaccine candidate.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Typhoid fever remains highly prevalent in developing countries, with an estimated 16 million new cases annually and 600,000 deaths (1, 2). The appearance of antibiotic-resistant Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, has added new urgency for the development of a typhoid vaccine that is well tolerated, highly immunogenic, and protective (1, 2). The only licensed attenuated live oral typhoid vaccine, S. Typhi strain Ty21a, is well tolerated and immunogenic, but requires three or four spaced doses of 2–6 x 10⁹ CFU each given every other day, an important practical shortcoming (1).

Relatively little information is available concerning the protective mechanisms against S. Typhi infection in humans. Some studies in volunteers immunized with Ty21a suggest that O Ab responses, either as IgA O Ab-secreting cell responses or as serum IgG O Abs, loosely correlate with protection (3). In recent clinical trials with other attenuated live oral typhoid vaccines, we were also able to demonstrate strong cell-mediated immunity (CMI)³ responses in subjects vaccinated with S. Typhi strain CVD 908 vaccine candidate (a aroC aroD mutant). Immunization with CVD 908 elicited increased proliferative responses and IFN- production by PBMC following stimulation with specific S. Typhi Ags (4), as well as CTLs directed against autologous targets expressing on their surface S. Typhi Ags (5). Recently, similar results were observed in volunteers immunized with S. Typhi strain Ty21a (6). We and others (7, 8, 9, 10) have also reported that immunization with Ty21a elicits the appearance of specific lymphocytes that proliferate and secrete IFN-, but not IL-4, following stimulation with S. Typhi Ags. More recently, we have shown that CD8⁺ T cells that secrete IFN- and kill S. Typhi-infected cells are also elicited by immunization of volunteers with Ty21a (6). Taken together, these studies suggest that type 1 CMI, as well as Abs, may play an important role in protection from typhoid fever.

CVD 908-htrA (a aroC aroD htrA mutant) is a leading new generation attenuated typhoid vaccine candidate. In the past few years, we have demonstrated that CVD 908-htrA is clinically safe and highly immunogenic in phase 1 and 2 human clinical trials after a single dose (11, 12). Oral immunization of volunteers with CVD 908-htrA induced significant anti-LPS and anti-S. Typhi flagella Ab responses, as well as IFN- production against S. Typhi Ags (11, 12). To further evaluate the suitability of this promising typhoid vaccine candidate, we studied its ability to induce CD4⁺ and CD8⁺ S. Typhi-specific responses in volunteers who participated in a double-blind crossover design phase II vaccine trial (12). Responses observed in volunteers immunized with a single oral dose of 4.5 x 10⁸ CFU (high dose) or 5 x 10⁷ CFU (low dose) of CVD 908-htrA were compared with those of volunteers that received placebo. Cytotoxic activity and secretion of IFN- to soluble S. Typhi flagella- and S. Typhi-infected targets were used to assess CD4⁺ and CD8⁺ T cell function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subjects

Eighty healthy adult volunteers, recruited from the Baltimore-Washington community, the University of Maryland, Baltimore campus, and the University of Maryland, College Park campus, participated in a randomized, placebo-controlled, double-blind crossover study (Protocol Typhoid CVD 27000) (12). Following appropriate screening, detailed explanation of the protocol, and obtaining informed consent, outpatient volunteers were randomized to receive with buffer a single oral dose of: 1) high dose CVD 908-htrA (4.5 x 10⁸ CFU) (n = 20); 2) low dose CVD 908-htrA (0.5 x 10⁸ CFU) (n = 20); 3) placebo preparation 1 (n = 20); or 4) placebo preparation 2 (n = 20). The placebo preparations were identical and consisted of buffer solution alone. On day 28, there was a crossover in which all volunteers received another preparation. Those volunteers who ingested CVD 908-htrA vaccine on day 0 received placebo; volunteers who received placebo on day 0 received CVD 908-htrA vaccine in either high or low dose. This trial has been described in detail elsewhere (11, 12).

In this work, specimens are designated by volunteer study number alone when collected from a volunteer who received either dose of CVD 908-htrA at day 0; specimens were designated by volunteer study number followed by an "a" if the specimen was collected after placebo at day 0 and by volunteer study number followed by a "b" if the specimen was collected after the crossover (day 28) in which the volunteer received low or high dose of vaccine.

PBMC from 30 volunteers from this trial, 21 men and 9 women, between 18 and 37 years of age (mean age, 26 years), were used in the studies reported in this work. A total of 11 were immunized with a single oral dose of 4.5 x 10⁸ CFU (high dose) and 9 with 5 x 10⁷ CFU (low dose) of CVD 908-htrA, and 10 volunteers received placebo.

