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The Journal of Immunology, 2004, 173: 2692-2698.
Copyright © 2004 by The American Association of Immunologists

Oral Administration with Papillomavirus Pseudovirus Encoding IL-2 Fully Restores Mucosal and Systemic Immune Responses to Vaccinations in Aged Mice1

Raja Fayad, Hongtao Zhang, Daniel Quinn, Yujun Huang and Liang Qiao2

Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Medical Center, Maywood, IL


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Infectious diseases are one of the major threats for the elderly because their immune system is often compromised, and vaccinations to prevent these infections are not effective. A major defect in their immune system seems to be the inability of T cells to produce IL-2. We used papillomavirus (PV) pseudoviruses (PSVs) as a model vaccine and a gene delivery vector to address how to enhance immune responses to vaccinations. We found that oral immunization with PV PSV induced minimal mucosal and systemic Abs and CTLs specific for the PSVs in aged mice compared with young adult mice. In addition, fewer specific Th cells were generated in the aged mice. When aged mice were immunized with PV PSVs encoding human IL-2, specific Th cells were generated, producing murine IL-2, IL-4, and IFN-{gamma}. Further, specific Abs and CTLs were induced, resulting in protection against mucosal viral challenge. Thus, this study provided a basis for clinical trials using PV PSVs encoding IL-2 for vaccination of the elderly.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infectious disease is one of the most common causes of death among the elderly. Pathogenic viruses and bacteria readily infect mucosal surfaces in the body, particularly the intestinal mucosa, which is one of the most important portals for infectious agents.

Most pathogens initiate their infectious processes by interacting with epithelial cells at mucosal surfaces and then spread systemically. The mucosal immune system plays a critical role in protecting hosts against mucosal pathogens. However, immune system functions decline with age. In aged hosts, there are defects in humoral and cellular immunity (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) as well as in NK cell function (19, 20, 21). Thus, aged hosts are unable to generate particular Ab isotypes or sufficient numbers of high-affinity Ab-forming cells (3, 8, 10, 12, 14). T cells of aged hosts produce less IL-2 (22, 23), but more IL-4 and IFN-{gamma} (23, 24). It was also found that there is reduction in the early events of signal transduction (25, 26), TLRs (27), and decreased cellular proliferation in response to TCR stimulation in both the absence and presence of costimulation (28). More importantly, intestinal mucosal immune response in aged hosts is deteriorated (1, 4, 5, 9, 17, 18). Thus, GALT has shown to be reduced in number generally and in CD4+ T cells particularly (29). It has been shown that vaccines that prevent severe infections such as influenza and streptococcal pneumonia are less protective in the elderly (14, 30, 31, 32, 33, 34). Currently, no successful approach exists to enhance immune responses of the elderly to vaccinations to protect them from fatal infectious diseases.

Papillomavirus (PV)3 major protein L1 can be assembled spontaneously into virus-like particles (VLPs) when expressed in cells (35, 36, 37, 38, 39, 40). VLPs can be used to package unrelated plasmids to form PV pseudovirus (PSV) (41, 42, 43). Because of their mucosa-tropic property, the PV PSVs, when orally given to mice, are able to deliver the gene they carry to the mucosal and systemic immune systems, and can induce mucosal and systemic immune responses (42). Thus, PV PSV can be used as a mucosal vaccine and gene therapy delivery vector. In this study, we used the PSVs encoding lymphocytic choriomeningitis virus (LCMV) gp33–41 as a model vaccine to analyze impairments in immune responses to mucosal vaccination in aged host. More importantly, we used the PV PSVs to deliver the IL-2 gene to the immune systems of aged hosts so that the mucosal vaccination could induce effective immune responses and provide protection in aged hosts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Three-month-old C57BL/6 mice (purchased from Harlan Breeders, Indianapolis, IN) and 12-mo-old C57BL/6 mice (purchased from National Institute on Aging, Bethesda, MD) were used. All mice were kept in pathogen-free conditions. The protocol was approved by Institutional Animal Care and Use Committee.

