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Originally published online as doi:10.1189/jlb.1004621 on April 7, 2005

Published online before print April 7, 2005
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(Journal of Leukocyte Biology. 2005;78:178-186.)
© 2005 by Society for Leukocyte Biology

Mucosal application of plasmid-encoded IL-15 sustains a highly protective anti-Herpes simplex virus immunity

Felix N. Toka*,{dagger} and Barry T. Rouse*,1

* Department of Microbiology, University of Tennessee, Knoxville; and
{dagger} Department of Preclinical Sciences, Immunology Laboratory, Faculty of Veterinary Medicine, Warsaw Agricultural University, Poland

1 Correspondence: Department of Microbiology, University of Tennessee, Walter’s Life Science Bldg. M409, 1414 Cumberland Ave., Knoxville, TN 37996. E-mail: btr{at}utk.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a DNA immunization against Herpes simplex virus (HSV), we examined the ability of plasmid-encoded interleukin-15 (pIL-15) to induce and maintain the mucosal B and T cell immune response. pIL-15 generated memory CD8+ T cell responses that were threefold higher and mainly maintained in the spleen, but high levels of immunoglobulin A antibodies were induced and maintained long-term in the vaginal mucosa. Both of these enhanced components of the immune responses were recalled rapidly upon challenge with a lethal dose of HSV McKrae, affording protection in mice immunized with codelivery of pIL-15. Our results show for the first time that intranasal administration of pIL-15 along with plasmid-encoded glycoprotein B of HSV leads to enhancement of primary and memory CD8+ T cell responses as well as humoral immune response. Therefore, a mucosal immunization strategy that incorporates a potent cytokine such as IL-15 as an adjuvant might induce protective mucosal immune responses that constitute the initial barrier at mucosal portals of pathogen entry.

Key Words: HSV • CD8+ T cells • IgG • IgA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mucosa is an entry route of many pathogens. Herpes simplex virus (HSV) and human immunodeficiency virus (HIV) are good examples that do not yet have an effective vaccine. Therefore, it is important that prospective vaccines impart maximal immunity at vulnerable mucosal sites, and a promising means to target the mucosa is through genetically engineered vaccines [1 ]. Mucosal administration of vaccine may not only be safe but may eliminate unwanted effects produced if certain adjuvants are delivered systematically [2 ]. Among various adjuvant candidates, there are many proinflammatory cytokines and chemokines that might be used to enhance the primary and memory immune responses to a level sufficient for protection (reviewed in ref. [3 ])

Although interleukin-15 (IL-15) was identified only recently, it appears to have been well-characterized in vitro [4 ], whereas in vivo studies about the immune modulation potential of IL-15 and its biological effects on the mucosal tissues are few. IL-15 is a 14- to 15-kD member of the {alpha}-helix bundle family of cytokines [5 ]. IL-15 signals through IL-15 receptors (IL-15R) ß and {gamma}, and the {alpha} subunit acts as a high-affinity receptor for binding the cytokine [6 , 7 ]. It shares many biological properties with IL-2, but unlike IL-2, which is produced by T cells, IL-15 is produced by macrophages, dendritic cells (DC), keratinocytes, and epithelial cells (reviewed in ref. [8 ]). This cytokine maintains the homeostasis of memory CD8+ T cells [9 ], and overexpression of IL-15 in vivo increased antigen-specific-driven memory CD8+ T cells after exposure to antigen [10 ].

The attractiveness of IL-15 as a candidate for genetic adjuvant is its ability to support generation and maintenance of memory CD8+ T cells [5 ]. Rubinstein et al. [4 ] reported that IL-15 administered together with a DC vaccine elicited an enhanced CD8+ T cell response. Furthermore, mice lacking IL-15 or IL-15R have a diminished population of memory CD8+ T cells, and such animals are not able to contain a virus infection [11 ].

