Intranasal IFN-g gene transfer protects BALB/c mice againstrespiratory syncytial virus infection

Mukesh Kumara, Aruna K. Beheraa, Hiroto Matsusea, Richard F. Lockeya, ShyamS. Mohapatraa,b,*

aThe Joy McCann Culverhouse Airway Disease Research Center, Division of Allergy and Immunology, The University of South Florida College of

Medicine and VA Hospital, 13000 Bruce B Downs Blvd., Tampa, FL 33612, USAbDepartment of Medical Microbiology and Immunology, The University of South Florida College of Medicine and VA Hospital, Tampa, FL 33612,

USA

Received 29 December 1998; received in revised form 14 April 1999; accepted 14 April 1999

Abstract

Respiratory syncytial virus (RSV) is a major respiratory pathogen in infants, young children and the elderly and causes severe

bronchiolitis and asthma. In an e�ort to develop a preventive IFN-g therapy against RSV infection, an intranasal gene transferstrategy was utilized. Intranasal administration of a plasmid expressing the IFN-g cDNA (pIFN-g) resulted in the expression ofIFN-g in murine lungs and decreased RSV replication. The mice administered with pIFN-g and then infected with RSVexhibited a signi®cant decrease in broncho-alveolar lavage lymphocyte and neutrophil counts. A signi®cant reduction in

epithelial cell damage, in®ltration of mononuclear cells in the peribronchiolar and perivascular regions, and thickening of thesepta was observed in the lungs of mice treated with pIFN-g when compared to controls. These results suggest that intranasalIFN-g gene transfer results in decreased RSV replication and pulmonary in¯ammation and may be useful against RSV

infection. # 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Gene transfer; RSV; Cytokine

1. Introduction

The respiratory syncytial virus (RSV) is the mostcommon cause of viral lower respiratory tract infec-tions leading to pneumonia and bronchiolitis in infantsand children [1±4]. In addition, emerging evidencepoints to RSV as a signi®cant pathogen in adults andthe elderly [5]. Data obtained from the NationalRespiratory and Enteric Virus Surveillance Systemdemonstrate a seasonal pattern of RSV infection,which has peak rates of 30±40% at the beginning ofeach year [6]. In the USA alone, RSV causes about4500 deaths per year, about 95 000 hospitalizations,and an estimated 4 million cases of respiratory tract

infections annually [4,7]. RSV is also a major public

health concern globally, with an estimated 5 million

deaths annually due to RSV infections [8,9]. The devel-

opment of a vaccine against RSV is considered import-

ant, since primary RSV infection renders limited

immunity to subsequent infection [10]. Vaccines devel-

oped in 1960's using formalin the inactivated RSV not

only failed but exacerbated the disease upon sub-

sequent RSV infection [11,12]. The development of an

attenuated, immunogenic and genetically stable live

RSV vaccine has not been successful.

RSV infection is also associated with recurrent epi-

sodes of bronchial obstruction and exacerbation of

established asthma in some children with atopic gen-

etic predisposition [13±15]. Acute viral respiratory

tract infections also enhance sensitization to inhalant

allergens in mice [16]. Although the mechanism under-

lying the potential synergy between RSV infection and

Vaccine 18 (2000) 558±567

0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.

PII: S0264-410X(99 )00185 -1

www.elsevier.com/locate/vaccine

* Corresponding author. Tel.: +1-813-974-8568; fax: +1-813-974-

8907.

E-mail address: smohapat@com1.med.usf.edu (S.S. Mohapatra)

allergic sensitization is not fully characterized, it maybe mediated via the T cell response. A polarized Thelper type-2 (Th2)-like immunity to diverse allergensis a distinct feature of asthma. Th2 cells produce cyto-kines such as IL-4, IL-5, IL-10, and IL-13 [17] that areeither important for IgE production or are involved inthe recruitment and activation of in¯ammatory cells.In marked contrast, Th1 cells produce cytokines IL-2and IFN-g. IL-4 and IFN-g reciprocally regulate IgEantibody production. A recent study in childrensuggests that RSV infection induces a Th2-like re-sponse [18]. Also formalin-inactivated RSV induces aTh2-like response and IgE antibody production inmice [19±21]. RSV infection, similar to that of the ex-posure to allergens, can augment IgE mediated in¯am-mation in individuals genetically predisposed todevelop atopic disease [18].