Isolation of PBMC and HLA typing

PBMC were isolated by density-gradient centrifugation and used immediately or cryopreserved in liquid N₂, as previously described (6). HLA typing was performed by microlymphocytotoxicity tests (13) using local and commercial serologic reagents and confirmed using molecular methods including sequence-specific oligonucleotide probes hybridization (14) and sequence-based typing (14, 15, 16) (Table I). CTL experiments were performed with freshly isolated or cryopreserved PBMC, as indicated in Results. When freshly obtained cells were used in CTL, PBMC obtained 2 days before immunization were HLA typed using microcytotoxicity assays, and HLA-matched EBV-transformed lymphoblastoid B cell lines (B-LCL) were used as targets in the assays (see Materials and Methods below and Table I). Cryopreserved PBMC were used in experiments involving the generation of supernatants to S. Typhi flagella (STF) stimulation and IFN- ELISPOT assays.

Two kinds of target/stimulator cells were used in this study: autologous blasts (for ELISPOT assays) (6) and homozygous B-LCL for CTL assays (5). Blasts were obtained by incubating 5–10 x 10⁶ PBMC with 1 µg/ml PHA type L (Sigma-Aldrich, St. Louis, MO) for 24 h in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, 2 mM L-glutamine, 2.5 mM sodium pyruvate, 10 mM HEPES buffer, and 10% heat-inactivated FBS (complete RPMI). PHA-activated PBMC were then washed three times with RPMI 1640, and cultured in complete RPMI supplemented with 20 IU/ml human rIL-2 (rhIL-2) (Boehringer Gmbh, Mannheim, Germany) for 5–6 days.

The EBV-transformed B-LCL used in these studies were from the Reference Cell Line Panel of the Tenth International Histocompatibility Workshop. HLA class I and class II Ags of these cell lines were characterized by several methods (17). B-LCL were maintained in culture in complete RPMI before being used in the experiments.

Infection of target/stimulator cells with S. Typhi

Target cells were incubated in RPMI (without antibiotics) for 3 h at 37°C in the absence or presence of wild-type S. Typhi strain ISP1820 (wild-type S. Typhi) at 20:1 multiplicity of infection (6). After infection, cells were washed in antibiotic-containing medium to kill any bacteria not associated with cells and incubated overnight at 37°C, 5% CO₂, in complete RPMI. In blast-containing cultures, 20 IU/ml of rhIL-2 was also added. For ELISPOT assays, the following day, autologous blasts were gamma irradiated (4000 rad) and used as stimulators. For CTL assays, infected and noninfected homozygous B-LCL were gamma irradiated (6000 rad) and used as stimulators to expand effector cells or labeled with 200 µCi of sodium chromate (⁵¹Cr) (Amersham Pharmacia Biotech, Piscataway, NJ), for 1 h at 37°C, washed three times, and used as targets.

To confirm that infection of target/stimulator cells has taken place, cells were stained with a FITC-labeled polyclonal Ab recognizing Salmonella common structural Ags (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and expression of bacterial Ags was measured on the cell surface by flow cytometry, as previously described (6).

Preparation of effector cells

For cytotoxic assays, effector cells were obtained using a modification of previously described techniques (5, 6). Briefly, PBMC were cocultured with stimulator cells at an effector to stimulator cell ratio of 7:1 in complete RPMI supplemented with 60 IU/ml of rhIL-2 for 7–8 days. Stimulator cells consisted of autologous blasts infected with S. Typhi and gamma irradiated (4000 rad), as described above. For IFN- ELISPOT assays, PBMC from immunized volunteers were used ex vivo as effector cells. In some experiments, PBMC were fractionated into CD4 or CD8 T cell subsets using immunomagnetic beads coated with goat anti-mouse IgG (Dynal, Great Neck, NY), following the manufacturer’s instructions. Briefly, cells were incubated with a cocktail of CD4 or CD8, CD14, CD19, and CD56 mAbs (BD Biosciences, San Diego, CA). The non-CD4 or non-CD8 T cells were then magnetically tagged using immunomagnetic beads coated with goat anti-mouse IgG. Excellent recoveries of CD8⁺ and CD4⁺ T cells were achieved by retaining the magnetically labeled non-CD4 or non-CD8 T cells using a magnet (Dynal). As determined by flow cytometric analysis, CD4⁺ and CD8⁺ T cell subsets were 80% CD4/0.2% CD8 and 40% CD4/50% CD8 T cells, respectively.