Generation of PV PSVs

To generate PSV-LCMV and PSV-IL-2, we constructed two mammalian expression plasmids: pCI-GLP-LCMV encoding the green lantern protein (GLP) fused with LCMV gp33 H-2Db-restricted epitope (amino acids KAVYNFATC) and pCDNA/purIL-2 encoding human IL-2. Bovine PV (BPV)-1 VLPs were disrupted, pCI-GLP-LCMV or pCDNA/purIL-2 plasmids were mixed with disrupted VLPs, and then VLPs were reassembled according to previously described methods (42). We confirmed that plasmids were inside the VLPs as depicted in our previous publication (42).

Immunization

Three- and 12-mo-old C57BL/6 mice were immunized orally by blunt stainless steel needle (Fisher Scientific, Pittsburgh, PA) with 40 µg of PSV-LCMV in 100 µl of PBS alone, or with 40 µg of PSV-IL-2, or 40 µg of control BPV-VLPs, or 100 µl of PBS. Three to six mice were used in each group. Two weeks later, the mice were boosted orally with the same prime vaccines and dosages. Two weeks after the boost, the mice were sacrificed, and splenic and mucosal cells were isolated for further study. For the vaccinia virus protection assay, the mice were immunized, and boosted 2 wk later. Three months after the boost, the mice were inoculated with 5 x 108 PFU of recombinant vaccinia virus encoding LCMV glycoprotein intrarectally. Six days later, spleen, ovaries, lamina propria (LP) lymphocytes, and Peyer’s patch (PP) lymphocytes were isolated for further study.

Cells isolation

Spleens were mechanically disassociated, and RBC were lysed in ACK lysing buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2) and filtered into a nylon strain. Cells were washed, centrifuged, and resuspended into 4 ml of 37°C complete cell culture medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin; Invitrogen Life Technologies, Carlsbad, CA), and incubated in a nylon wool column at 37°C and 5% CO2 for 45 min. The cells then were run into the nylon wool column and washed with 50 ml of complete cell culture medium. The cells were centrifuged, resuspended in complete cell culture medium, and stored on ice until used.

LP lymphocytes were isolated by removing the small intestines and flushed their contents with cold PBS. After PPs were excised, intestines were opened longitudinally, minced into 5- to 10-mm pieces, and washed extensively with cold PBS. The mucosal pieces were then digested twice for 30 min each time at 37°C with 5 mM EDTA (Sigma-Aldrich, St. Louis, MO) and 10% FCS in PBS. After each digestion with EDTA, mucosal pieces were washed with cold PBS and the supernatants were discarded. The remaining tissues were then digested for four 30-min intervals in a buffer containing 100 U/ml collagenase (Sigma-Aldrich), 25 mM HEPES, 7 mM CaCl2, and 20% FCS in DMEM. After each 30-min interval, the released cells were centrifuged, washed, and stored in complete cell culture medium on ice, and the mucosal pieces were replaced in the collagenase buffer. After the fourth interval, the digestion buffer was supplemented with an additional 100 U/ml collagenase and 10% FCS, and the mucosal pieces were digested for two additional 30-min intervals. Large debris was eliminated from the cell suspension by passage through nylon wool columns at room temperature. Column elutant was diluted in RPMI 1640 medium supplemented with 5% FCS containing 0.3 mg/ml DTT (Invitrogen Life Technologies), and viable cells were isolated by centrifugation over Lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada). After centrifugation, cells were collected from the interface and washed. PPs were excised and homogenized through a 70-µm cell strainer (BD Biosciences, San Jose, CA), and washed with cold PBS. Viable cells were isolated using Lympholyte-M as in the LP lymphocytes preparation.

