HUMAN GENE THERAPY 13:1415–1425 (August 10, 2002) © Mary Ann Liebert, Inc. Intranasal Gene Transfer by Chitosan–DNA Nanospheres Protects BALB/c Mice Against Acute Respiratory Syncytial Virus Infection MUKESH KUMAR,1 ARUNA K. BEHERA,1 RICHARD F. LOCKEY,1 JIAN ZHANG, 1 GURAMAN BHULLAR,1 CRISTINA PEREZ DE LA CRUZ,2 LI-CHEN CHEN,3 KAM W. LEONG,2 SHAU-KU HUANG, 3 and SHYAM S. MOHAPATRA 1 ABSTRACT Respiratory syncytial virus (RSV) infection is often associated in infancy with life-threatening bronchiolitis, which is also a major risk factor for the development of asthma. At present, no effective prophylaxis is available against RSV infection. Herein, we describe an effective prophylactic intranasal gene transfer strategy utilizing chitosan–DNA nanospheres (IGT), containing a cocktail of plasmid DNAs encoding all RSV antigens, except L. A single administration of IGT (25 mg/mouse) induces expression of the mRNA and proteins of all antigens in the lung and results in a significant reduction of viral titers and viral antigen load after acute RSV infection of these mice. IGT-administered mice show no significant change in airway reactivity to methacholine and no apparent pulmonary inflammation. Furthermore, IGT results in significant induction of RSVspecific IgG antibodies, nasal IgA antibodies, cytotoxic T lymphocytes, and interferon-g production in the lung and splenocytes compared with controls. Together, these results demonstrate the potential of IGT against acute RSV infection. OVERVIEW SUMMARY We describe an intranasal gene transfer approach against RSV infection, using chitosan nanospheres. Intranasal administration of chitosan nanospheres, containing plasmid DNAs encoding nine different RSV antigens, into the mouse lung reduced RSV titers and induced the production of antiRSV antibody with neutralizing properties. The therapy also enhanced interferon-g production in spleen and lung and generated cytotoxic T lymphocyte responses against RSV. This prophylactic gene expression therapy also reduced RSV-induced lung inflammation. We conclude that intranasal gene transfer utilizing chitosan nanospheres may be useful against acute RSV infection. INTRODUCTION R (RSV), the most common cause of viral lower respiratory tract infections in infants ESPIRATORY SYNCYTIA L VIRUS and children, affects about 4 million children globally and causes about 100,000 hospitalizations and 4500 deaths per annum in the United States alone (Centers for Disease Control and Prevention, 1999). Acute RSV infection is associated with episodes of bronchiolitis, wheezing, and exacerbation of asthma in children (Chanock et al., 1992). In the 1960s, children administered a formalin-inactivated RSV vaccine developed exaggerated disease when subsequent RSV infection occurred (Chanock et al., 1992). The development of a protective RSV vaccine has been a high priority at a global level. No effective vaccine is currently available for RSV infection. Experimental vaccines have included subunit, peptide, attenuated-live, and RSV DNA vaccines, and some have progressed to clinical trials (Hall, 1994; Brandenburg et al., 2001). Immunization with plasmid DNAs can potentially lead to more efficient antigen processing that induces a strong protective cellular and humoral immune response, as well as greater safety and cost-effectiveness (Cohen et al., 1998; Donnelly et al., 1998). Intramuscular injection of pDNA encoding the RSV-F or RSV-G protein was effective in mice (Li et 1Division of Allergy and Immunology-Joy McCann Culverhouse Airway Disease Center, James A. Haley Veterans Administration Hospital and University of South Florida College of Medicine, Tampa, FL 33612. 2Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205. 3Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224. 1415 1416 KUMAR ET AL. al., 1998, 2000); however, the quantity of DNA used per unit body mass, as much as 10 mg/kg, and the route of administration chosen are inconvenient for infants and are suboptimal for inducing mucosal immunity against a pulmonary infection (Guy et al., 2001). Most, if not all, of the RSV antigens are immunogenic in humans and mice, although only the F and G antigens have been shown to induce neutralizing antibodies against RSV (Connors et al., 1991; Wyatt et al., 1999). An analysis of the cytotoxic T lymphocyte (CTL) repertoire in humans indicates that the N, F, P, M2, and NS2 proteins are strong target antigens (Nicholas et al., 1990). Similarly, in BALB/c mice, the F, N, and especially the M2 proteins have been shown to be the major target antigens of CTL activity (Domachowske and Rosenberg, 1999). Both serum and mucosal antibodies and MHC class I-restricted CTLs are considered to protect against RSV infection (Brandenburg et al., 2001). Because the airway epithelium is the major target of RSV infection, we reasoned that DNA vaccines capable of mounting a mucosal immunity against RSV might be more effective. However, the development of such mucosal DNA vaccines has been hindered by inefficient transgene expression of pDNAs in the airway epithelium. Chitosan, a biodegradable, biocompatible, low-toxic polysaccharide has been used as a gene carrier to mucosal sites such as the gastrointestinal tract (Artursson et al., 1994; Richardson et al., 1999; Roy et al., 1999) and by the nasal route (Illum et al., 2001), which induces higher transfection efficiency and ensures more sustained expression of the vaccine antigens. Further, DNA vaccines induce elevated production of interferon-g (IFN-g), which has an antiviral effect against RSV (Kumar et al., 1999). To test potential