Cytotoxicity assay (⁵¹Cr release test)

Cytotoxicity was determined by a 4-h ⁵¹Cr release assay, as previously described (5, 6). Spontaneous release was determined from wells containing medium alone; maximum release was determined from wells to which 2% Triton X-100 (Sigma-Aldrich) was added. All cultures were set up in quadruplicate. Lysis (%) was calculated as follows: (experimental release - spontaneous release)/(maximum release - spontaneous release) x 100. Specific cytotoxicity mediated by effector cells was calculated by subtracting the lysis of uninfected targets from the lysis of S. Typhi-infected targets. The cutoff for positive responses in CTL assays was defined as >10% specific lysis above the mean lysis of effector PBMC when cultured with noninfected target cells, as previously described (18).

In experiments directed to evaluate the ability of anti-CD4 (clone RPA-T4; BD PharMingen, San Diego, CA) or anti-CD8 mAb (clone B9.11; Beckman Coulter, Fullerton, CA) to block CTL activity, effector cells were incubated for 60 min at 37°C with 10 µg/well of the appropriate mAb and used immediately in the cytotoxicity assays.

IFN- ELISPOT assay

The frequency of IFN--secreting cells was quantified by using a modified IFN- ELISPOT assay, as previously described (6). Briefly, anti-human IFN- mAb (5 µg/ml, clone 2G1; Endogen, Woburn, MA) was used to coat MultiScreen-HA filtration plates (MAHA S4510; Millipore, Bedford, MA). After overnight incubation at 4°C, plates were washed, and unoccupied sites were blocked with 200 µl/well PBS/5% BSA for 2 h at room temperature. After washing, stimulator:effector cells at a 1:7 ratio were seeded in 200 µl/well and incubated in a humidified 37°C, 5% CO₂ incubator. Effector cells cultured without stimulator cells or with CD3/CD28 beads (0.6 µl/ml; Dynal) were used as negative and positive controls, respectively. Target cells uninfected or infected with S. Typhi without effector cells were also used as controls. After 16 h of undisturbed incubation, wells were washed and incubated for 2 h at room temperature with 100 µl/well of biotin-labeled anti-human IFN- mAb (clone B133.5, 2 µg/ml in PBS/1% BSA, ELISPOT buffer; Endogen). After washing, avidin-peroxidase (Sigma-Aldrich) was added and incubated at room temperature for 2 h. Wells were then washed and developed with TrueBlue (Kirkegaard & Perry Laboratories). Spots were enumerated using a stereomicroscope. Net frequencies of IFN- spot-forming cells (SFC) were calculated by using the following formula: (number of SFC in effector cell populations incubated with S. Typhi-infected targets) - (number of SFC in effector cell populations incubated with uninfected targets + number of SFC in cultures containing S. Typhi-infected target cell populations alone). The cutoff for positive responses in IFN- ELISPOT assays (99 SFC/10⁶ PBMC) was established by calculating the average frequency of IFN--producing cells/10⁶ PBMC when cultured with noninfected target cells + 2 SE.

IFN- chemiluminescence ELISA

Supernatants were collected after a 3-day stimulation of PBMC with STF Ag. STF was purified from the rough serovar Typhi strain Ty2R by a bulk shearing method (4, 12). The STF preparation used in these studies consisted of a single flagellin band of 55 kDa in SDS-PAGE. Less than 24 pg/ml LPS was present in the purified S. Typhi flagella preparation, as determined by using chromogenic Limulus amebocyte lysate kits (Associates of Cape Cod, Falmouth, MA; level of sensitivity = 24 pg/ml). Levels of IFN- were determined by chemiluminescence ELISA, as previously described (12). Briefly, PBMC from control and immunized volunteers were incubated with medium alone or with 2 µg/ml of STF Ag for 3 days, and supernatants were assayed for IFN- by chemiluminescence ELISA. Anti-IFN- and biotinylated anti-IFN- mAbs were used as capture and detection reagents, respectively (clone 4S.B3; BD PharMingen). Complexes were detected by incubation with avidin-peroxidase conjugate (Sigma-Aldrich), and chemiluminescence ELISA reagent was used as a substrate. rIFN- (BD PharMingen) was used for preparation of standard curves. Each sample was tested in duplicate. The cutoff for positive responses in IFN- ELISPOT assays (31 pg/ml) was established by calculating the average concentration of IFN- released by effector PBMC from all volunteers when cultured in medium alone + 3 SE.