Our study examined the ability of IL-15 to generate and sustain antigen-specific memory CD8+ T cells in the genital tract of mice after intranasal DNA encoding glycoprotein B of HSV (gBDNA) immunization followed by administration of IL-15-encoding plasmid (pIL-15). In this study, we assess T and B cell responses in the vaginal tract of mice, as this route constitutes an important portal of entry of HSV. Establishing intact defense mechanisms at this mucosal site would reduce incidence of genital HSV transmission. Our data show that pIL-15 maintained a good level of HSV-specific CD8+ memory T cells in the systemic compartment. It is important that pIL-15, given intranasally, maintained the memory level of mucosal immunoglobulin (Ig)A and IgG in the genital tract. These data strongly suggest that IL-15 modulates humoral and cellular immune responses that are maintained at sufficient levels required for protection against infection. Results are discussed in the context of rational vaccine design.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and viruses
C57BL/6 female mice were purchased from Harlan Sprague Dawley (Indianapolis, IN) and used at the age of 6–7 weeks. The Institutional Animal Care and Use Committee approved the experimental procedures performed on these animals. HSV McKrae strain was propagated in Vero cells and used as plaque-forming units (pfu)/ml.

Plasmid DNA and immunization
Human IL-15-encoding plasmid (hIL-15) was a kind gift of Dr. Perera Liyanage at National Insitutes of Health (NIH; Bethesda, MD) [12 ]. Plasmid DNA was prepared as described earlier [13 ] with a slight modification in the endotoxin-removal procedure. Briefly, following precipitation with polyethylene glycol, the plasmid DNA was further subjected to endotoxin removal by adding 3 M solution of sodium acetate at a ratio of 1:10 and was brought to a total volume of 1.0 ml with endotoxin-free water. The plasmid DNA was then incubated on ice for 5 min. Triton X-114 (0.03 vol, Sigma Chemical Co., St. Louis, MO) was added to the samples and thoroughly mixed and then incubated at 50°C for 5 min. The aqueous phase containing DNA was removed following centrifugation at 14,000 rpm in an Accuspin microfuge. DNA samples were subjected to another round of Triton X-114 and finally precipitated with 96% ethanol. The Limulus amoebocyte lysate test (Charles River Endosafe, Charleston, SC) was 0.13 EU/ml.

Plasmid encoding hIL-15 was tested for the effective expression of IL-15 in 293 free-style cells (Invitrogen, Carlsbad, CA). Transfection of the 293 cells was done with Superfect (Qiagen, Valencia, CA) transfection reagent, as per the manufacturer’s instructions. Supernatants were tested for the presence of IL-15 in an enzyme-linked immunosorbent assay (ELISA) DuoSet kit (R&D Systems, Minneapolis, MN), following the manufacturer’s instructions. Results are shown in Figure 1A .



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  		Figure 1. (A) Production of IL-15 by 293 free-style cells following transfection with pIL-15 used in studies described in this report. ELISA was performed as described in Materials and Methods. (B) Administration of pIL-15 in the primary immunization increases the number of SSIEFARL-specific CD8+ T cells. Mice were immunized as described in Materials and Methods, and cells were isolated at 9 days post-immunization. Spleen cells (1x106) were incubated in the presence of 2.5 µg HSV-gB498–505, 50 U IL-2, and GolgiPlug for 5 h and were subsequently stained with anti-CD8+-fluorescein isothiocyanate (FITC) and anti-interferon-{gamma} (IFN-{gamma})-phycoerythrin antibodies. FITC rat anti-IgG was used for isotype control (data not shown). Cytometry of IFN-{gamma}-producing cells and data analysis was performed in FACScan and CellQuest software (Becton Dickinson, San Jose, CA), respectively. Figures show representative data from two independent experiments. The responses are presented in each plot as mean ± SE percentage of IFN-{gamma}-producing CD8+ T cells obtained from four mice in each group. *P ≤ 0.05. (C) Proliferation of CD8+ T cells isolated from spleens of mice immunized or not and coimmunized with pIL-15 or not. T cell proliferation assessment was done as described in Materials and Methods. ß-gal, ß-Galactosidase; pgBDNA, plasmid gBDNA; cpm, counts per minute; CLN, cervical lymph nodes; MLN, mesenteric lymph nodes.