mucosal genetic immunization, in this study, we utilized a strategy involving an intranasal gene transfer, referred to as IGT, with chitosan–DNA nanospheres containing a cocktail of plasmid DNAs (pDNAs) encoding nine immunogenic RSV antigens, against acute RSV infection in a BALB/c mouse model. The effectiveness and mechanism of this IGT strategy were investigated. Results demonstrate that IGT is safe and effective against RSV and significantly attenuates pulmonary inflammation induced by RSV infection. Vaccine development and protocol Individual RSV cDNAs were amplified from an RSV-infected mouse lung cDNA library by polymerase chain reaction (PCR), using Vent polymerase (New England BioLabs, Beverly, MA), and cloned in the mammalian expression vector pVAX (Invitrogen, San Diego, CA). The resulting plasmids were propagated in Escherichia coli DH5a cells. Large-scale plasmid DNA was prepared with a Qiagen kit (Qiagen, Chatsworth, CA), according to the manufacturer’s specifications. This produced sufficiently pure DNA. Equal quantities of pDNAs were mixed to make a cocktail of RSV cDNAs. DNA–chitosan nanospheres were generated as described previously (Roy et al., 1999), with the total DNA concentration in the solution equally contributed by the nine RSV plasmids; the resulting product is referred to as IGT. Mice were intranasally administered IGT (25 mg of total DNA per mouse) under light anesthesia. Control mice received either phosphate-buffered saline (PBS), equivalent quantities of naked DNA, empty vector complexed in chitosan nanospheres (chitosan plus pVAX), or chitosan alone. Sixteen days after vaccination, mice were infected intranasally with 1 3 106 PFU of the human RSV A2 strain (ATCC) in a 50-ml volume. Five days postinfection, mice were killed and their lungs and spleens were collected aseptically for reverse transcriptase (RT)-PCR, histopathological studies, and cytokine and viral plaque analyses. On day 21 after vaccination mice were bled to obtain serum. Nasal washes were also collected on day 21 for IgA antibody assays as described previously (Matsuo et al., 2000). Quantitation of RSV titers and antigen in lung To quantify RSV titers in mouse lung, whole lungs were first weighed and placed immediately in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS). Lungs were homogenized and centrifuged at 10,000 rpm for 10 min at 4°C, and the clear supernatants were used for plaque assays by the shell vial technique (Domachowske and Bonville, 1998). RSV antigen load was determined as described previously (Kumar et al., 1999). RNA extraction and RT-PCR analysis MATERIALS AND METHODS Animals Six-week old female BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained under pathogen-free conditions at the animal center. All procedures were reviewed and approved by the University of South Florida and James A. Haley Veterans Administration Medical Center Committee on Animal Research (Tampa, FL). Cells and virus HEp-2 (ATCC CCL-23) cells and RSV A2 Long strain (VR1302) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HEp-2 is an epithelial-like cervical carcinoma cell line and is used for propagating RSV. Cells were grown and viral stocks were prepared as described previously (Behera et al., 2001). Total cellular RNA was isolated from lung tissue with TRIzol reagent (Life Technologies, Gaithersburg, MD), according to the manufacturer’s instructions. RT-PCR was carried out for different RSV genes, as described previously (Behera et al., 2001). The primers used for individual cDNA amplification and their corresponding sizes are listed in Table 1. Pulmonary function To evaluate the pulmonary function in vaccinated and control groups, mice were administered IGT, as described previously. Three days later, airway responsiveness was assessed noninvasively in conscious, unrestrained mice in a whole body plethysmograph (Buxco Electronics, Troy, NY) as previously described (Matsuse et al., 2000). With this system, the volume changes that occur during a normal respiratory cycle are recorded as the pressure difference between an animal-containing chamber and a reference chamber. The resulting signal is used to calculate respiratory frequency, minute volume, tidal 1417 GENE THERAPY FOR RSV INFECTION TABLE 1. PRIMER SEQUENCES cDNA NS1 NS2 M SH F M2 N G P FOR RSV cDNAs Primer sequence Sense: Antisense: Sense: Antisense: Sense: Antisense: Sense: Antisense: Sense: Antisense: Sense: Antisense: Sense: Antisense: Sense: Antisense: Sense: Antisense: 59-ctg acg gga tcc gaa ttc agg atg ggc agc aat tca ttg-39 59-ggc att ctc gag tta tgg att aag atc aaa tcc aag taa-39 59-ttg tcc gga tcc acc atg gac aca acc cac aa-39 59-ggc att ctc gag tta tgg att gag atc ata ctt gt-39 59-gtc ggc gga tcc aat atg gaa aca tac gtg aac-39 59-ggc att ctc gag tta atc ttc cat ggg ttt gat tgc a-39 59-cac act gta ccc aca atg gaa aat aca tcc ata aca ata g-39 59-gac aga ctg cag cta tgt gtt gac tcg agc tct tgg taa ctc-39 59-ggg ccg gga tcc aca atg gag ttg cta atc ctc aaa-39 59-cta tgt cga ctt agt tac taa atg caa tat tat tta-39 59-gtg tgc gga tcc aat atg tca cga agg aat cct tgc a-39 59-ggc atg ctc gag tta tga cac taa tat ata tat tgt ata-39 59-gtg tgg gga tcc aag atg gct ctt agc aaa gtc-39 59-ggc att ctc gag tca aag ctc tac atc att atc t-39 59-gtg tgc gga tcc aac atg tcc aaa aac aag gac caa cgc-39 59-gtt gtc gac taa cta ctg gcg tgg tgt gtt-39 59-ttg tgg gga tcc atc atg gaa aag ttt gct cct gaa-39 59-ggc atg ctc gag tca gaa atc ttc aag tga tag atc at-39 volume, and enhanced pause (Penh). Penh was used as the measure of bronchoconstriction and was calculated according