Analysis of intracellular levels of IFN- by flow cytometry

Identification of the effector cell populations secreting IFN- following exposure to STF Ag was performed by multicolor flow cytometry, as previously described (6). Briefly, ex vivo PBMC effectors were cultured with different concentrations of STF plus mAb to costimulatory molecules CD49d (clone 9F10) and CD28 (clone 37.51) (2 µg/ml each; BD PharMingen). Effector cells cultured in medium alone or with anti-CD3/CD28 beads (0.6 µl/ml; Dynal) were used as negative and positive controls, respectively. After 16 h of incubation at 37°C, cytokine secretion was blocked by the addition of brefeldin A (Sigma-Aldrich) at a final concentration of 10 µg/ml for 5–6 h. Cells were then harvested, washed with PBS, and surface stained with CD3-allophycocyanin (clone UCHT1; Beckman Coulter), CD4-ECD (clone SFCl12T4011, PE-Texas Red; Beckman Coulter), CD8-PerCP (clone SK1; BD Biosciences), and CD56-FITC (clone B159; BD Biosciences) mAbs, or the corresponding isotype controls. After washing, cells were fixed with PBS/4% formaldehyde (Sigma-Aldrich) for 30 min at room temperature. Cells were then washed with PBS and incubated for 10 min with 150 µl PBS/1% BSA/0.5% saponin (Sigma-Aldrich; permeabilization buffer). Optimal concentrations of an anti-IFN- PE mAb (clone 4S.B3; BD Biosciences) or a mouse IgG1-FITC isotype control (BD Immunocytometry Systems) were then added and incubated at room temperature. After 30 min, cells were washed twice with permeabilization buffer and once with PBS. Samples were analyzed by flow cytometry (Epics Elite ESP flow cytometer cell sorter system; Beckman Coulter) by evaluating the percentage of CD3⁺CD4⁺CD8^-CD56^- or CD3⁺CD4⁺CD8⁺CD56^- populations coexpressing IFN-⁺.

Measurement of serum anti-S. Typhi LPS Ab responses

Total serum IgG against S. Typhi LPS was determined by ELISA, as previously described, with minor modifications (12). Briefly, 96-well Immulon II plates (ThermoLabsystems, Franklin, MA) were coated with S. Typhi LPS (Difco, Detroit, MI) at 10 µg/ml in carbonate buffer, pH 9.6. Samples were tested in eight 2-fold dilutions in 10% dried milk in PBS Tween 20, 0.05% (PBSTM), in duplicates. Specific IgG was detected with phosphatase-labeled goat anti-human IgG (Kirkegaard & Perry Laboratories) diluted 1/5000 in PBSTM. The substrate solution used was p-nitrophenyl phosphate (Sigma-Aldrich). After a 30-min incubation, the reaction was stopped by the addition of 50 µl of NaOH 3N, and the OD₄₅₀ were measured in an ELISA microplate reader (Multiskan Ascent; ThermoLabsystems). Negative and positive control sera were included in each assay. Linear regression curves were plotted for each serum sample to calculate Ab titers. Titers are reported as ELISA U/ml, defined as the serum dilution that produces an OD₄₀₅ of 0.2 above the blank. A 4-fold rise in titer between prevaccination and peak postvaccination samples was considered a positive response.

Statistical analysis

All tests were performed using SigmaStat software (version 2.03, SPSS Science Software Products, Chicago, IL). Values of p < 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CTL activity against S. Typhi-infected targets following oral immunization with CVD 908-htrA

S. Typhi-specific CTL responses were evaluated in eight CVD 908-htrA vaccinees and two placebo recipients. Five vaccinees were immunized with a single oral dose containing 4.5 x 10⁸ CFU (volunteers 23b, 31, 35, 48b, and 51b) (high dose), and the remaining three vaccinees received 5 x 10⁷ CFU (volunteers 22b, 27, and 34) (low dose) of CVD 908htrA. PBMC were expanded and evaluated against wild-type S. Typhi-infected homozygous B-LCL targets. Specific lysis of S. Typhi-infected targets was observed in six of eight vaccinees (four high dose, Vol 31, 35, 48b, and 51b; two low dose, Vol. 27 and 34) at one or more of the times evaluated (14, 28, 42, and 56 days) after immunization. Noninfected target cells exhibited only background lysis. Of note, PBMC from only one time point (day 14) were available from the high dose vaccinee that was negative for CTL activity (Vol. 23b; data not shown). PBMC from two placebo controls (volunteers 22a and 23a) failed to exhibit specific lysis up to day 28 (Fig. 1).