 
Unless otherwise stated, mice were immunized intranasally with pgBDNA twice, at days 0 and 21. Mice were sedated with avertin 25 µg/ml, and then 50 µg hIL-15 DNA and 50 µg gBDNA [admixed in 50 µl phosphate-buffered saline (PBS)] were instilled slowly into both nostrils. Mice were placed in a dorsal position for 10 min and later returned to cages for recovery. For some experiments, primary immune responses were measured at 9 days post-initial immunization, and memory responses were determined at 60+ days. To assess the potential of hIL-15 to modulate generation and maintenance of memory CD8+ T cells, pIL-15 was administered together with the immunizing agent or at the memory stage only (60+ days post-initial immunization).

Preparation of vaginal mucosa T cells
The vaginas from test and control mice were excised, placed in Hanks’ balanced salt solution (HBSS), cut longitudinally, and washed at least three times in HBSS. The tissue was later minced with sharp, surgical scissors and placed in RPMI 1640 without serum but containing 0.5 mg/ml Collagenase D. The tubes containing the tissue were incubated at 37°C on a shaker for 45 min–1 h and then forced through a metal sieve with a syringe plunger. The cell suspension was collected and washed in RPMI 1640 without serum three times and then resuspended in the same medium containing 10% serum. Cells were later used in enzyme-linked immunospot (ELISPOT) assay.

Intracellular staining for IFN-{gamma}
Spleen or lymph node cells were isolated from immunized or nonimmunized mice at appropriate times. Cells (1x106) were plated in U-bottomed microwell plates supplemented with 50 U IL-2, stimulated with HSV-1 immunodominant peptide SSIEFARL or unrelated peptide SIINFEKL (negative control), and placed at 37°C. Brefeldin A was added after 1 h. The cells were incubated for a total of 5 h. IFN-{gamma} was detected as described earlier [14 ]. Flow cytometry was performed in a Becton Dickinson FACScan, and data were corrected in CellQuest software.

Single-cell ELISPOT assay
ELISPOT was used to quantify IFN-{gamma}-producing CD8+ T cells at a single-cell level. MultiScreen-HA sterile plates (Millipore, Bedford, MA) were coated with capture antibody against IFN-{gamma} overnight in PBS at 4°C. Cells (1x106) were plated in the first well and then twofold-diluted to a final density of 1.25 x 105. Stimulator cells previously pulsed with SSIEFARL peptide and X-irradiated were added at a ratio of 1:5. Cells were cultured for 48–72 h. Detection of the spot-forming cells (SFC) was performed as described elsewhere [15 ].

Tetramer staining
Cells (106) were suspended in fluorescence-activated cell sorter buffer and stained with anti-CD8 monoclonal antibody and SSIEFARL-specific tetramers. Cells were then incubated for 45 min at 37°C, and cytometric analysis was performed using Becton Dickinson FACScan and CellQuest software.

Proliferation assay
CD8+ T cell proliferation was evaluated after in vitro stimulation of splenocytes with murine recombinant IL-15 (rIL-15; R&D Systems). Briefly, splenocytes isolated from immunized mice were plated on 96-well plates at 5 x 104 per well and incubated with varying concentrations of rIL-15 for 3 days. [3H]Thymidine (1 µCi/well) was added to each well 18 h before harvest. A ß-scintillation counter (Inotech) was used to determine the level of 3[H]thymidine incorporation, as a readout of proliferative capability of the tested cells.

Cytolytic T lymphocyte (CTL) assay
Spleen or lymph node cells isolated from test animals were expanded in culture for 5 days, and CTL measurement was done as described earlier [15 ]. Data were corrected by the formula (experimental release–spontaneous release)/(total release–spontaneous release) x 100.

Antibody ELISA
Genital tract washings were collected by flushing the vaginal vault several times with 200 µl PBS and frozen at –80°C until use. Costar enzyme immunoassay plates (Corning Inc., Corning, NY) were coated with gB protein, kindly supplied by Chiron (Emerville, CA) in phosphate buffer overnight at 4°C and then blocked for 2 h with 3% bovine serum albumin (BSA; Roche, Germany). A 1:20 dilution (in 1% BSA) of samples was added to the protein-coated wells and incubated overnight at 4°C. Later, horseradish peroxidase-conjugated, goat anti-mouse IgG, IgG1, IgG2a, and IgA were added to appropriate wells and incubated for 2 h at 37°C. Finally, the plates were washed five times, and detection buffer [2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] diammonium salt in 0.1 M citric acid and 3% hydrogen peroxide was added for 5–10 min. Plates were then read, and optical densities were corrected for concentration values with SpectraMax 340 ELISA reader and SoftMax Pro software (Molecular Devices, Sunnyvale, CA), respectively.