IFN-g down-regulates the Th2-like response and IgEproduction in allergic individuals [22,23]. It is alsoreported to attenuate the respiratory rhinovirus infec-tion of epithelial cells [24]. However, IFNs per se areinadequate for therapy because of their adverse e�ects.We reasoned that local transient over-expression ofIFN-g delivered in the form of a gene therapy mightprovide e�ective protection against RSV infection.Such gene therapy has been used in various modelseither to correct the genetic de®ciency or to modify thecourse of the immune response [25]. To test this possi-bility, we devised an intranasal gene transfer strategyutilizing a plasmid expressing IFN-g cDNA, referredto as pIFN-g, in a murine model of RSV infection.The BALB/c mouse provides a good model to studyRSV infection, in which the time course of infectionand pulmonary pathology closely resembles to those ofthe humans [26,27]. The results demonstrate thatpIFN-g gene transfer results in the over-expression ofIFN-g, which prevents RSV replication in the lungs ofmice. Consequently, gene transferred mice show a sig-ni®cant reduction in pulmonary in¯ammation, includ-ing a reduction in eosinophils in the lungs of mice anda shift in the local cytokine production pro®le fromTh2-like to a Th1-like.

2. Materials and methods

2.1. Animals

Six-week old female BALB/c mice were purchasedfrom the Jackson laboratory (Bar Harbor, ME) andmaintained in pathogen free conditions at the animalcenter. All procedures were reviewed and approved bythe University of South Florida and the James AHaley VA Medical Center Committee on AnimalResearch.

2.2. Gene construct and gene transfer

The mouse IFN-g cDNA was ampli®ed from themouse spleen cDNA library by polymerase chain reac-tion (PCR) using Vent polymerase (New EnglandBiolabs, Beverly, MA). The sequence of the sense andanti-sense oligonucleotide primers were as follows:sense, 5 '-dTCT GGA TCC ATG AAC GCT ACACAC TG-3 '; and anti-sense, 5 '- dCAC CTC GAGTCA GCA GCG AC-3 ', and the primers contained aBamHI and a XhoI site, respectively. Following ampli-®cation, the PCR product was eluted from the agarosegel by the freeze squeeze method [28], puri®ed andthen digested with BamHI and XhoI. The digested pro-duct was ligated to the mammalian expression vectorpcDNA3.1(+) (Invitrogen, San Diego, CA) at theidentical sites. The resulting plasmid, referred to aspIFN-g, was propagated in E. coli DH5a cells. Large-scale plasmid DNA was prepared using a Qiagen kit(Qiagen, Chatsworth, CA) following the manufac-turer's speci®cations. This produced su�ciently pureDNA with minimum endotoxin contamination. PBS,lipofectAMINE alone and pcDNA3.1 were used ascontrols. For intranasal gene transfer, mice wereinoculated intranasaly thrice at two day intervals, witha mixture of 100 mg of pIFN-g DNA complexed withthe cationic lipid, lipofectAMINE (Life Technologies,Gaithersburg, MD), in a 50 ml volume.

2.3. Viral infection of animals and tissue collection

Mice were infected intranasaly with RSV A2 strain(ATCC, Rockville, MD) with an infectious dose of1 � 106 pfu 7 days after the last intranasal DNA ad-ministration. Animals were sacri®ced on day four post-infection (p.i.) and their lungs removed for RT±PCRand histopathological studies.

2.4. Quantitation of RSV antigen in lung

RSV antigen in the mouse lung was quantitated byELISA. Whole lungs were homogenized in 2 ml oflysis bu�er (0.5% Triton X-100, 150 mM NaCl, 15mM Tris, 1 mM CaCl2 and 1 mM MgCl2, pH 7.4).Homogenates were held on ice for 30 min, and thencentrifuged at 3000 rpm for 10 min. Supernatants werecollected, passed through a 0.2 mm ®lter (GelmanSciences, Ann Arbor, MI) and used for ELISA.Brie¯y, supernatants were ®rst incubated in goat anti-human RSV antibody (AB1128, Chemicon, Temecula,CA) coated ELISA plates (Costar, Cambridge, MA)and then incubated with mouse anti human RSV poly-clonal antibody (NCL-RSV3, Vector Laboratories).Following incubation with peroxidase labeled goatanti-mouse IgG antibody (Boehringer Manheim,Indianapolis, IN), immune complexes were detected

M. Kumar et al. / Vaccine 18 (2000) 558±567 559

using tetra methyl benzidine (TMB) as the substrate.Optical densities were read at 450 nm using an ELISAreader.