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Kinetics of induction of CTL activity in volunteers immunized with CVD 908-htrA. PBMC were obtained from six volunteers: two received placebo, and two were immunized with low dose (5 x 107 CFU) and two were immunized with high dose (4.5 x 108 CFU) of CVD 908-htrA. PBMC were collected before (day 0) and at days 14, 28, 42, and 56 after immunization and restimulated in vitro for 8 days with S. Typhi-infected B-LCL targets before testing. Specific cytotoxic activity was determined by 51Cr release of labeled target cells at varying E:T cell ratios and calculated as described in Materials and Methods.

To identify the CTL effector cell populations, cryopreserved PBMC from two vaccinees (high dose, Vol. 48b and 51b) obtained at 28 days after immunization were preincubated with anti-CD4 or anti-CD8 blocking mAb before being used in CTL assays. Killing was abrogated with anti-CD8, but not by anti-CD4 mAb, suggesting that CTL activity is mediated by CD8⁺ T cells (data not shown). These results, demonstrating that CD8⁺ T cells are responsible for the observed cytotoxicity, confirm and extend previous observations in volunteers immunized with an earlier typhoid vaccine candidate (CVD 908) (5) and Ty21a typhoid vaccine (6).

Taken together, these findings demonstrate that B-LCL targets expressing S. Typhi Ags are susceptible to lysis by specific effector CD8⁺ T cells from volunteers immunized with S. Typhi strain CVD 908-htrA.

Frequency of IFN--secreting cells assessed by IFN- ELISPOT assay

We have previously shown in volunteers immunized with the Ty21a typhoid vaccine that the frequency of S. Typhi-specific CD8⁺ T lymphocytes can be evaluated by an ex vivo IFN- ELISPOT assay and that increases in IFN- SFC correlate with specific CTL activity (6, 19). This ELISPOT technique was used to examine whether immunization with CVD 908-htrA elicits the appearance of effector cells able to secrete IFN- in response to stimulation with S. Typhi-infected blasts. Volunteers showing increases in IFN- SFC greater than the cutoff (99 SFC/10⁶ PBMC) were considered responders. An increase in the net frequency of IFN- SFC following immunization was observed in 75% of volunteers in the high dose group (six of eight) and in 50% of volunteers of low dose group (three of six) (Table II). Mean increases were 233 ± 87 SFC/10⁶ PBMC and 221 ± 41 SFC/10⁶ PBMC in the low and high dose groups, respectively. None of the control volunteers showed specific increases in IFN- SFC as compared with preimmunization levels (Table II). Significant differences were observed between the net increases in the frequency of IFN- SFC between the low dose and the control group (p = 0.016) and between the high dose and the control group (p = 0.006) (one-way ANOVA test). No differences were observed between the increases in net frequency of IFN- SFC between low and high dose groups (p = 0.982). Controls were as follows (in SFC/10⁶ PBMC): anti-CD3/CD28 beads (mean ± SE: 3752 ± 536, range 1587–6880); medium alone (mean ± SE: 3 ± 2, range 0–110); and uninfected blasts (mean ± SE: 25 ± 7, range 0–160).

View this table: [in this window] [in a new window]   Table II. Frequency of SFC in the presence of S. Typhi-infected stimulators detected by an ex vivo IFN- ELISPOT assay

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Identification of T cell populations secreting IFN- to S. Typhi-infected blasts. PBMC from volunteers 48b, 65b, and 66b, collected on days 0 and 14 after immunization, were depleted of CD8+ or CD4+ cells by negative selection using immunomagnetic beads and stimulated with autologous blasts infected with S. Typhi. After 16 h, IFN- SFC were detected using a modified ELISPOT assay. Net frequencies of IFN- SFC (±SD) were calculated as described in Materials and Methods. Values of p represent the significance of the difference between the mean values before and after immunization.