to the following formula: Penh 5 pause 3 (peak expiratory pressure/peak inspiratory pressure), where pause is the ratio of time required to exhale the last 30% of tidal volume relative to the total time of expiration. Mice were placed in the plethysmograph and the chamber was equilibrated for 10 min. They were exposed to aerosolized PBS (to establish baseline) followed by incremental doses (6, 12.5, 25, and 50 mg/ml) of methacholine (Sigma Chemicals, St. Louis, MO). Each dose of methacholine was aerosolized for 5 min, and respiratory measurements were recorded for 5 min afterward. During the recording period, an average of each variable was derived from every 30 breaths (or 30 sec, whichever occurred first). The maximum Penh value after each dose was used to measure the extent of bronchoconstriction. Bronchoalveolar lavage, spleen cell culture, and assay for IFN-g Bronchoalveolar lavage (BAL) were performed on IGT-administered and control mice, as described previously (Kumar et al., 1999). For spleen cell culture, single-cell suspensions were prepared from the spleens of BALB/c mice and cultured in wells coated with anti-CD3 antibody (1 mg/ml, clone 17A2; PharMingen, San Diego, CA). IFN-g was assayed from BAL fluid and 24-hr spleen cell culture supernatant, using an ELISA kit (R&D Systems, Minneapolis, MN). ELISA for antibodies Microtiter plates were coated overnight at 4°C with proteins (500 ng/well) from a purified RSV or HEp-2 cell protein preparation. The plates were washed and then blocked at room temperature for 30 min with 100 ml of PBS containing 10% FBS. This solution was replaced with 2-fold serial dilutions of immune sera or of nasal washes prepared in PBS containing 10% PCR product (bp) 419 374 770 194 1724 825 1175 896 725 FBS and 0.2% (v/v) Tween 20. The plates were incubated for 2 hr at room temperature and washed three times. For the evaluation of IgA titers, 100 ml of a 1:1000 dilution of biotinylated anti-mouse IgA antibody (556978; PharMingen) was added and the plates were incubated for another 2 hr. After three washes, 100 ml of avidin–peroxidase conjugate (1:10,000; Sigma) was added and the plates were incubated for another 1 hr. For the evaluation of IgG, anti-mouse IgG–peroxidase conjugate was added at a dilution of 1:10,000 (Boehringer, Mannheim, Germany). After three washes, substrate was added and absorbance was read at 450 nm. Virus neutralization assay Different dilutions of serum obtained on day 21 were mixed with 100 ml of RSV inoculum and incubated at 37°C for 1 hr. This was used to infect HEp-2 cell cultures growing in 48-well culture plates. RSV titer was determined as described previously. Immunoblotting Lung tissues from uninfected and mice infected with RSV were homogenized in 1 ml of lysis buffer (0.5% Triton X-100, 150 mM NaCl, 15 mM Tris, 1 mM CaCl2, 1 mM MgCl2 [pH 7.4], 1 mM phenylmethylsulfonyl fluoride [PMSF], aprotinin [1 mg/ml], leupeptin [1 mg/ml], and pepstatin [1 mg/ml]). The homogenates were incubated on ice for 30 min and centrifuged at 3000 rpm for 10 min. Clear supernatants were collected and total protein was estimated with bicinchoninic acid (BCA) reagent (Pierce, Rockford, IL). Seventy-five micrograms of protein extract was fractionated on a 4–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel, transferred to nitrocellulose membrane, and processed as described previously (Behera et al., 1998). Briefly, the membrane was blocked and incubated overnight at 4°C with a 1:250 dilution of pooled serum from IGT-administered and control mice. The membrane 1418 was washed four times in washing buffer and incubated with anti-mouse IgG peroxidase conjugate for 1 hr at room temperature. After four more washes, the blot was developed and exposed to X-ray film. Histology and scoring for airway inflammation Histological staining and a semiquantitative analysis of airway inflammation in the lungs of IGT and control groups of mice were performed as described previously (Kumar et al., 1999). Lung inflammation was assessed after staining the sec- KUMAR ET AL. tions with hematoxylin and eosin (H&E) and scoring for severity on a scale of 0–3, indicating the degree of inflammation. The entire lung section was reviewed, and pathological changes were evaluated for epithelial damage, peribronchovascular cell infiltrate, and interstitial–alveolar cell infiltrate for mononuclear cells and polymorphs. CTL studies Splenocytes (2.5 3 106 cells/ml) from mice administered IGT and from control groups were incubated in complete RPMI FIG. 1. (A) Expression of RSV cDNAs after IGT administration. BALB/c mice were intranasally administered a cocktail of RSV cDNAs cloned in the plasmid vector pVAX (IGT) and complexed as chitosan nanospheres. Each mouse was instilled with a total of 25 mg of cocktail DNA. Animals were killed 3 days after IGT administration and RT-PCR was performed on total lung RNA. The products were electrophoresed. Marker lane, 1-kb DNA marker; lanes NS1, NS2, M, SH, F, M2, N, G, and P, PCR products corresponding to the RSV cDNAs. (B) Immunoblot analysis. Serum samples were collected from PBS- and IGT-administered mice, pooled, and used to detect RSV antigens from RSV-infected (lanes 2, 4, and 5) and uninfected (lanes 1 and 3) murine lungs on Western blots. Data from one of two experiments with similar results are shown. 