2.5. RNA extraction

Total cellular RNA was isolated from the lung tissueusing TRIZOL reagent (Life Technologies,Gaithersburg, MD) following the manufacturer'sinstructions. One ml of Trizol reagent was added to50±100 mg of lung tissue and homogenized. Lunghomogenate was suspended by pipeting and allowed tostand at room temperature (RT) for 5 min for lysis.Chloroform (200 ml) was added to each tube andmixed thoroughly. After 5 min, samples were centri-fuged at 12 000 rpm for 15 min at 15±208C. The clearaqueous supernatant was transferred to a fresh tubeand an equal volume of iso-propanol was added,mixed well, and centrifuged at 12 000 rpm for 15 minat 15±208C. The RNA pellet was washed with 70%ethanol, air-dried and dissolved in diethyl-pyrocarbo-nate-treated water.

2.6. Reverse transcription±polymerase chain reaction(RT±PCR)

RSV replication in murine lungs was monitored bychecking the mRNA expression of RSV N gene. Thesense and anti-sense oligonucleotide sequences were asfollows: 5 '- dGCG ATG TCT AGG TTA GGA AGAA-3 ' and 5 '- dGCT ATG TCC TTG GGT AGT AAGCCT-3 '. The following oligonucleotide primers wereused to check the mRNA expression of mouse IFN-g,5 '-dTCT GGA TCC ATG AAC GCT ACA CAC TG-3 ' and anti-sense, 5 '- dCAC CTC GAG TCA GCAGCG AC-3 '. The primers for IL-5 were 5 '-d AAGGAT GCT TCT GCA CTT GA-3 ' and 5 '-dACACCA AGG AAC TCT TGC A-3 '. The oligonucleotideprimers, 5 '-dGAC ATG GAG AAG ATC TGG CAC-3 ' and 5 '-dTCC AGA CGC AGG ATG GCG TGA -3 ', were used to examine the expression of the mouseb-actin which was used as an internal control. For thesynthesis of ®rst strand cDNA, 1 mg of total RNA wasmixed with 150 ng of random primer and heated to708C for 10 min, immediately chilled on ice for 5 min,followed by the addition of 9 ml of reverse transcrip-tion mixture prepared in a total volume of 25 ml con-taining 5 ml of 5X ®rst strand bu�er, 2.5mM of eachdNTP, 8 mM DTT, and 100 units of SuperscriptRNAse Hÿ reverse transcriptase (Life Technologies,Gaithersburg, MD). The reaction was further incu-bated at 428C for 50 min. The reverse transcriptasereaction was terminated by the incubation of the reac-tion tubes sample at 708C for 10 min and the ®rststrand cDNA was cooled to 48C.

PCR ampli®cation was carried out with a 20 ml reac-

tion volume consisting of a PCR bu�er (LifeTechnologies, Gaithersburg, MD) containing 1.5 mMMgCl2, 0.2 mM of each dNTP, 50 pM of each primer,and 1unit of recombinant Taq DNA polymerase (LifeTechnologies, Gaithersburg, MD). The reaction mix-ture was denatured at 958Cfor 1 min, annealed at therespective annealing temperature for 1 min. andextended at 728C for 1 min. The cDNA was ampli®edfor 25±40 cycles, followed by an extension step of 7min at 728C to extend the partially ampli®ed products.The resultant PCR products were analyzed by electro-phoresis on 1.5% agarose gel and the products visual-ized by staining with ethidium bromide.