IFN- production to S. Typhi STF evaluated by ELISA

We have previously shown that PBMC from volunteers immunized with attenuated S. Typhi strains CVD908 (4) and CVD908htrA (12) exhibited increased levels of IFN- production to STF soluble Ag, suggesting that CD4⁺ cells, in addition to CD8⁺ T cells, might be an important source of IFN- production during S. Typhi infection. To investigate this possibility, we measured by ELISA IFN- levels released in supernatants after 3 days of stimulation of PBMC with STF by CD4⁺ and CD8⁺ T cell subsets. These studies involved PBMC obtained from 9 placebos, as well as from 19 immunized volunteers (8 low dose and 11 high dose). Volunteers showing increases in IFN- production greater than the cutoff (31 pg/ml) as compared with preimmunization values were considered responders. Increases in IFN- production following immunization in response to STF soluble Ag occurred in 82% of volunteers and ranged from 67 to 1399 pg/ml (mean ± SE: 417 ± 130) in the high dose group (Fig. 3). Increases in the low dose group occurred in 38% of volunteers and ranged from 41 to 529 pg/ml (mean ± SE: 225 ± 94). IFN- production in the placebo group ranged from 0 to 87 pg/ml (mean ± SE: 21 ± 10) (Fig. 3). IFN- production levels were significantly higher in the high dose or low dose group as compared with the placebo group (p < 0.001 and p = 0.042, respectively; Mann-Whitney rank sum test). No significant differences were observed between the low dose and high dose groups (p = 0.355; Mann-Whitney rank sum test), most likely because of the relatively small number of volunteers evaluated.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. IFN- induction by soluble STF stimulation in volunteers following immunization with CVD 908-htrA. PBMC from 9 placebo volunteers as well as from 19 immunized volunteers (8 low dose and 11 high dose) were incubated with medium alone or with 2 µg/ml of STF Ag for 3 days. Supernatants were collected and assayed for IFN- by ELISA. Bars represent the mean concentration (picograms per milliliter) and SE observed for each group. The dashed line represents the cutoff (31 pg/ml) for ELISA. Values of p represent the significance of the difference in the mean values between the indicated groups.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Identification of the T cell populations secreting IFN- to STF Ag. PBMC from volunteer 66b were collected on days 0 and 28 after immunization and stimulated ex vivo with S. Typhi flagella Ag (10, 5, or 2.5 µg/ml) for 16 h. IFN- production was evaluated by intracellular staining with anti-IFN- PE-labeled mAbs and five-color flow cytometric analysis using Abs against CD3, CD4, CD8, and CD56, conjugated to the appropriate fluorochromes, as described in Materials and Methods. Data are presented as percentage of IFN--containing cells in CD3+CD4+CD8-CD56-- or CD3+CD4-CD8+CD56--gated populations. Total cells collected ranged from 144,000 to 247,000 per sample. Results shown are based on 10,000–18,000 CD3+CD4-CD8+CD56-- and 20,000–27,000 CD3+CD4+CD8-CD56--gated cells.

Correlations between IFN- secretion detected by ELISA and ELISPOT, CTL activity, and Ab levels

To evaluate whether the frequency of cells that secrete IFN- in response to stimulation with autologous S. Typhi-infected cells correlates with the concentration of IFN- released by PBMC in response to stimulation with STF soluble Ags, we compared the results obtained by ELISPOT with those obtained by ELISA. No significant correlation was found between the magnitude of the responses measured by ELISPOT and those determined by ELISA (r = 0.068, p = 0.387; linear regression analysis). In contrast, a strong correlation was found between responders and nonresponders assessed by these techniques (r = 0.822, p < 0.001; Pearson product moment correlation) (Table III).

View this table: [in this window] [in a new window]   Table III. Comparison between the frequency of IFN- SFC in the presence of S. Typhi-infected stimulator cells detected by an ex vivo ELISPOT assay, IFN- production in the presence of purified S. Typhi flagella assessed by ELISA, and S. Typhi LPS Ab levels in volunteers immunized with S. Typhi strain CVD 908-htrA

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Correlation between the peak concentrations of IFN- detected by ELISA and cytolytic activity of CTL effectors at E:T ratios of 30:1 (•; dashed line) and 20:1 (; solid line) detected by 51Cr release assays. PBMC from two placebos (22a and 23a), three low dose (22b, 27, and 34), and three high dose (23b, 31, and 35) volunteers were used for this analysis. Significance values of these correlations are shown.

Taken together, these results showing a close association between IFN- responses detected by ELISPOT and those detected by ELISA, and between ELISA and CTL activity, suggest that immunization with attenuated S. Typhi strains elicits concomitant IFN- responses by both CD4 and CD8 T cell subsets, as well as CTL activity by CD8⁺ T cells.