Secretory IgA (sIgA) detection

After immunization, the mice were sacrificed, and the small intestine contents were flushed with 5 ml of cold PBS containing a Protease inhibitor set (Roche Applied Science, Penzberg, Germany) in each mouse. The intestine contents were vortexed for 30 s and centrifuged for 10 min to remove insoluble material. The resulting extract was passed through a 0.45-µm filter (Millipore, Bedford, MA), and aliquots were stored at –20°C. ELISA was used to detect IgA specific for BPV-VLPs. BPV-VLPs (200 ng/100 µl/well) were incubated in 96-well plates at room temperature overnight or with PBS alone as a control. Blocking solution (PBS with 1% BSA, 200 µl/well) was incubated at room temperature for 1 h, then intestinal extracts were diluted in blocking solution and incubated in the plates (100 µl/well) for 2 h. The intestinal extracts were washed, and 50 µl of biotinylated anti-mouse IgA (Sigma-Aldrich) (1:1000 in 1% BSA/PBS, 2 mg/ml) was added to the wells and incubated for 1–2 h at room temperature and then washed. Streptavidin-HRP (DakoCytomation, Carpinteria, CA) was incubated in a volume of 100 µl/well (1:5000 in PBS) for 30 min at room temperature, then washed. The substrate 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) was added (100 µl/well), and the reaction was stopped by adding 100 µl of 2 N H2SO4 per well. The ODs at 450 nm were measured by an ELISA reader.

Systemic IgG detection

After immunization, the mice were sacrificed, the blood was withdrawn from the heart and centrifuged, and serum was collected. BPV-VLPs (200 ng/100 µl/well) were incubated overnight in 96-well plates at room temperature or with PBS alone as a control. Blocking solution (200 µl/well) was incubated at room temperature for 1 h, then the sera were diluted in blocking solution and incubated in the plates (100 µl/well) for 2 h. The sera were washed, and 50 µl of (1:1000 in 1% BSA/PBS, 2 mg/ml) biotinylated anti-mouse IgG (Sigma-Aldrich) was added to the wells and incubated for 1–2 h at room temperature and then washed. Streptavidin-HRP (DakoCytomation) was incubated in the volume of 100 µl/well (1:5000 in PBS) for 30 min at room temperature, then washed. The substrate 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) was added (100 µl/well), and the reaction was stopped by adding 100 µl of 2 N H2SO4 per well. The OD at 450 nm was measured by an ELISA reader.

Cytotoxicity assay

Target cells (1 x 106 RMA cells, murine T cell lymphoma cell line) were labeled with 51Cr (100 µCi) for 1 h at 37°C and washed three times. The RMA cells were pulsed with 5 µg/ml LCMV gp33–41 peptide or human PV (HPV)-16 E7 peptide aa 49–57 (RAHYNIVTF, H-2Db-restricted epitope) as control. Splenic lymphocytes were cultured with LCMV gp33–41 peptide (5 µg/ml) and T-stim (IL-2 culture supplement without Con A; BD Biosciences) for 5 days at 37°C and 5% CO2, and then used for the CTL assay, whereas mucosal lymphocytes (LP and PP lymphocytes) were mixed and immediately used in the assay. Target cells (2000 cells/well) were then incubated with effector cells at different E:T ratios in V-bottom 96-well microtiter plates for 6 h at 37°C. Supernatant was collected and 51Cr release was quantified by gamma counter (Valeant Pharmaceuticals, Costa Mesa, CA). Specific lysis was calculated according to the formula: specific lysis (%) = [(experimental release – spontaneous release)/(maximum release – spontaneous release)] x100. Spontaneous release was determined in control microcultures containing 51Cr-labeled target cells in culture medium with no effector cells. Maximum release was determined by lysing 51Cr-labeled target cells with 0.5% (v/v) Nonidet P-40.

T cell proliferation

After immunization, LP, PP, and splenic lymphocytes were isolated from each mouse. Spleen feeder cells from untreated C57BL/6 mice were isolated and gamma irradiated (50 Gy). Mucosal lymphocytes (LP and PP lymphocytes) or splenic lymphocytes (105) were cultured with different dosages of VLPs and with irradiated spleen feeder cells (104) in round-bottom microtiter plates in complete cell culture medium in a total volume of 200 µl/well. Three days after incubation at 37°C and 5% CO2, the cells were pulsed with [3H]thymidine (1 µCi/ml; Amersham Biosciences, Piscataway, NJ) for 16 h at 37°C and 5% CO2. The cultures were harvested with a PHD harvester (Cambridge Technology, Cambridge, MA), and the [3H]thymidine incorporation was measured in a liquid scintillation spectrometer (Packard Bioscience, Cincinnati, OH).