Virus challenge
Mice were challenged in the memory phase (65+ days post-secondary immunization). Depo-provera (medroxyprogesteron acetate, Pharmacia & Upjohn, Kalamazoo, MI) at 2 mg/ml was injected subcutaneously in female mice previously immunized as described earlier. Five days later, the mice were infected intravaginally with HSV McKrae at a dose of 1 x 107pfu/ml. Mice were scored for genital lesions, and vaginal washings were collected until day 15. Virus titration was determined on Vero cells as pfu/ml.

Statistical analysis
Where appropriate, significant differences were calculated with Student’s t-test. P values ≤0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mucosally given pIL-15 and gBDNA induce a higher CD8+ T cell response
We investigated the potential of pIL-15, applied intranasally, to modulate the immune response generated by immunization with plasmid DNA encoding gBDNA, also applied mucosally. Figure 1B shows the difference in CD8+ T cell responses in animals immunized with codelivery of pIL-15 and animals immunized without. At 9 days post-immunization, the IFN-{gamma} responses were 2.4- and 2.7-fold higher in the CLN and spleen, respectively, of pIL-15-treated mice compared with the nontreated. MLN, assessed at this time-point, also showed the presence of SSIEFARL-specific CD8+ T cells, although the difference (1.4-fold) between the pIL-15-treated and nontreated was not as marked as in the CLN or spleen. The exception here was the distal mucosal site (vaginal tract) in which no SSIEFARL-specific CD8+ T cells were detected at 9 days post-immunization (data not shown). The negative-control mice, given ß-gal DNA, did not show any response to SSIEFARL stimulation. These results confirmed the findings of others [9 , 10 , 16 ] that IL-15 also supports generation of the effector CD8+ T cell response.

pIL-15 supports bystander proliferation of CD8+ T cells
As application of pIL-15 influenced the acute responses to gBDNA immunization, we investigated whether the cytokine affected the responding CD8+ T cells directly. At 9 days after initial immunization, spleen CD8+ T cells were purified on separation columns (R&D Systems) and incubated with murine IL-15 (R&D Systems) for 72 h, followed by determination of [3H]thymidine incorporation. Figure 1C shows that IL-15 induced proliferation of CD8+ T cells, but there was almost no difference between mice immunized with codelivery of pIL-15 or not. The activity of IL-15 was not evident on naïve CD8+ T cells and only slightly above background in ß-gal-treated mice. However, ß-gal CD8+ T cells examined at 21 days post-immunization showed a proliferative response when exposed to IL-15, although the magnitude was less than that obtained from gB-specific CD8+ T cells (data not shown). We speculate that the difference in T cell reactivity between ß-gal and gB-treated mice may be a result of a low frequency of the immunodominant CD8 T cell epitope in ß-gal. Also, this could be a result of the nature of the plasmid encoding ß-gal. The increase in cpm was dose-dependent. This result suggested that IL-15 acted directly on CD8+ T cells that are initially activated by antigen. This could mean that the receptor for IL-15 is up-regulated on activated CD8+ T cells. Liu et al. [17 ] previously described similar proliferation results, as did Carrio et al. [18 ] and Berard et al. [9 ] recently.