1419 GENE THERAPY FOR RSV INFECTION containing interleukin 2 (IL-2, 10 U/ml) and persistently RSVinfected mitomycin (Sigma)-treated fibroblasts (BCH4 cells, 2.5 3 106 cells/ml) (Fernie et al., 1981). Cultures were tested on day 6 for antigen-specific lysis by adding varying numbers of effector cells to 51 Cr-labeled syngeneic fibroblasts, either persistently RSV-infected (BCH4) or uninfected (BC) target cells (1 3 104). After 5 hr of incubation at 37°C, cell supernatants were harvested for the determination of 51Cr in a g counter. The percentage of specific lysis was calculated as [(experimental cpm 2 spontaneous cpm)/(total cpm 2 spontaneous cpm)] 3 100. Spontaneous release and total release were determined from target cells incubated with medium alone or after the addition of 2.5% Triton X-100, respectively. Statistical analysis Pairs of groups were compared by Student t test. Differences between groups were considered significant at p , 0.05. Values for all measurements are expressed as means 6 SD. RESULTS IGT induces expression of viral antigens To examine whether IGT administration results in efficient expression of the constituent RSV antigens, lung tissues of mice were examined for the presence of mRNA and proteins by RTPCR and Western blot analysis, respectively. IGT-administered mice show mRNA expression in the lung tissue within 3 days, as revealed by RT-PCR products from the corresponding nine different cDNAs amplified with specific primers (Table 1 and Fig. 1A). Evidence that these mRNAs are translated to produce sufficient immunogens was obtained by testing pooled sera (n 5 4) of the IGT mice but not of the control (PBS-administered) mice; the pooled sera reacted with a number of RSV antigens present in RSV-infected murine lung homogenate in a Western blot analysis (Fig. 1B). These results indicate that IGT induces the production of RSV antigens, which elicit an antibody response. C. IGT is safe and effective To test whether IGT administration induces airway hyperreactivity, the percent baseline enhanced pause (Penh) was measured in different groups of mice. Mice receiving IGT exhibited a similar response to methacholine challenge when compared with naked DNA, PBS alone (Fig. 2A), chitosan, and chitosan plus pVAX (data not shown) control groups. These results suggest that the IGT treatment by itself does not induce any significant change in airway hyperreactivity. To test the effectiveness of IGT, mice were administered a single dose of either IGT (a total of 25 mg of DNA), PBS, chitosan, empty plasmid vector complexed as chitosan nanospheres (chitosan plus pVAX), or naked DNA in saline. Analysis of lung virus titers after acute, live RSV infection shows a significant (100-fold, p , 0.01) reduction in RSV titers in IGT mice compared with controls (Fig. 2B). Examination of total RSV antigen load in the immunized and control groups of mice shows that immunized mice exhibit a 77% decrease (p , 0.01) in antigen load when compared with PBS controls (Fig. 2C). These results indicate that IGT constitutes an effective prophylaxis against RSV infection. FIG. 2. IGT decreases viral titers and viral antigen load. (A) Determination of methacholine responsiveness of mice intranasally administered PBS, naked DNA, and IGT, as measured by a whole-body plethysmograph. Methacholine responsiveness was measured as percent baseline enhanced pause (Penh). The experiments were repeated with similar results. (B and C) Mice were administered IGT, naked DNA, chitosan plus pVAX, chitosan, or PBS as described previously, infected with RSV on day 16, and killed 4 days later (day 21). Lungs were removed and their homogenates were used for RSV plaque assays and antigen loads. (B) RSV titers from lungs of mice intranasally administered IGT, naked DNA, chitosan plus pVAX, chitosan, or PBS. (C) Antigen load of RSV (measured by ELISA) from lungs of mice intranasally administered IGT, naked DNA, chitosan plus pVAX, chitosan, or PBS. The experiment was repeated and similar results were obtained. The values represent means 6 SD (n 5 4), compared with various control groups. **p , 0.01. 1420 IGT decreases RSV infection-induced pulmonary inflammation Lung inflammation was examined in different groups of mice. Mice treated with chitosan alone (Fig. 3A), chitosan plus pVAX (Fig. 3C), naked DNA (Fig. 3B), or PBS (data not shown) on acute RSV infection exhibit disruption of the epithelium and cellular infiltration. Representative pathological features reveal that groups of mice receiving IGT (Fig. 3D) exhibit less epithelial damage and reduced mononuclear cell (MNC) and polymorphonuclear cell (PMNC) infiltrates in the interstitial and peribronchovascular regions, as compared with controls (Fig. 3A–C). These results suggest that IGT protects mice from RSV infection-induced pulmonary inflammation. A semiquantitative analysis using a scoring system for inflammation in the lung is shown in Table 2. Groups of mice that received IGT exhibit reduced epithelial damage (p , 0.01 compared with PBS; and p , 0.05, compared with other control groups) and pulmonary inflammation compared with controls. The group of mice that received IGT exhibit reduced (p , 0.001 compared with PBS and p , 0.05 compared with chitosan and KUMAR ET AL. chitosan plus pVAX) interstitial alveolar infiltrate and peribronchovascular infiltrate (p , 0.05 compared with PBS and chitosan plus pVAX). The scores for epithelial damage or infiltration among the different control groups were not found to be significant. These results suggest that IGT protects mice from RSV infection-induced pulmonary inflammation. IGT induces both serum and mucosal anti-RSV antibody response Both serum and mucosal responses are important components of an effective gene transfer prophylaxis. Secreted IgA antibody is considered to be protective against mucosal pathogens, as