ELISPOT

Ninety-six-well filtration plates (Millipore; Fisher Scientific) were coated overnight at 4°C with 100 µl of purified anti-mouse-IL-2, anti-IL-4, or anti-IFN-{gamma} Abs (2 µg/ml) (BD Pharmingen, San Diego, CA). Three-fold dilutions of splenic lymphocytes or mucosal lymphocytes (LP and PP lymphocytes) in complete cell culture medium were added to the wells along with 105 gamma-irradiated (50 Gy) feeder spleen cells plus 3 µg/ml BPV-VLPs. After 48-h incubation, the plates were washed, followed by incubation with 100 µl of biotinylated anti-mouse IL-2, IL-4, or IFN-{gamma} Abs (2 µg/ml) (BD Pharmingen). Streptavidin-HRP (1 µg/ml; DakoCytomation) was incubated in a volume of 100 µl/well (1:1000 in 1% Tween 20/PBS) for 1–2 h at room temperature. Spots were developed using freshly prepared substrate buffer (0.33 mg/ml 3-amino-9-ethyl-carbazole and 0.015% H2O2 in 0.1 M sodium acetate, pH 5).

Vaccinia virus expressing LCMV gp33–41

The LCMV glycoprotein gene was amplified by PCR and cloned into the SmaI site of the plasmid pSC11 (44) by blunt end ligation. The resulting pSC11 plasmid, containing LCMV glycoprotein in the correct orientation, was introduced into the thymidine kinase gene of the WR strain of vaccinia virus by homologous recombination (45). The resultant recombinant vaccinia virus encoding LCMV glycoprotein was plaque-purified four times, and the identity of the glycoprotein gene in the recombinant virus was confirmed by sequencing.

Vaccinia virus protection assay

Three months after the boost with PSV-LCMV alone or PSV-LCMV plus PSV-IL-2, or control PV-VLPs, the mice were challenged by intrarectal inoculation with 5 x 108 PFU of vaccinia virus encoding LCMV glycoprotein. Six days after viral challenge, the mice were sacrificed, and both ovaries of each mouse were removed, homogenized, and resuspended in DMEM supplemented with 5% FCS at a concentration of one ovary per milliliter. Samples from individual mice were kept at –70°C. To measure virus titers, samples were thawed, sonicated, and assayed by plating serial 10-fold dilutions on CV-1 cells in 24-well plates. Plates were incubated for 48 h and stained with crystal violet (0.1% w/v crystal violet in 20% v/v ethanol). Individual plaques at each dilution were counted. The limit of detection of virus plaques by this assay was 10 PFU.

Statistical analysis

Data are reported as means ± SE or means ± SD, and were analyzed using the SPSS statistical software (SPSS 8.0; SPSS, Chicago, IL). Data were also analyzed by one-way ANOVA, followed by a least significance difference test. A p value < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mucosal vaccination with PV PSV does not effectively induce specific Abs, CTLs, or protection

We initially determined whether oral immunization with PV PSV-LCMV induced mucosal and systemic immune responses in aged mice. Young adult (3 mo old) and aged mice (1 year old) were immunized orally with PSV-LCMV. We used 1-year-old mice because they were reported to present immune dysfunction, such as decrease in Ab production (4, 5). The PSV-LCMV was made of BPV-VLPs that were packaged with a plasmid encoding an H-2Db-restricted LCMV glycoprotein epitope (aa 33–41). Two weeks after the oral boost, sIgA and serum IgG specific for BPV-VLPs were measured by ELISA, and mucosal and splenic CTL responses to the LCMV gp33–41 epitope were determined by a 51Cr-release assay. Compared with those in young adult mice, minimal BPV-VLP-specific sIgA and serum IgG were found in aged mice immunized with PSV-LCMV (Fig. 1). Moreover, mucosal and systemic LCMV-specific CTL responses were significantly lower in aged mice compared with those in young adults immunized with PSV-LCMV (Fig. 2). Oral administration with BPV-VLPs alone did not induce LCMV-specific mucosal and systemic immune responses (data not shown). Thus, the mucosal vaccine did not induce strong mucosal and systemic immune responses in aged mice, which is similar to the effects of many other vaccines in the elderly.