pIL-15, given mucosally, maintains a high-systemic CD8+ T cell response
As there were no SSIEFARL-specific CD8+ T cells in the vaginal tract following primary immunization, we extended the observation into the memory phase to assess whether administration of pIL-15 mucosally would induce memory responses at this distal mucosal site. Mice were immunized at day 0 with pgBDNA and pIL-15 and boosted on day 21. One group was given a second dose of pIL-15 at the time of boost. Spleen, lymph node, and the vaginal tract cells were examined in vitro at 7 and 60 days after the boost. As shown in Figure 2A , at 7 days post-boost, IFN-{gamma}-producing, SSIEFARL-specific CD8+ T cells were detected in the vaginal tracts of mice, irrespective of whether the mice received pIL-15 or not. However, there were differences in the level of this response in various experimental groups. The group that was not given pIL-15 at all had the lowest number of SSIEFARL-specific CD8+ T cells, and the group that received a second dose of pIL-15 had the highest, with a 1.7-fold difference compared with non-pIL-15-treated mice. Single administration of pIL-15 did not contribute substantially to the increase of the CD8+ T cell response after boost, as only a 1.1-fold difference was observed between non-pIL-15-treated and the group that was treated once with pIL-15. When the vaginal tracts were examined at the memory phase (60 days post-boost), a diminished CD8+ T cell response was observed (Fig. 2B) , irrespective of pIL-15 treatment. This result suggested that the use of pIL-15 as a response modifier allowed increase and detection of antigen-specific CD8+ T cells in the vaginal tract, but these cells could not be maintained at higher levels for a longer period at this distal mucosal site.



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  		Figure 2. Secondary and memory responses in vaginal tract, CLN, MLN, and spleen. Mice were immunized at day 0 and boosted at day 21 with pgBDNA and given pIL-15 or not. Cells were isolated 7 days later to determine the number of IFN-{gamma}-producing CD8+ T cells following boost and 60 days to assess memory responses. (A) Appearance of CD8+ T cells in the vaginal tract after secondary stimulation. (B) Memory level of SSIEFARL-specific CD8+ T cell response. (C) Number of IFN-{gamma}-producing CD8+ T cells in the CLN, spleen, and MLN after boosting with pgBDNA. (D) Memory response in CLN, spleen, and MLN at 60 days post-boost. *, P ≤ 0.05, in comparison with non-pIL-15-treated mice; **, P > 0.05, in comparison with non-pIL-15-treated mice. (E) Percentage of SSIEFARL-positive CD8+ T cells in immunized mice during the memory phase. Spleen cells were isolated at 300 days after boost from immunized mice previously treated or not treated with pIL-15 and stained for SSIEFARL tetramer-positive CD8+ T cells. Flow cytometry was performed in a Becton Dickinson FACScan. *P≤ 0.05. Data shown were derived from two independent experiments. Each group contained four mice. Mean ± SE.

 
Spleen cells from mice treated with pIL-15 twice showed, at 7 days post-boost, an enhanced CD8+ T cell response (threefold) as compared with the non-pIL-15-treated, and the group that received pIL-15 at initial priming was only 2.1-fold higher compared with the non-pIL-15-treated mice (Fig. 2C) . A similar trend was observed in MLN responses, when the pIL-15-treated and non-pIL-15-treated animals were compared (Fig. 2C) . However, large differences were found when memory CD8+ T cell responses in the spleen were analyzed during the memory phase (60 days post-boost). In the pIL-15-treated, the CD8+ T cell response was 3.6-fold higher than in the non-pIL-15-treated mice (Fig. 2D) . Few antigen-specific CD8+ T cells or none at all were detected in the MLN at this time-point, and differences between mice given pIL-15 and mice that did not receive pIL-15 were not significant (P>0.05). These results showed that the memory responses in pIL-15-treated mice were maintained at a reasonably high level in comparison with mice that were not given the plasmid-encoded cytokine. Although the cytokine-encoding plasmid was administered mucosally, it did not preferentially support the mucosal CD8+ T cell memory in the distal mucosal site. Therefore, these data suggest that mucosal administration of pIL-15 supported the maintenance of the systemic pool of memory SSIEFARL-specific CD8+ T cells more than their mucosal counterparts. Retention of memory CD8+ T cells in the systemic compartment was still evident when the mice were examined at 300 days after boost. As shown in Figure 2E , cells isolated from the spleen of mice treated twice with pIL-15 still possessed SSIEFARL tetramer-positive CD8+ T cells, an indication that the cells were indeed antigen-specific, memory CD8+ T cells. Administration of pIL-15, only in the memory phase (60 days post-boost), did not enhance the subsequent memory CD8+ T cell response significantly (data not shown).