the nasal passage is the main site of entry for RSV (Godding et al., 2000). To examine whether IGT induces specific antibodies in mice, RSV-specific antibody titers were measured in IGT-administered and control mice. Animals administered IGT exhibit significantly higher serum IgG antibody titers than controls (Fig. 4A). Incubation of RSV with the serum obtained from IGT mice reduces virus infection of HEp-2 cells, indicating the production of neutralizing antibodies after gene FIG. 3. Histological analysis of lung after gene transfer. Mice were treated as described previously, and infected with RSV on day 16. Four days later, these mice were killed, their lungs were removed, and histological sections were stained with hematoxylin and eosin (H&E). IGT-administered mice (D) showed less epithelial damage and cellular infiltration than the chitosan (A), naked DNA (B), and chitosan plus pVAX (C) control groups. Arrows indicate epithelial damage and cellular infiltration. 1421 GENE THERAPY FOR RSV INFECTION TABLE 2. SEMIQUANTI TATIVE ANALYSIS Lung pathology Epithelial damage Interstitial–alveolar infiltrate Peribronchovascular infiltrate OF LUNG PATHOLOGY a PBS Chitosan Chitosan plus pVAX Naked DNA IGT 2.53 6 0.17 2.66 6 0.21 2.24 6 0.12 2.45 6 0.35 2.39 6 0.16 2.47 6 0.19 2.25 6 0.30 2.36 6 0.33 1.4 6 0.52b–e 1.76 6 0.35c,d,f 2.01 6 0.20 1.89 6 0.25 2.16 6 0.11 1.81 6 0.57 1.46 6 0.23d,g a Each value represents the mean 6 SD of five random fields from six individual lung sections from each mouse in a group (n 5 4). b p , 0.01 compared with PBS control. cp , 0.05 compared with chitosan control. d p , 0.05 compared with chitosan plus pVax control. e p , 0.05 compared with naked DNA control. fp , 0.001 compared with PBS control. g p , 0.05 compared with PBS control. transfer (Fig. 4B). IGT mice show significantly higher neutralizing titers compared with control groups. The levels of IgA antibody in nasal wash were measured to verify whether this class of antibody was changed as a result of vaccination. Animals administered IGT exhibit significantly higher RSV-specific IgA antibody titers than controls (Fig. 4C). These results indicate that IGT induces increased production of neutralizing antibodies in serum and nasal IgA. IGT generates RSV-specific CTL and IFN-g production To test whether IGT also induces a CTL response, mice were analyzed for the presence of splenic, RSV-specific CTLs using persistently RSV-infected BCH4 cells as the target and RSVnegative BC cells as the control. PBS, naked DNA, chitosan, and empty vector complexed in chitosan nanosphere controls do not elicit a detectable CTL response. In contrast, mice administered IGT generate CTL responses (Fig. 5A), and these CTLs are CD81 and MHC class I restricted (data not shown). IFN-g is considered to be the major antiviral cytokine. Thus, in order for a prophylaxis to be effective against RSV, it must induce IFN-g expression. IFN-g was assayed from the anti-CD3 antibody-stimulated cultured spleen cells and the BAL fluid of IGT-administered and control groups of mice. In both cases, IGT-administered mice exhibit significantly higher IFN-g production (Fig. 5B and C). DISCUSSION This study concerns the development of an effective and safe prophylaxis against RSV, utilizing a mucosal gene transfer approach, which may provide for the protection of infants 2 to 6 months of age, who are among the most susceptible to RSV infection. Furthermore, a mucosal gene transfer approach is considered more appropriate for developing immunity in the lungs of these infants, who may have an immature immune system. The results of this study are deemed significant, because of the lack of a safe and effective vaccine against RSV. Currently, passive immunization with anti-RSV antibodies or with a hu- manized antibody to the RSV-F antigen (Kneyber et al., 2000), at a monthly interval, is one of the options or often the only option available to certain infants, who are at high risk of developing RSV infection. These passive therapies are inconvenient, expensive, and only partially effective. The results demonstrate that the IGT utilized in this study for mucosal gene transfer is both safe and effective. The safety is demonstrated by the lack of change in methacholine responsiveness between vaccinated and control mice. IGT also induced a significant decrease in overall lung inflammation accompanying acute RSV infection, which presumably contributes to the lung pathology in bronchiolitis and exacerbation of asthma. This issue is important in view of the previous failure of the formalin-inactivated vaccine, which exacerbated the disease (Chanock et al., 1992). The results of the semiquantitative analyses of epithelial damage and of perivascular, peribronchial, and interstitial infiltrating cells indicate that IGT significantly reduces cellular infiltration and epithelial damage compared with naked DNA and unvaccinated mice. This demonstrates that IGT markedly attenuates inflammation while rendering protection against RSV infection. This is most likely due to the effect of IGT on decreasing the virus titers, which limits the spread of infection and consequently results in less virusinduced inflammation. IGT has two distinct components: a plasmid DNA cocktail conferring vaccine potency and chitosan conferring adjuvant activity. Both of these components add intrinsic value to the efficacy and safety of this vaccine. A core feature of IGT is that it comprises a cocktail of pDNAs encoding all potentially immunogenic RSV antigens, increasing the immune response and, thus, the efficacy of the