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  		FIGURE 1. BPV-VLP-specific IgA and IgG production in young adult and aged mice. Twelve- and 3-mo-old B6 mice (three per group) were immunized orally with PSV-LCMV alone or PSV-LCMV plus PSV-IL-2 or PBS (control) and boosted orally. Two weeks after the boost, intestinal washings and sera were obtained as described in Materials and Methods, and BPV-VLP-specific sIgA (A) and serum IgG (B) were determined by ELISA. The data are the mean value of OD450 from each group, and the SD was ≤8%, representing one experiment of three performed. *, and **, p ≤ 0.002 for the OD450 value of sIgA and serum IgG from aged mice immunized with PSV-LCMV plus PSV-IL-2 vs that from aged mice immunized with PSV-LCMV alone.

 


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  		FIGURE 2. Mucosal and systemic LCMV-specific CTL responses in young adult and aged mice. Twelve- and 3-mo-old B6 mice (five per group) were immunized orally with PSV-LCMV alone or PSV-LCMV plus PSV-IL-2 or PBS (control) and boosted orally. Mucosal and splenic lymphocytes were isolated. LCMV-specific cytolytic activity was determined among mucosal lymphocytes (A) and splenic lymphocytes (B) by 51Cr-release assay. Target cells were RMA cells pulsed with HPV-16E7 peptide (control) or LCMV gp33–41 peptide. The data are the mean value of the cpm from each group, and the SD was ≤11%, representing one experiment of three performed. *, p ≤ 0.036; **, p ≤ 0.02; §, p ≤ 0.017; and §§, p ≤ 0.003 vs the percentage of specific lysis in aged mice immunized with PSV-LCMV alone.

 
To determine whether oral immunization of PSV-LCMV can protect mice against mucosal viral challenge, we immunized young adult and aged mice orally with PSV-LCMV or control PV-VLPs and boosted them orally. Three months after the boost, the mice were infected rectally with 5 x 108 PFU of recombinant vaccinia virus encoding LCMV glycoprotein. Six days later, the mice were sacrificed. The ovaries from the mice were obtained, and the viral titers were measured by plaque assay. The young adult mice immunized with PSV-LCMV cleared the virus (Log titer = 0), whereas those immunized with PV-VLPs (control) did not. In contrast, in the aged mice immunized with PSV-LCMV, the immunization did not provide full protection (Log titer = 2.9) (Fig. 3).



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  		FIGURE 3. Protection of the aged mice against mucosal challenge with vaccinia virus encoding LCMV glycoprotein. Twelve- and 3-mo-old B6 mice were immunized orally with PSV-LCMV alone or PSV-LCMV plus PSV-IL-2 or control PV-VLPs, and boosted orally. Three months after the boost, the mice were challenged once with recombinant vaccinia virus encoding LCMV glycoprotein rectally, and 6 days later were sacrificed. The viral titers were determined from the ovaries of each mouse. The data are the mean value ± SE of viral titer in each group (the total numbers of mice immunized with PSV-LCMV plus IL-2 are six mice per group; the other groups have four mice per group). *, p < 0.02 for the vaccinia virus titer from aged mice immunized with PSV-LCMV plus PSV-IL-2 vs that from aged mice immunized with PSV-LCMV only.