2.7. Bronchial alveolar lavage and cytokine assay

Mice were sacri®ced on day 4 p.i. by an overdoseinjection of pentobarbital [Nembutal (AbbottLaboratories, North Chicago, IL)] (0.6g/kg) and thethorax was opened in each mouse. The lung vascularbed was ¯ushed with 2 to 3 ml of chilled saline. Thetrachea was exposed and canulated with a 26G needleconnected to a tuberculin syringe. The lung was thenlavaged thrice with 0.5 ml of PBS and the bronchioal-veolar lavage ¯uid (BALF) was pooled. RecoveredBAL ¯uid volumes ranged between 75% and 85% ofinstilled PBS. There was no statistically signi®cantdi�erence in recovered BAL ¯uid volumes betweencontrol and experimental groups. Supernatant was col-lected following centrifugation of the BAL and storedat ÿ708C until it was assayed for cytokines. The cellpellet was suspended in 200 ml of RPMI 1640 mediasupplemented with 10% FBS and a small aliquotcounted using the hemocytometer. The remaining cellsuspension was centrifuged onto a glass slide using acytospin centrifuge at 1500 rpm for 5 min at roomtemperature. Cytocentrifuged cell smears were airdried and stained by LeukostatTM (Fisher Scienti®c,Atlanta, GA). At least 300 cells were examined in ablinded fashion for a di�erential cell count by micro-scopic observation. IFN-g and IL-5 assay from BALFwas carried out using ELISA kits from R&D systems(Minneapolis, MN) and Endogen (Woburn, MA) re-spectively, following the manufacturer's speci®cations.

2.8. Histology and scoring for airway in¯ammation

Lungs were in¯ated with intratracheal injections ofPBS followed by 10% neutral bu�ered formalin sol-ution (Sigma Chemicals, St Louis, MO) to preserve thepulmonary architecture in an expanded state. Lungswere transferred to 80% ethanol after 1 h and thenembedded in para�n. The sections were stained withhematoxylin and eosin.

A semi-quantitative evaluation of in¯ammatory cellsin the lung sections, including alveolar spaces, bronch-

M. Kumar et al. / Vaccine 18 (2000) 558±567560

ovascular bundles and interstitium, was performed in ablinded fashion. In¯ammatory in®ltrates were assessedmorphologically for location, thickness, and cell com-position. The sections were scored as follows: for epi-thelial damage: 0=no damage, 1=increased epithelialcell cytoplasm without desquamation, 2=epithelialdesquamation without bronchial exudate composed ofin¯ammatory cells, 3=bronchial exudate composed ofdesquamated epithelial cells and in¯ammatory cells;for interstitial cellularity: 0=no in®ltrate, 1=mild,generalized increase in cellularity of the alveolar septawithout thickening of the septa or signi®cant airspaceconsolidation, 2=dense septal mononuclear in®ltrates

with thickening of septa, 3=signi®cant alveolar conso-lidation in addition to interstitial in¯ammation; forperibronchovascular in®ltrates: 0=no in®ltrate,1=in®ltrate upto 4 cells thick in most vessels, 2=in®l-trate from ®ve to seven cells thick in most vessels,3=in®ltrate greater than 7 cells thick in most vessels.Data are expressed as means2standard errors.

2.9. Statistical analysis

Histological scores were expressed as mean2stan-dard error of mean and statistical comparisons in thetwo groups (controls and vaccinated) were made withone way analysis of variance (ANOVA). The meanvalues were further compared using Fisher's PLSDtest. Di�erences between groups were considered sig-ni®cant at p-values less than 0.05. All statistical ana-lyses were performed with Statview II software(Abacus Concepts, Berkley, CA).

3. Results

3.1. Expression of IFN-g in mouse lung following pIFN-g gene transfer

Mouse IFN-g cDNA was ampli®ed as a 486 bpBamHI-XhoI cassette, which had transcription in-itiation and termination codons. Ligation of this PCRproduct to the mammalian expression vectorpcDNA3.1 put IFN-g gene under the transcriptionalcontrol of the cytomegalovirus late gene promoter fol-lowed by the bovine poly A sequences and terminationsignal. The resulting plasmid pcDNA3.1-IFN-g,referred to as pIFN-g, was transfected into NIH-3T3cells to ascertain its expression (data not shown). Thelung expression of IFN-g was analyzed in mice follow-ing administration of the pIFN-g by assessing BALFcollected at 7 days post-treatment from four di�erentgroup of mice (4 mice/group). Naive mice receivedeither PBS, pcDNA 3.1 (mock; 100 mg/mouse), pIFN-g or lipofectamine. As shown in Fig. 1A, mice admi-nistered with pIFN-g expressed high levels of IFN-g(62.19214.6 pg/ml). IFN-g levels were lower than thedetection limit in all other groups of mice used as con-trols. The time course of IFN-g expression was ana-lyzed in pIFN-g administered mice by assessing BALFcollected at di�erent days post-treatment. As shown inFig. 1B, the highest levels of IFN-g (254.76220.37pg/ml) were observed at day seven post-treatment.Expression of IFN-g transcripts and the presence ofthe pIFN-g constructs in the transduced mice werealso con®rmed by RT±PCR (data not shown).