Another important consideration in establishing the breadth of the immune responses elicited by CVD 908-htrA is to attempt to correlate, on a volunteer-by-volunteer basis, Ab and CMI responses. No correlations were observed among these immunological responses (LPS Ab vs IFN- ELISPOT, p = 0.400; LPS Ab vs IFN- ELISA, p = 0.851; Pearson product moment correlation).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Consensus is emerging that protection to S. Typhi infection requires the induction of broad humoral and cellular immune responses (3). In recent phase I and II vaccine trials, we have shown that oral immunization with a single oral dose of CVD 908-htrA elicits strong Ab responses and IFN- production to soluble S. Typhi Ags (11, 12). However, heretofore, no studies had been performed to specifically assess the contribution of effector immune responses mediated by CD4⁺ and CD8⁺ T cells in volunteers immunized with CVD 908-htrA. Thus, to further evaluate the suitability of this promising typhoid vaccine candidate, we investigated its ability to induce broad CD4⁺ and CD8⁺ S. Typhi-specific responses, as well as the frequency of IFN--producing cells, in volunteers who participated in a double-blind crossover design phase II vaccine trial (12).

We observed that immunization with CVD 908 htrA elicits both CD4- and CD8-mediated CMI. These responses are evidenced by both CTL and IFN- production in response to S. typhi-infected targets mediated by CD3⁺CD8⁺ cells, as well as IFN- production to soluble S. typhi flagella mediated by CD3⁺CD4⁺ lymphocytes. Of note, we observed that IFN- production is not mediated by NK cells, because the responding populations were consistently CD3⁺CD56^-.

Our results indicate that S. Typhi-specific responses from CD8⁺ and CD4⁺ T cells are quantitatively different between volunteers who received low vs high doses. Considerably higher responses were observed in volunteers vaccinated with the high dose, both in terms of the percentage of responders, as well as in the magnitude of the responses. These observations suggest that doses of at least 5 x 10⁸ CFU of CVD 908-htrA will be required for successful immunization particularly if a single dose immunization schedule is pursued. Notably, their immune responses persisted until at least 56 days after immunization (the last time evaluated), adding support for the continuing investigation of this typhoid vaccine candidate.

An important factor to consider in evaluating the potential of a vaccine candidate is its ability to induce broad immunity in individual volunteers (20, 21). Thus, in the present study, we evaluated whether significant correlations were observed between IFN- production (in response to soluble S. Typhi flagella- or S. Typhi-infected targets) and CTL activity in individual vaccinees. Despite the relatively small number of volunteers studied, a significant correlation was observed between the volunteers that exhibit significant increases in IFN- secretion by ELISPOT (CD8 mediated) and increased IFN- released by ELISA (CD4 mediated), as well as between CTL activity and IFN- released by CD4⁺ T cells. Unfortunately, because of limited cell numbers, we could not directly compare the frequency of IFN--secreting cells as measured by ELISPOT and CTL responses. These results strongly suggest that immunization with CVD 908-htrA elicits broad CMI characterized by both CD4⁺ and CD8⁺ T cell responses, which might be required to eliminate S. Typhi infections. Moreover, these results extend our previous observations with Ty21a vaccinees, demonstrating a positive correlation between the frequency of IFN--secreting cells and cytotoxic activity (6). These results are in agreement with the observations in other human clinical trials that demonstrated the induction of both CD4⁺ and CD8⁺ T cell responses following immunization with other intracellular organisms, such as Plasmodium falciparum (20, 21).

Both CTL activity and IFN- production have been shown to be key mechanisms in protection against infection by intracellular pathogens, including bacterial, viral, and parasitic microorganisms (6, 22, 23, 24). The results presented in this work further support and expand previous data showing that intracellular bacteria, including S. Typhi (4, 6, 7, 9), can stimulate IFN- production by specific T cells. This, in turn, can lead to the recruitment and/or activation of the microbicidal activities of macrophages, neutrophils, and/or NK cells, as well as increases in the expression of immunologically relevant molecules such as those involved in Ag presentation (e.g., HLA class I molecules) (6, 22, 23, 24). Moreover, it has been proposed in viral infection models that the induction of CD4⁺ T cells is critical for maintaining functionality and full differentiation of CTL (25, 26). Although only limited information is available on the mechanisms by which T cells might protect from Salmonella infection (4, 5, 6, 11, 12, 27), the findings reported in this work on the concomitant induction of CD4- and CD8-mediated responses raise the possibility that similar mechanisms might be operative in infections with intracellular bacteria such as S. Typhi.