pIL-15 maintains a cytotoxic CD8+ T cell population
Next, we determined the cytotoxic potential of the pIL-15-generated CD8+ T cells to measure their functionality. A conventional chromium release assay was performed on cells isolated from the spleen during the memory phase (60+ days). Figure 3 shows that these cells were capable of lysing targets pulsed with SSIEFARL peptide. Although the cells were expanded for 5 days in vitro, the percentage lysis by splenocytes from pIL-15-treated mice was higher than that of non-pIL-15-treated. Taken together, pIL-15 maintained cytotoxic CD8+ T cells. However, the data do not allow a conclusion that pIL-15 increased the cytolytic capacity of CD8+ T cells against cells presenting the target antigen.



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  		Figure 3. (A) SSIEFARL-specific CD8+ T cell cytolytic activity in mice vaccinated intranasally with gBDNA and/or without codelivery of pIL-15. Splenocytes were isolated at 60 days post-boost (memory phase) from vaccinated and control mice and expanded in vitro with syngeneic-irradiated splenocytes pulsed with gB498–505 [specific for major histocompatibility complex (MHC) class I-restricted CD8+ T cells] for 5 days, followed by 51Cr release assay using MHC-matched MC38 (mouse colon adenocarcinoma) cells pulsed with gB498–505 as targets. MC38 pulsed with SIINFEKL was used as negative control. Data were corrected for percent cytolysis with the formula described in Materials and Methods. E:T, Effector:target ratio. (B) Number of IFN-{gamma}-producing CD8+ T cells in the vaginal tract at 3 days post-challenge, determined by ELISPOT assay, as described in Materials and Methods. (C) Percent of IFN-{gamma}-producing CD8+ T cells in the iliac lymph nodes (ILN) at 3 days post-challenge, determined by intracellular staining for IFN-{gamma}, as described in Materials and Methods. Data were derived from two independent experiments. Each experimental group contained six animals. *, P ≤ 0.05. Mean ± SE.

 
pIL-15 enhances antibody production by B cells
Protection of the mucosal surfaces is usually mediated through antigen-specific antibodies found in mucosal secretions. In mice, infection with HSV type 1 induces anti-HSV antibody production. Therefore, we assessed the antibody levels in vaginal vault fluid and serum after mucosal immunization with gBDNA and pIL-15. The samples were tested in an ELISA assay at 64 days post-boost. Although the levels of IgG in serum were not significantly high, there was still a 1.2-fold difference between pIL-15-treated and the nontreated (Table 1 ). Serum IgG responses were higher compared with vaginal tract IgG levels. An interesting observation, however, was the elevation of the IgA levels in the vaginal tract of pIL-15-treated mice versus nontreated (fourfold difference). This result is in agreement with the data reported by Hiroi et al. [19 ]. The level of IgA did not reduce substantially at 90 days and 300 days after immunization (data not shown). IgG levels in pIL-15-treated also remained elevated in comparison with non-pIL-15-treated mice. Naturally, the vaginal fluid inhibited virus replication when incubated together with virus before infection of Vero cells (data not shown). The results described above suggest that IL-15 influenced the activity of antigen-specific B cells, leading to heightened production of IgA. As the level of the two Igs remained reasonably stable for a long period, it is possible that IL-15 sustains B cell activity under normal conditions.


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  		Table 1. Concentrations of Anti-gB Antibodies in Mice Immunized with pgBDNA and pIL-15