Fig. 1. (A) Expression of murine IFN-g following gene transfer in

BALB/c mice. Groups of mice were intranasaly administered either

with pIFN-g (100 mg+lipofectamine), Lipofectamine alone (50 ml),PBS (50 ml) or pcDNA3.1 (100 mg) alone thrice with two day interval

in a 50 ml volume. Seven days after the last intranasal adminis-

tration, IFN-g expression was detected from BALF by ELISA. Bars

represent mean2SD (n= 4). (B)Time course of IFN-g expression.

IFN-g expression was estimated from BALF by ELISA on days

1,3,5 and 7 following last intranasal delivery. IFN-g expression was

not detected in any of the control groups of mice. Bars represent

mean2SD (n = 4).

M. Kumar et al. / Vaccine 18 (2000) 558±567 561

3.2. pIFN-g gene transfer attenuates RSV replication

BALB/c mice, intranasaly administered with RSV,developed severe RSV infection, including lesions intheir lungs by day 4 (data not shown). RSV infectionin mouse lungs could be detected by RT±PCR by day2 but not in mice administered with PBS or UV-inacti-vated RSV (data not shown). To examine the e�ect of

pIFN-g inoculation, mice were infected with 1 � 106

pfu of RSV, 7 days after pIFN-g administration. RSVreplication was detected in lung tissues by a RT±PCRassay of the RSV N gene (364 bp) on day 4 of infec-tion in control and treated mice. Fig. 2A, panel 1,lanes 1±4 and lanes 11±14 show RSV replication incontrol animals that received PBS and lipofectamine,respectively. RSV replication was also detected in ani-mals treated with pcDNA3.1 alone (data not shown).In marked contrast, mice treated with pIFN-g showedeither minimal (Fig. 2A, panel 1, lanes 5 and 6) or noRSV replication (Fig. 2A, panel 1, lanes 7±10).Treated mice also showed an 82.75% reduction inRSV antigen load as compared to controls (Fig. 2B),which received PBS only (the values were correctedwith the basal antigen level obtained from that of UV-inactivated RSV). These results indicate that ex-pression of IFN-g following gene transfer resulted insigni®cant reduction of RSV replication and total RSVantigen load in the lungs of mice inoculated with RSV.

3.3. pIFN-g gene transfer alters in¯ammatory cellpopulation in the RSV-infected airway and lung

The cellular composition of the airway and lung fol-lowing RSV infection was examined by a BAL celldi�erential; the results are shown in Fig. 3. A signi®-cant ( p < 0.05) decrease (27.3%) in the percentageof lymphocytes was observed in the BALF of micetreated with pIFN-g as compared to the controls.There was also an increase in the percentage of neutro-phils following RSV infection in all groups comparedto uninfected mice, however, no signi®cant change wasobserved among the control groups and pIFN-g admi-nistered mice. Percentage of eosinophils in all thegroups was low. Naive uninfected and pIFN-g admi-nistered and RSV infected mice showed a signi®cant( p < 0.05) reduction in the percentage of eosinophilswhen compared only to the vector control. There wasa signi®cant reduction in the percentage of macro-phage cells ( p < 0.05) in control groups as comparedto pIFN-g administered and naive uninfected groupsof mice. No signi®cant change was found in the cell

Fig. 2. (A) Detection of RSV infection in the lungs of BALB/c mice.

RSV-N gene expression was checked by RT±PCR. Mice were treated

with PBS alone (lanes 1±4), pIFN-g+lipofectamine (lanes 5±10) or

lipofectamine alone (lanes 11±14) as described, under methods and

subsequently infected with RSV a week after the last treatment. RSV

N gene expression was detected in all the PBS control mice (lanes 1±

4) and also in the mice treated with lipofectamine alone (lanes 11±

14); whereas in pIFN-g treated mice, RSV- N gene expression was

not detected (lanes 7 ± 10) or minimally detected (lanes 5 and 6).