Another issue to be considered in the development of successful live oral typhoid vaccines is their ability to elicit potent immune responses in individuals of diverse genetic background. Host factors, such as highly polymorphic MHC HLA class I and II gene products on APC, play a key role in regulating immune responses to infection by presenting peptides to T cells (28). Thus, it would stand to reason that the higher the number and diversity of S. Typhi-derived peptides that can be presented by diverse HLA molecules following immunization, the higher the likelihood that a particular attenuated S. Typhi strain would become a viable vaccine candidate. Although the number of volunteers is to date limited, the preliminary results indicate that the CD4⁺ and CD8⁺ T cell responses elicited in CVD 908-htrA vaccinees are not restricted by specific HLA molecules. These results suggest that: 1) CVD 908-htrA immunization elicits S. Typhi-specific CMI responses using diverse HLA molecules that might present multiple naturally occurring S. Typhi epitopes, and 2) CVD 908-htrA immunization is likely to elicit S. Typhi-specific immune responses in a large fraction of the population.

The magnitude of Th1-type responses (levels of IFN- secretion to S. Typhi-infected targets by ELISPOT and CTL activity) in CVD 908 htrA vaccines is, in general, somewhat lower than those observed in Ty21a vaccinees (6). However, overall, the immune responses recorded with CVD 908-htrA compare favorably with those observed with Ty21a, taking into account that: 1) CVD 908-htrA was given as a single dose instead of three or four spaced doses required by Ty21a; 2) high dose volunteers received 5 x 10⁸ CFU CVD 908-htrA instead of the 2–6 x 10⁹ CFU/dose of Ty21a; and 3) similar percentages of responders were observed with both vaccines (66–80% of high dose CVD 908-htrA and Ty21a in the various effector CMI responses). Of note, the magnitude of increase in IFN- production to S. Typhi flagella in CVD 908-htrA vaccinees was similar to those reported in volunteers immunized with other attenuated S. Typhi strains (4) or three to four doses of Ty21a (7, 9, 10).

Interestingly, no correlations were observed between these CMI responses and serum Abs to S. Typhi LPS. These results confirm and extend our previous findings depicting the lack of a correlation on a volunteer-by-volunteer basis between the induction of humoral (serum Abs and Ab-secreting cells) and CMI (lymphoproliferation and IFN- production) responses to S. Typhi Ags in individuals immunized with various attenuated S. Typhi vaccine strains (4, 12). The current results confirm that there is a wide variation among individuals in the predominance of the humoral and CMI responses elicited by vaccination with attenuated strains of S. Typhi. Although the reason for this discrepancy is unclear, it could be the result of several factors, including: 1) the relatively small number of volunteers evaluated; 2) the use of lymphocytes isolated from peripheral blood rather than from mucosal tissues; and/or 3) the likely possibility that individual volunteers respond preferentially to B or T cell epitopes. In any event, currently available evidence argues that serum and mucosal Ab responses, as well as CMI, contribute to protection. However, the relative contribution of each of these effector immune mechanisms in protection can only be ascertained in efficacy trials or experimental challenge with wild-type S. Typhi in which both Ab and CMI responses are exhaustively investigated in a volunteer-by-volunteer basis.

In summary, the results presented in this study constitute the first evidence that oral immunization of volunteers with a single dose of CVD 908-htrA elicits specific concomitant IFN- production by CD4⁺ and CD8⁺ lymphocytes and CD8-mediated CTL. Moreover, they provide an estimate of the frequency of IFN--secreting cells induced by vaccination, which can be a useful tool in comparing the activity of various live oral typhoid vaccine candidates. Finally, the observed immune responses strongly support the further evaluation of CVD 908-htrA as a leading typhoid vaccine candidate.

	Acknowledgments

 
We are indebted to the volunteers who allowed us to perform these studies. We also thank the staff of Adult Clinical Studies Section at the Center for Vaccine Development, including Kathleen Palmer, Catherine Black, Ron Grochowski, Brenda Berger, Theresa Mowry, Elizabeth Peddicord, and Elisa Sindall. We also thank Regina Harley for excellent technical assistance in flow cytometric determinations.

	Footnotes

 
¹ This work was supported by Grants R01-AI-36525 (to M.B.S.) and NO1-AI-45251 (to M.M.L.) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

² Address correspondence and reprint requests to Dr. Marcelo B. Sztein, Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore Street, HSF 480, Baltimore, MD 21201. E-mail address: msztein{at}medicine.umaryland.edu

³ Abbreviations used in this paper: CMI, cell-mediated immunity; B-LCL, EBV-transformed lymphoblastoid B cell line; rhIL-2, human rIL-2; SFC, spot-forming cell; STF, S. Typhi flagella.

Received for publication August 22, 2002. Accepted for publication December 30, 2002.

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