 
Mucosal pIL-15 treatment affords protection against challenge with pathogenic HSV-1
We tested the efficacy of the memory SSIEFARL-specific CD8+ T cells to protect against challenge at 9 weeks from secondary immunization. Mice were given Depo-provera and 5 days later, infected intravaginally with HSV McKrae. Table 2 shows the number of surviving mice, virus titers, and illness score. Infection of the vaginal epithelium was observed in all mice challenged, irrespective of immunization status, but ß-gal-immunized mice developed extensive ulceration of the external vagina and were killed or died by 8–9 days. Immunized mice resisted progression of infection with the least resistance observed in mice that did not receive pIL-15 (two out of six immunized). Five out of six mice survived in the pIL-15-treated group. Following challenge, some mice from each experimental group were killed at days 3 and 5, and cells were isolated from the vaginal tracts and ILN. Figure 3B and 3C (day 5, data not shown), shows rapid appearance of IFN-{gamma}-producing CD8+ T cells in the vaginal tract and enhanced production of IFN-{gamma} from ILN cells. Although high responses were evident in all vaccinated groups, the pIL-15-treated mice showed the highest responses in the lymph node and the vaginal tracts. Therefore, the immune responses induced by pIL-15 were protective.


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  		Table 2. Challenge of Mice Mucosally Immunized with pgBDNA and Codelivery of pIL-15

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mucosal immunity against HSV has been studied extensively in mice, but still, no conclusive view has prevailed about the memory status of the mucosal responses following immunization. In this study, we have investigated an immunomodulator, IL-15, that is well-known for its properties to induce and sustain memory CD8+ T cell responses [5 , 9 , 10 , 16 ], mostly in systemic settings. However, much less data are available about the potential effects of IL-15 on the mucosal immunity. A plasmid construct of IL-15 was used together with plasmid DNA encoding an antigen to immunize mice intranasally. We found, as previously reported by others [5 ], that pIL-15 supported generation of primary CD8+ T cells upon exposure to microbial antigen and maintained an elevated level of antigen-specific CD8+ T cell response for a long period. However, although the immunization was done through the nasal route, the maintained T cell memory was found in the spleen and was minimally present in the distal mucosal site. It is interesting that the distal mucosal site showed an elevated IgA antibody level in mice that had been treated with pIL-15. Mucosal administration of vaccine together with IL-15 as an immunomodulator induced a rather transient cellular immune response but long-term humoral immune responses at the distal mucosal site. Overall, this strategy of immunization induced a strong, systemic immune response.

In IL-15–/– mice, 40–50% reduction in generation of primary CD8+ T cell response was observed by Schluns et al. [5 ] during infection with vesicular stomatitis virus (VSV). In the same studies, IL-15R{alpha}–/– mice behaved similarly, although to a lesser extent. In our studies, it is likely that the observed increase of primary CD8+ T cells could have been a result of augmented expression of the IL-15R{alpha} or -ß in the presence of overexpressed pIL-15. Support for this comes from the proliferation data, which showed the sensitivity of activated CD8+ T cells upon incubation with IL-15 in vitro, as shown in other reports [16 ]. However, we did not determine the level of IL-15R expression on the activated CD8+ T cells. Studies by Schluns et al. [5 ] showed that VSV-activated CD8+ T cells up-regulated the IL-15R{alpha}, but whether this was responsible for the increase in CD8+ T cell reactivity is not exactly clear. Recent reports [21 , 22 ], however, appear to argue against such a notion and show that IL-15R{alpha}-mediated presentation of IL-15 in trans may be the primary mechanism by which IL-15R{alpha} functions in vivo. Upon IL-15 treatment, a large number of genes are up-regulated; among them are genes such as Bcl-2, which contribute to the long-lived nature of memory CD8+ T cells [10 ]. It is the expression of such prosurvival molecules that endows memory CD8+ T cell persistence for long periods. Vaccination of IL-15 transgenic mice with bacillus Calmette-Guerin increased the memory CD8+ T cells that protected mice from challenge at 24 weeks after immunization [23 ]. Similarly, CD8+ T cells, generated in IL-15 transgenic mice, rapidly counteracted infection of mice with Listeria monocytogenes [24 ]. Also, administration of rIL-15 to mice on 6 consecutive days after infection with HSV-2 increased the number of natural killer, CD8+ T cells as well as enhanced their capacity to produce IFN-{gamma}, which led to reduced mortality by 80% [25 ]. Although the CD4+ T cells are also important in the protection of mice against HSV, none of these studies reported the enhancing effect of IL-15 on these cells. Therefore, IL-15 may selectively maintain the memory CD8+ T cells, which are responsible for the cytolytic activity and virus clearance during an infection.