vaccine. The mucosal gene transfer approach has not previously been investigated for RSV. Differences in composition, species, dosage, and route of administration preclude a direct comparison of results reported in this paper with those published previously. Nonetheless, the results of this study show that a single dose of IGT, with a total of 25 mg of DNA per mouse, given intranasally, induces a significant reduction (100-fold or 2 orders of magnitude) in viral titers after an acute infection. Vaccines studied to date consist of a subunit, peptide, or DNA vaccine made up of the RSV-F, RSV-G and/or RSV-M2 protein. In a mouse model, pDNAs encoding 1422 KUMAR ET AL. FIG. 4. IGT induces increased antibody titers to virus. (A) Anti-RSV antibody response after IGT administration. BALB/c mice were vaccinated as described. Sera were collected from mice on day 21 after vaccination, and anti-RSV antibody titers were measured by ELISA. (B) Determination of RSV neutralizing antibody titers after vaccination. RSV suspension was incubated with various dilutions (0.01, 0.1, and 1) of sera and neutralization was carried out as described. (C) IgA antibody response after vaccination from nasal washes. RSV-specific IgA antibody levels from nasal washings, collected on day 21 after vaccination, were measured by ELISA. The experiment was repeated and similar results were obtained. The values represent means 6 SD (n 5 4). *p , 0.05, compared with control groups. GENE THERAPY FOR RSV INFECTION 1423 FIG. 5. IGT increases CTL response and IFN-g production. (A) Characterization of RSV-specific CTLs induced by IGT vaccination. Mice were vaccinated as described. Three weeks later, immune splenocytes were stimulated with the persistently RSVinfected fibroblast cell line BCH4. CTL activity was assessed in a standard 5-hr 51Cr release assay, using uninfected BC cells and RSV-infected BCH4 fibroblast as targets. (B) Determination of IFN-g levels in BAL fluid. Groups of mice treated as described above were infected with RSV on day 16. BAL was performed on these mice on day 21, and IFN-g levels were measured by ELISA. (C) Determination of IFN-g levels in splenocyte cultures. Groups of mice vaccinated as described above were infected with RSV on day 16. Mice were killed on day 21 and their splenocytes were cultured in vitro on anti-CD3 antibodycoated plates, and IFN-g levels in the culture supernatants were measured by ELISA. The experiment was repeated and similar results were obtained. The values represent means 6 SD (n 5 4). *p , 0.05, compared with different control groups. F or G antigen (100 mg/mouse) administered via the intramuscular route were effective (Li et al., 1998, 2000). In a cotton rat model, an F–G vaccine induced neutralizing antibody titers, which were one to two orders of magnitude lower compared with live RSV (Prince et al., 2000). The results of the abovedescribed studies are comparable to the mucosal gene transfer approach with IGT, although this study examined protection from RSV challenge only after day 16 of gene transfer, when the primary immune response to viral antigens is at its peak. The persistence of this protection remains to be established. The second important feature of IGT is its formulation with chitosan, a biodegradable and biocompatible natural biopolymer that increases nasal absorption of the vaccine without any adverse effects. Chitosan allows increased bioavailability of the DNA because of protection from degradation by serum nucleases in the matrix (Richardson et al., 1999). Chitosan has also been found to have antiinflammatory (Otterlei et al., 1994) and immunostimulatory activity (Nishimura et al., 1984), and it is capable of modulating immunity of the mucosa and bronchusassociated lymphoid tissue. The results of this study of IGT in the form of chitosan nanoparticles, which significantly induces specific neutralizing IgG antibody titers, nasal IgA titers, and IFN-g levels in the lung compared with various controls, suggests that chitosan increases the immunologic potency of IGT. Although the detailed mechanisms underlying chitosan potentiation of an antiviral immunity is unclear, chitosan increases transcellular and paracellular transport across the mucosal epithelium (Artursson et al., 1994) and, thus, facilitates mucosal gene delivery and may modulate immunity of the mucosa and bronchus-associated lymphoid tissue. Chitosan also has been reported to bind via CD14 to macrophages and activate them (Otterlei et al., 1994; Bianco et al., 2000). Synergistic cooperation between soluble chitosan oligomers and IFN-g resulted in increased NO synthesis, which may contribute antiviral ac- 1424 KUMAR ET AL. tivity (Seo et al., 2000). The mechanism of chitosan-induced immune potentiation remains to be investigated. In an effort to unravel the protective mechanisms underlying the efficacy of IGT, induced humoral and cellular immunity was investigated. A notable finding is that IGT significantly augmented levels of both neutralizing serum and mucosal IgA antibodies compared with naked DNA-vaccinated and unvaccinated control groups. Previously, passive administration of neutralizing serum antibodies was shown to decrease the risk of RSV disease in animal models and in humans (Groothuis et al., 1991; Hemming et al., 1995). Although the secreted IgA antibody provides protection against pathogens that enter via the mucosal route (Godding et al., 2000), the role of secretory IgA in protection against RSV is poorly understood. Because RSV is an obligatory intracellular mucosal pathogen affecting both the upper and lower