Lower panel shows the RT±PCR of b-actin gene, used as the in-

ternal control. Data are from two independent sets of experiments.

(B) pIFN-g gene transfer decreases RSV antigen load in mouse

lungs. Groups of mice (n= 6) were intranasaly administered with

100 mg of pIFN-g complexed with lipofectamine three times on two

day intervals and infected with RSV seven days later. On day four

p.i. their lungs were taken out, homogenized and RSV antigen was

estimated by ELISA. Results represent the mean2SD of 6 mice/

group.

Table 1

Semi-quantitative analysis of the lung damage by histopathological scoresa

Treatment group Epithelial damage Interstitial change Peri-bronchovascular change

Control + RSV 220.2 1.420.2 1.920.2

pIFN-g+ RSV 1.120.2� 0.320.2�� 0.920.1��

Uninfected 0.820.2� 0.3320.3�� 0.820.1��

a Each value represents the mean2SEM of 6 ®elds from 5 individual lung sections from each mouse in a group (n= 12 for pIFN-g treated

and n = 4 for each control groups; the average value for all the control groups is shown as no statistically signi®cant di�erence was observed

among the control groups). Statistical di�erences compared to control are indicated.� Value signi®cant at p< 0.05.�� Value signi®cant at p< 0.01.

M. Kumar et al. / Vaccine 18 (2000) 558±567562

di�erential between mice treated with pIFN-g followedby RSV infection when compared to naive uninfectedmice.

3.4. pIFN-g gene transfer decreases RSV induced lungpathology

Para�n-embedded sections of the lungs were stainedwith hematoxylin-eosin (HE) and examined to deter-mine the degree of RSV-induced in¯ammation (Fig. 4).The group of mice treated with pIFN-g showed nor-mal lung histology (Figs. 4C and D) similar to that ofthe uninfected control (Figs. 4A and B). Control miceinfected with RSV showed various features of in¯am-mation. The epithelial cells of a�ected bronchioleswere swollen and occasionally, an exudate of desqua-mated epithelial cells was present in the lumen of someof the bronchioles (Figs. 4E and F). Some of thealveolar spaces were densely in®ltrated with mono-nuclear cells (Fig. 4H). Alveolar exudate and enlargedseptal cells (Fig. 4H) were also present. Peri-vascular(Fig. 4G) in®ltration was observed in the controlgroup of mice.

Semi-quantitative analyses of the lung sections basedon histologic scoring for airway in¯ammation are sum-marized in Table 1. Sections were scored for epithelialdamage, interstitial changes including both in®ltration

and thickness and peri-bronchovascular in®ltration.Signi®cant changes for all the above features werefound among infected controls and IFN-g adminis-tered and RSV infected mice.

3.5. pIFN-g gene transfer down-regulates IL-5 mRNAexpression

Expression of IL-5 and IFN-g was examined byRT±PCR and ELISA in lungs of mice treated withpIFN-g and infected with RSV seven days later. Fourout of six mice treated with pIFN-g (Fig. 5A, panel 2,lanes 6 and 8±10) did not express mRNA for IL-5.Mice intranasaly administered with PBS or lipofecta-mine as controls showed IL-5 mRNA expression (Fig.5A, panel 2, lanes 1±4 and 11±14). Five of six micewhich were treated with pIFN-g expressed IFN-gmRNA as detected by the RT±PCR analysis (Fig. 5A,panel 1, lanes 6±10). Control mice did not show anyIFN-g mRNA expression (Fig. 5A, lanes 1±4 and 11±14). The pIFN-g treated mouse which did not expressIFN-g mRNA also showed no reduction in IL-5mRNA. Expression of IL-5 mRNA and absence ofIFN-g transcripts was also observed in control micetreated with pcDNA3.1 alone (data not shown).