Despite the reduced presence of cellular response at the distal mucosal site before challenge, the mice were protected against lethal challenge with HSV McKrae. Parr and Parr [26 ] made similar observations in their studies on intranasal immunization with attenuated HSV-2. In their study, the least response was observed in intranasally immunized mice, which, however, survived challenge equally well as those immunized intravaginally. A recent study [27 ] showed that overexpression of IL-15 led to resistance of infection with HSV-2 and that the anti-HSV antibody levels were higher compared with control mice. In another study, mortality of mice injected with rIL-15 for 6 days, after infection with HSV-2, was reduced by at least 80% [25 ]. However, both studies did not investigate the impact of the cytokine on memory T cell response. Our studies show that memory CD8+ T cell responses against HSV are maintained at considerably higher levels when IL-15 is overexpressed at initial priming and during antigenic boost.

The fact that antibody responses were enhanced by overexpression of pIL-15 indicated that this cytokine may be directly or indirectly influencing B cells. Some reports suggest that this could be mediated through helper T cells, as Loser et al. [27 ] showed increased activation of CD4+ T cells when IL-15 was overexpressed in keratinocytes. However, the nature of the interaction between IL-15 and CD4+ T cells remains ill-explained. The studies performed by Hiroi et al. [19 ] appear to explain our observations. They found that IL-15 and IL-15R were important in the development of B-1 cells that eventually differentiated into IgA-producing cells. Upon fractionation into B-1 and B-2 cells, the former highly expressed IL-15R mRNA. Addition of IL-15 to B-1 cells enhanced proliferation of B-1 cells and induced production of IgA, and addition of anti-IL-15 in vivo led to inhibition of mucosal IgA. Therefore, according to Hiroi’s report [19 ] and our findings, IL-15 is an important cytokine that can modulate mucosal IgA and IgG responses. What our study does not explain is whether the IgA levels observed were a result of a high number of mucosal-resident, IgA-producing cells or rather transudation from the bloodstream, as others have reported that B-1 cells mainly produce IgA in the circulation that prevent systemic infection by intestinal bacteria [28 ]. It is possible that the IgA found in the vaginal vault could be produced at a site further up the reproductive tract, as suggested by Parr and Parr [26 ]. However, we share the view that there is preferential homing of IgA-producing B cells to the mucosal tissue, which implies that there exist adhesion molecules that selectively mediate trafficking of mucosal IgA-producing precursor cells [29 ]. Further studies are required to localize the vaginal IgA-producing cells. Although we did not determine the presence of IgA-producing B cells, the fact that there was a prolonged presence of mucosal antibodies suggested that IL-15 acted on B cells through a mechanism similar to that in CD8+ T cells, i.e., through the Bcl-2 pathway, as suggested by Yajima et al. [10 ].

In our studies, there were few resident SSIEFARL-specific memory CD8+ T cells in the vaginal mucosa, but protective CD8+ T cells were recruited rapidly to the vaginal epithelium upon challenge with virus. This cause of events upon challenge has been reported by many [30 , 31 ], which seems to suggest that design of immunization strategies should concentrate on immune modulation that could enhance mucosally generated systemic memory and local mucosal antibody production. As a result of the fact that IL-15 enhances the survival, activation, and IFN-{gamma} production and has an important role in regulating CD8+ T cell homeostasis in vivo, others have proposed its trial in HIV immunotherapy [32 ]. It has been shown that there is defective production of IL-15 in AIDS patients, and this likely accounts for the impaired innate and adaptive immune responses during HIV infection [33 ].

In summary, our study shows that pIL-15 induced and maintained two layers of immune protection when administered intranasally with vaccine: a strong, systemic cellular immunity and a high humoral immunity at the distal mucosal surface. The enhanced immune responses ensuing from this strategy of immunization were protective upon lethal challenge with HSV-1. Therefore, IL-15 is a potent immune response modifier that may enhance the memory CD8+ T cell response and their effector function, leading to effective, long-term immune memory.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH Grant RO1 AI 4646 201.

Received October 29, 2004; accepted March 14, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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