respiratory tract, it is likely that mucosal IgA might provide protection against severe RSV disease by precluding its entry into mucosa and/or inhibiting the cell–cell syncytial spread of RSV (Mazanec et al., 1992; Bomsel et al., 1998). The protective role of IgA requires further investigation. Virus-specific CTLs play a major role in the clearance of RSV infection (Graham et al., 1991). Passive transfer of RSVspecific T cells has been shown to effectively clear RSV (Cannon et al., 1987). The results reported in this paper demonstrate that IGT delivery generates a significantly stronger CTL response compared with naked DNA and other controls. These results, which are in agreement with other experimental approaches, support a role for vaccine-induced CTLs in virus clearance. Several studies indicate that the protective effect of CTLs against cytopathic viruses is dependent on their production of cytokines such as IFN-g (Hsu et al., 1998; Okamoto et al., 1999). Indeed, IGT significantly enhanced the production of IFN-g, which may be useful in fighting RSV infection. IFNg has a direct antiviral effect and is particularly important in stimulating the cytolytic activity of natural killer (NK) cells and CD81 CTLs, which play a critical role in the control of RSV infection in a murine model (Hsu et al., 1998) and in humans (Aberle et al., 1999). Collectively, these data demonstrate that IGT represents a novel gene transfer approach against RSV infection, which, at a single dose of about 1 mg/kg body weight, is capable of decreasing viral titers by 2 orders of magnitude (100-fold) on primary infection. The immunologic mechanisms for the effectiveness of this prophylaxis include the induction of high levels of both serum IgG and mucosal IgA antibodies, the generation of an effective CTL response, and elevated lung-specific production of IFN-g with antiviral action. Although as a singledose vaccine IGT is effective, it is possible that dose escalation and prime–booster strategies might further enhance its effectiveness. In addition, IGT significantly decreases pulmonary inflammation and does not alter airway hyperresponsiveness, thus making it a safe vaccine. ACKNOWLEDGMENTS This study was supported by the grants from the VA Merit Review Award and the American Heart Association, Florida Affiliate award to S.S.M. and by generous support from the Joy McCann Culverhouse endowment to the Division of Allergy and Immunology. REFERENCES ABERLE, J.H., ABERLE, S.W., ALLISON, S.L., STIASNY, K., ECKER, M., MANDL, C.W., BERGER, R., and HEINZ, F.X. (1999). A DNA immunization model study with constructs expressing the tick-borne encephalitis virus envelope protein E in different physical forms. J. Immunol. 163, 6756–6761. ARTURSSON, P., LINDMARK, T., DAVIS, S.S., and ILLUM, L. (1994). Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Pharm. Res. 11, 1358–1361. BEHERA, A.K., KUMAR, M., MATSUSE, H., LOCKEY, R.F., and MOHAPATRA, S.S. (1998). Respiratory syncytial virus induces the expression of 5-lipoxygenase and endothelin-1 in bronchial epithelial cells. Biochem. Biophys. Res. Commun. 251, 704–709. BEHERA, A.K., MATSUSE, H., KUMAR, M., KONG, X., LOCKEY, R.F., and MOHAPATRA, S.S. (2001). Blocking intercellular adhesion molecule-I on human epithelial cells decreases respiratory syncytial virus infection. Biochem. Biophys. Res. Commun. 280, 188– 195. BIANCO, I.D., BALSINDE, J., BELTRAMO, D.M., CASTAGNA, L.F., LANDA, C.A., and DENNIS, E.A. (2000). Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages. FEBS Lett. 466, 292–294. BOMSEL, M., HEYMAN, M., HOCINI, H., LAGAYE, S., BELEC, L., DUPONT, C., and DESGRANGES, C. (1998). Intracellular neutralization of HIV transcytosis across tight epithelial barriers by antiHIV envelope protein dIgA or IgM. Immunity 9, 277–287. BRANDENBURG, A.H., NEIJENS, H.J., and OSTERHAUS, A.D. (2001). Pathogenesis of RSV lower respiratory tract infection: Implications for vaccine development. Vaccine 19, 2769–2782. CANNON, M.J., STOTT, E.J., TAYLOR, G., and ASKONAS, B.A. (1987). Clearance of persistent respiratory syncytial virus infections in immunodeficient mice following transfer of primed T cells. Immunology 62, 133–138. CENTERS FOR DISEASE CONTROL AND PREVENTION (1999). Update: Respiratory syncytial virus activity—United States, 1998– 1999 season. MMWR 48, 1104. CHANOCK, R.M., PARROTT, R.H., CONNORS, M., COLLINS, P.L., and MURPHY, B.R. (1992). Serious respiratory tract disease caused by respiratory syncytial virus: Prospects for improved therapy and immunization. Pediatrics 90, 137–143. COHEN, A.D., BOYER, J.D., and WEINER, D.B. (1998). Modulating the immune response to genetic immunization. FASEB J. 12, 1611– 1626. CONNORS, M., COLLINS, P.L., FIRESTONE, C.Y., and MURPHY, B.R. (1991). Respiratory syncytial virus (RSV) F, G, M2 (22K), and N proteins each induce resistance to RSV challenge, but resistance induced by M2 and N proteins is relatively short-lived. J. Virol. 65, 1634–1637. DOMACHOWSKE, J.B., and BONVILLE, C.A. (1998). Overnight titration of human respiratory syncytial virus using quantitative shell vial amplification. Biotechniques 25, 644–647. DOMACHOWSKE, J.B., and ROSENBERG, H.F. (1999). Respiratory syncytial virus infection: Immune response, immunopathogenesis, and treatment. Clin. Microbiol. Rev. 12, 298–309. DONNELLY, J.J., ULMER, J.B., and LIU, M.A. (1998). DNA vaccines. Dev. Biol. Stand. 95, 43–53. FERNIE, B.F., FORD, E.C., and GERIN, J.L. (1981). The development of BALB/c cells persistently infected with respiratory syncytial virus: Presence of ribonucleoprotein on the cell surface. Proc. Soc. Exp. Biol. Med. 167, 83–86. 