 
Fewer specific Th cells were generated by the vaccine in the aged mice

The reduced levels of BPV-VLP-specific sIgA, serum IgG, and mucosal and systemic CTL immune responses to LCMV glycoprotein epitope could be due to the lack of PSV-specific Th cells in aged immunized mice. To test that hypothesis, we determined whether mucosal and splenic lymphocytes from mice immunized with PSV-LCMV proliferated in response to BPV-VLPs in vitro. Mucosal T cell proliferation from aged mice immunized with PSV-LCMV in response to in vitro stimulation with BPV-VLPs was significantly lower than that in young adults (Table I, Mucosal lymphocytes). In addition, splenic T cell proliferation from aged mice immunized with PSV-LCMV was lower than that in young adult mice (Table I, Splenic lymphocytes). We further determined whether BPV-VLP-specific Th cells were generated in the aged mice by using the ELISPOT assay. As expected, both mucosal and systemic BPV-VLP-specific IL-2, IL-4, and IFN-{gamma}-secreting T cell numbers in aged mice were fewer than those in young adult mice (Fig. 4), suggesting that both BPV-VLP-specific Th cells were not efficiently expanded.


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  		Table I. Mucosal- and splenic-specific Th cell proliferation in immunized micea

 


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  		FIGURE 4. Quantification of BPV-VLP-specific T cells by ELISPOT. Twelve- and 3-mo-old B6 mice (five per group) were immunized orally with PSV-LCMV alone or PSV-LCMV plus PSV-IL-2 or PBS (control) and boosted orally. Two weeks after the boost, mucosal and splenic lymphocytes were isolated and BPV-VLP-specific T cells producing IL-2, IL-4, and IFN-{gamma} were determined by ELISPOT as described in Materials and Methods. A, Mucosal T cells, B, splenic T cells. The data are the mean value ± SE from each group, representing one experiment of two performed. * and §, p ≤ 0.049; and {dagger}, p ≤ 0.05 for aged mice immunized with PSV-LCMV plus PSV-IL-2 vs aged mice immunized with PSV-LCMV alone.

 
Oral administration of PV PSV encoding IL-2 augmented the generation of specific Th cells

Because the reduction in IL-2 production by T cells in aged hosts may be a critical deficiency, we hypothesized that oral administration with PSV-encoding IL-2 (PSV-IL-2) plus PSV-LCMV in aged mice would result in expression of IL-2 in lymphoid tissue where the PSV reached. We expected that the IL-2 would initiate activation of specific T cells so that they might further produce IL-2 by themselves. To test this, aged and young adult mice were immunized with PSV-LCMV plus PSV-IL-2. The number of BPV-VLP-specific T cells producing IL-2, IL-4, and IFN-{gamma} was then determined by ELISPOT. Immunization with PSV-LCMV and PSV-IL-2 in aged mice generated mucosal and systemic BPV-VLP-specific T cells producing IL-2, IL-4, and IFN-{gamma} (Fig. 4). Further, young adult mice immunized with PSV-LCMV and PSV-IL-2 had higher numbers of T cells producing IL-2, IL-4, and IFN-{gamma} both mucosally and systemically compared with that in young adult mice immunized with PSV-LCMV only (Fig. 4). The data demonstrate that specific Th cells were generated.

We then examined mucosal and systemic T cell proliferation in response to BPV-VLPs in vitro in the aged mice immunized orally with PSV-LCMV only or with PSV-LCMV plus PSV-IL-2. We found that mucosal T cell proliferation in response to BPV-VLP stimulation was higher in aged mice immunized with PSV-LCMV plus PSV-IL-2 compared with that in the same age mice immunized with PSV-LCMV only (Table I, Mucosal lymphocytes). Similarly, splenic T cell proliferation in response to BPV-VLPs stimulation was higher in aged mice immunized with PSV-LCMV plus PSV-IL-2 compared with that in aged mice immunized with PSV-LCMV only (Table I, Splenic lymphocytes).