1425 GENE THERAPY FOR RSV INFECTION GODDING, V., VAERMAN, J.P., and SIBILLE, Y. (2000). Secretory mucosal immune mechanisms. Acta Otorhinolaryngol. Belg. 54, 255–261. GRAHAM, B.S., BUNTON, L.A., WRIGHT, P.F., and KARZON, D.T. (1991). Role of T lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice. J. Clin. Invest. 88, 1026–1033. GROOTHUIS, J.R., LEVIN, M.J., RODRIGUEZ, W., HALL, C.B., LONG, C.E., KIM, H.W., LAUER, B.A., and HEMMING, V.G. (1991). Use of intravenous gamma globulin to passively immunize high-risk children against respiratory syncytial virus: Safety and pharmacokinetics. RSVIG Study Group. Antimicrob. Agents Chemother. 35, 1469–1473. GUY, B., PASCAL, N., FRANCON, A., BONNIN, A., GIMENEZ, S., LAFAY-VIALON, E., TRANNOY, E., and HAENSLER, J. (2001). Design, characterization and preclinical efficacy of a cationic lipid adjuvant for influenza split vaccine. Vaccine 19, 1794–1805. HALL, C.B. (1994). Prospects for a respiratory syncytial virus vaccine. Science 265, 1393–1394. HEMMING, V.G., PRINCE, G.A., GROOTHUIS, J.R., and SIBER, G.R. (1995). Hyperimmune globulins in prevention and treatment of respiratory syncytial virus infections. Clin. Microbiol. Rev. 8, 22–33. HSU, S.C., OBEID, O.E., COLLINS, M., IQBAL, M., CHARGELEGUE, D., and STEWARD, M.W. (1998). Protective cytotoxic T lymphocyte responses against paramyxoviruses induced by epitopebased DNA vaccines: Involvement of IFN-g. Int. Immunol. 10, 1441–1447. ILLUM, L., JABBAL-GILL, I., HINCHCLIFFE, M., FISHER, A.N., and DAVIS, S.S. (2001). Chitosan as a novel nasal delivery system for vaccines. Adv. Drug Deliv. Rev. 51, 81–96. KNEYBER, M.C., MOLL, H.A., and DE GROOT, R. (2000). Treatment and prevention of respiratory syncytial virus infection. Eur. J. Pediatr. 159, 399–411. KUMAR, M., BEHERA, A.K., MATSUSE, H., LOCKEY, R.F., and MOHAPATRA, S.S. (1999). Intranasal IFN-g gene transfer protects BALB/c mice against respiratory syncytial virus infection. Vaccine 18, 558–567. LI, X., SAMBHARA, S., LI, C.X., EWASYSHYN, M., PARRINGTON, M., CATERINI, J., JAMES, O., CATES, G., DU, R.P., and KLEIN, M. (1998). Protection against respiratory syncytial virus infection by DNA immunization. J. Exp. Med. 188, 681–688. LI, X., SAMBHARA, S., LI, C.X., ETTORRE, L., SWITZER, I., CATES, G., JAMES, O., PARRINGTON, M., OOMEN, R., DU, R.P., and KLEIN, M. (2000). Plasmid DNA encoding the respiratory syncytial virus G protein is a promising vaccine candidate. Virology 269, 54–65. MATSUO, K., MACLAUGHLIN, F.C., MUMPER, R.J., WANG, J., TAGLIAFERRI, J.M., GILL, I., HINCHCLIFFE, M., and ROLLAND, A.P. (2000). Induction of innate immunity by nasal influenza vaccine administered in combination with an adjuvant (cholera toxin). Vaccine 18, 2713–2722. MATSUSE, H., BEHERA, A.K., KUMAR, M., RABB, H., LOCKEY, R.F., and MOHAPATRA, S.S. (2000). Recurrent respiratory syncytial virus infections in allergen-sensitized mice lead to persistent airway inflammation and hyperresponsiveness. J. Immunol. 164, 6583–6592. MAZANEC, M.B., KAETZEL, C.S., LAMM, M.E., FLETCHER, D., and NEDRUD, J.G. (1992). Intracellular neutralization of virus by immunoglobulin A anti-bodies. Proc. Natl. Acad. Sci. U.S.A. 89, 6901–6905. NICHOLAS, J.A., RUBINO, K.L., LEVELY, M.E., ADAMS, E.G., and COLLINS, P.L. (1990). Cytolytic T-lymphocyte responses to respiratory syncytial virus: Effector cell phenotype and target proteins. J. Virol. 64, 4232–4241. NISHIMURA, K., NISHIMURA, S., NISHI, N., SAIKI, I., TOKURA, S., and AZUMA, I. (1984). Immunological activity of chitin and its derivatives. Vaccine 2, 93–99. OKAMOTO, I., KOHNO, K., TANIMOTO, T., IKEGAMI, H., and KURIMOTO, M. (1999). Development of CD81 effector T cells is differentially regulated by IL-18 and IL-12. J. Immunol. 162, 3202– 3211. OTTERLEI, M., VARUM, K.M., RYAN, L., and ESPEVIK, T. (1994). Characterization of binding and TNF-a inducing ability of chitosans on monocytes: The involvement of CD14. Vaccine 12, 825–832. PRINCE, G.A., CAPIAU, C., DESCHAMPS, M., FABRY, L., GARCON, N., GHEYSEN, D., PRIEELS, J.P., THIRY, G., VAN OPSTAL, O., and PORTER, D.D. (2000). Efficacy and safety studies of a recombinant chimeric respiratory syncytial virus FG glycoprotein vaccine in cotton rats. J. Virol. 74, 10287–10292. RICHARDSON, S.C., KOLBE, H.V., and DUNCAN, R. (1999). Potential of low molecular mass chitosan as a DNA delivery system: Biocompatibility, body distribution and ability to complex and protect DNA. Int. J. Pharm. 178, 231–243. ROY, K., MAO, H.Q., HUANG, S.K., and LEONG, K.W. (1999). Oral gene delivery with chitosan–DNA nanoparticles generates immunologic protection in a murine model of peanut allergy. Nat. Med. 5, 387–391. SEO, W.G., PAE, H.O., KIM, N.Y., OH, G.S., PARK, I.S., KIM, Y.H., KIM, Y.M., LEE, Y.H., JUN, C.D., and CHUNG, H.T. (2000). Synergistic cooperation between water-soluble chitosan oligomers and interferon-g for induction of nitric oxide synthesis and tumoricidal activity in murine peritoneal macrophages. Cancer Lett. 159, 189–195. WYATT, L.S., WHITEHEAD, S.S., VENANZI, K.A., MURPHY, B.R., and MOSS, B. (1999). Priming and boosting immunity to respiratory syncytial virus by recombinant replication-defective vaccinia virus MVA. Vaccine 18, 392–397. Address reprint requests to: Dr. Shyam S. Mohapatra Joy McCann Culverhouse Airway Disease Center Division of Allergy and Immunology, Department of Internal Medicine University of South Florida and VA Hospital 12901 Bruce B. Downs Blvd. Tampa, FL 33612 E-mail: smohapat@hsc.usf.edu Received for publication February 1, 2002; accepted after revision June 17, 2002. Published online: July 17, 2002.