PSV-LCMV plus PSV-IL-2 induced PSV-specific CTLs, Abs, and protected aged mice against mucosal viral challenge

Young adult and aged mice were immunized orally with PSV-LCMV alone or PSV-LCMV plus PSV-IL-2 and boosted once. In aged mice immunized with PSV-LCMV plus PSV-IL-2, mucosal BPV-VLP-specific sIgA and systemic IgG levels were significantly higher (p ≤ 0.002; Fig. 1) than in same-age mice that were immunized with PSV-LCMV only. In young adult mice, BPV-VLP-specific sIgA and IgG levels were slightly higher in mice immunized with PSV-LCMV and PSV-IL-2 than those in young adult mice immunized with PSV-LCMV only (Fig. 1). Furthermore, in aged mice immunized with PSV-LCMV plus PSV-IL-2, the mucosal and systemic CTL responses to LCMV gp33–41 were higher than those immunized with PSV-LCMV only (Fig. 2). The mucosal and systemic CTL responses in young adult mice immunized with PSV-LCMV plus PSV-IL-2 were also higher than those immunized with PSV-LCMV only (Fig. 2). Our data demonstrate that immunization with PSV-LCMV plus PSV-IL-2 induced mucosal and systemic immune responses in aged mice to a level similar to that in young adult mice.

To determine whether immunization with PSV-LCMV plus PSV-IL-2 provided protection for aged mice against a mucosal viral challenge, we immunized young adult and aged mice orally with PSV-LCMV or PSV-LCMV plus PSV-IL-2 and boosted them 2 wk later. Three months after the boost, the mice were infected rectally with 5 x 108 PFU of recombinant vaccinia virus encoding LCMV glycoprotein. Six days later, the mice were sacrificed. The viral titers in ovaries were measured. The PSV-LCMV plus PSV-IL-2 immunization significantly improved the protection against the viral challenge in the aged mice (Fig. 3). We also determined the CTL responses to LCMV in the aged mice after the challenge. The LCMV-specific CTL response was present significantly in the aged mice immunized with PSV-LCMV plus PSV-IL-2, but not with PV-VLPs or PSV-LCMV, indicating that PSV-specific memory CTLs were induced by PSV-LCMV plus PSV-IL-2 (Fig. 5).



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  		FIGURE 5. Mucosal LCMV-specific memory CTLs in immunized mice. Twelve- and 3-mo-old B6 mice were immunized orally with PSV-LCMV alone (n = 4 per group) or PSV-LCMV plus PSV-IL-2 (n = 6 per group) or control PV-VLPs (n = 4 per group), and boosted orally. Three months after the boost, the mice were challenged intrarectally with vaccinia virus encoding LCMV glycoprotein for challenge test. Six days after the challenge, mucosal and splenic lymphocytes were isolated. CTL responses were determined in 51Cr-release assay. Target cells were RMA cells pulsed with HPV-16E7 peptide (control) or LCMV gp33–41 peptide. PV-VLPs did not induce specific cytolytic activity in both aged and young adult mice (data not shown). The data are the mean value of cpm from each group, and the SD was ≤15%. *, p ≤ 0.008; and **, p ≤ 0.05 for percentage of specific lysis in aged mice immunized with PSV-LCMV plus PSV-IL-2 vs that in aged mice immunized with PSV-LCMV alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we used PSV-LCMV (plasmids encoding LCMV glycoprotein aa 33–41, an H-2Db-restricted CTL epitope, packaged in BPV-VLPs) to determine whether oral immunization with this vaccine induced an effective immune response and protection in aged mice. We have previously shown that this PSV, when given orally, can induce mucosal and systemic CTL responses and provide protection in young adult mice (42). Because dysfunction of mucosal lymphoid tissues appeared earlier than that of systemic lymphoid tissues, we tested whether intestinal mucosal immune system responded to oral immunization with this vaccine. Thus, we analyzed the immune responses to different components of the PSV, i.e., Ab and Th responses to VLPs, and CTL responses to the LCMV gp33–41 epitope, in aged mice after immunization. We demonstrated that oral vaccination with PSV-LCMV did not induce effective cellular and humoral immune responses and protection in aged mice. Thus, the system serves as a model to address the important issue of how to develop an effective vaccination approach that could induce strong cellular and humoral immune responses and provide protection in the aged host.

In contrast to the young adult mice, the aged mice produced very low levels of mucosal and systemic BPV-VLP-specific Abs and LCMV-specific CTLs after oral immunization with PSV-LCMV. Furthermore, there were fewer BPV-VLP-specific Th cells that could produce IL-2, IL-4, and IFN-{gamma}, and proliferate after in vitro stimulation. Because generation of Ab and CTL responses in general require activation of Th cells, the reduced Ab and CTL responses to the vaccination were most likely due to inability of the Th cells to proliferate in response to the immunogen. It has been shown that IL-2 and its receptor were lower in aged hosts than in younger hosts (22, 31, 46). It appears that the defect in IL-2 production might be the only critical deficiency of aged naive CD4 T cells because T cells could be activated in vitro in the presence of exogenous IL-2 (22, 47, 48). Thus, the reduced proliferation of Th cells to PV PSVs may be due to an inability of those cells to produce IL-2.

We have previously shown that oral administration of PV PSV can deliver a gene, e.g., GLP packaged inside the PSV to mucosal and systemic lymphoid tissues (42). The GLP was expressed in the lymphoid tissues for about 2 wk and then disappeared. Because of this feature of transient expression, it is suitable to deliver certain genes to lymphoid tissues so that they are expressed only temporarily and no harm would be caused. Thus, we used PV PSV to deliver the human IL-2 gene to the lymphoid tissues together with the mucosal vaccine. In this way, IL-2 would be expressed in the lymphoid tissues so that it could promote proliferation of Th cells. Indeed, we found that when aged mice were immunized with PSV-LCMV plus PSV-IL-2, BPV-VLP-specific Th cells were generated; these T cells produced IL-2, IL-4, and IFN-{gamma} in response to BPV-VLPs stimulation in vitro. The IL-2 produced by the murine T cells was not derived from the PSVs because we used the human IL-2 gene in the PSV, and we could not detect the mRNA of human IL-2 in the murine T cells (data not shown). It has been shown that dendritic cells are functionally intact in aged hosts (49, 50); we believe that human IL-2 was initially expressed in these cells and that IL-2 helped activate specific Th cells to produce murine IL-2 by themselves. Because the murine T cells produced IL-2 by themselves, it appears that in aged mice there is a defect in activation of the T cells to produce IL-2. Once activated, these T cells could fully function as Th cells, as evidenced by the induction of specific CTLs and Abs. Thus, future studies will address how exogenous IL-2 helps activate T cells in aged hosts.

Due to the activation and proliferation of specific Th cells, significant PSV-specific CTL and Ab responses were augmented in the aged mice to levels similar to those in young adult mice. Three months after immunization, aged mice were significantly protected against rectal mucosal challenge with vaccinia virus encoding for LCMV glycoprotein, suggesting that specific memory T cells were generated. Although this challenge system only tested the arm of cellular immune response, the Ab response would be equally efficient in protecting the host based on the levels of serum IgG and mucosal IgA.

This study demonstrated a novel approach to induce mucosal and systemic immune responses in aged mice by using PV PSV encoding IL-2. The PSV encoding IL-2 could be used to enhance not only PV PSV vaccines but also other preexisting vaccines (e.g., influenza) in elderly who do not respond well to vaccinations. Thus, this study provides the basis for future clinical trials to develop effective vaccination techniques to protect the elderly from infectious diseases.


   Acknowledgments
 
We thank Dr. Wei Shi for his technical advice, and Amanda Knop for critical reading of this manuscript.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Research Scholar Grant #RSG-02-247-01-MBC from the American Cancer Society, and by National Institutes of Health Grant CA81254. Back

2 Address correspondence and reprint requests to Dr. Liang Qiao, Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153. E-mail address: lqiao{at}lumc.edu Back

3 Abbreviations used in this paper: PV, papillomavirus; PSV, pseudovirus; VLP, virus-like particle; LCMV, lymphocytic choriomeningitis virus; sIgA, secretory IgA; BPV, bovine PV; LP, lamina propria; GLP, green lantern protein; PP, Peyer’s patch; HPV, human PV. Back

Received for publication February 11, 2004. Accepted for publication June 15, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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