IFN-g and IL-5 were also assayed by ELISA fromthe BALF of treated and control mice after RSV infec-

Fig. 3. Analysis of bronchial alveolar lavage. Groups of mice (n= 6) were treated as described in Fig. 1A and infected with RSV seven days

later. BAL was collected from the lungs of mice on day 4 p.i. cytocentrifuged and stained. BAL cell di�erential from pIFN-g treated and control

mice is shown. Bars represent mean (n = 6)2SD. p< 0.05 compared to a: naive uninfected, b: pIFN-g administered and RSV infected mice.

M. Kumar et al. / Vaccine 18 (2000) 558±567 563

Fig. 4. E�ect of pIFN-g gene transfer on lung pathology. A and B are tissue sections from uninfected mice. C and D are tissue sections from

pIFN-g treated and RSV infected mice showing normal lung histology. The control mice with no IFN-g treatment showed epithelial denudation

(E) and bronchial exudate composed of in¯ammatory cells (F). Peri-vascular in®ltration (G) and enlarged septal cells with mono-nuclear cell in®l-

tration (H) were also observed in control groups. Arrows indicate the di�erent signs of in¯ammation in control mice as described in the text.

M. Kumar et al. / Vaccine 18 (2000) 558±567564

tion. All control mice showed increased IFN-g ex-pression after RSV infection (Fig. 5B), which was notdetected earlier in an uninfected state. There was nosigni®cant di�erence of IFN-g expression among thevarious control groups. However, the control groupsexpressed 2.5±6 fold higher IFN-g expression after theRSV infection. The pIFN-g treated group, when com-pared to di�erent controls showed signi®cantly( p < 0.05) higher levels of IFN-g expression.However, we could not detect IL-5 in the BALF ofthese groups of mice by ELISA.

4. Discussion

In certain diseases where IFN-g constitutes a poten-tial therapeutic modality, it is deemed necessary toadministrate it in high doses repeatedly to achievedesired therapeutic e�cacy because of the short half-life of IFN-g. However, in high doses IFN-g is cyto-toxic. Thus, the systemic use of IFN-g protein to treatrespiratory tract infections or allergic disease is con-sidered to be of limited value. In an e�ort to circum-

vent the drawbacks inherent with the use of IFN-gprotein, in the present study, an IFN-g gene transferapproach was used in a murine model to prevent RSVinfection. IFN-g gene transfer resulted in a signi®cantincrease in IFN-g levels in BAL ¯uids. Evidence fromthe studies of RSV replication, BAL, and lung histo-pathology of mice indicate that IFN-g gene transfer ise�ective in inhibiting RSV replication and conse-quently, infection-associated in¯ammation. The resultsalso show IFN-g gene transfer leads to the suppressionof Th2-like cytokine production in the lung, which istypically found in allergic in¯ammation and asthma.This latter ®nding is consistent with the inhibition ofallergic in¯ammation by IFN-g gene transfer [23].

A major ®nding of this study is the signi®cantdecrease in RSV replication detected in the pIFN-gadministered mice using RT±PCR and ELISA. Thetranscript encoding the RSV N protein was almost notdetectable in majority of the pIFN-g treated mice,which suggests that overproduced IFN-g inhibitedRSV replication. There was an 82.75% reduction inthe RSV antigen load in mice treated with pIFN-gconstruct. However, the mechanism by which, pIFN-ggene transfer inhibits RSV replication is unclear. IFNsare known for their anti-viral property [29]. Exposureof epithelial cells to IFN-g triggers the cellular signaltransduction pathways and activates several genesinvolved in anti-viral mechanisms, including the induc-tion of 2 '±5 ' oligoadenylate synthetase [30]. WhetherpIFN-g gene transfer-induced protection is mediatedby similar mechanisms remains to be investigated.

RSV infection induces in¯ammatory changes in thelung, which is readily detectable in BAL ¯uid. In nor-mal or sham infected mice most recovered cells inBAL ¯uid are macrophages with less than 5% consti-tuting lymphocytes. RSV speci®c lymphocytes isolatedfrom the lungs of RSV infected mice exhibit CTL ac-tivity, which peaks at days 7 to 9, post infection [31].The proportion of lymphocytes increases to about20% between 10 and 16 days after infection anddecreases thereafter [32]. Consistent with these ®nd-ings, BAL from the lungs of pIFN-g treated mice had27% less lymphocytes compared to control groups. Asmall number of eosinophils were also detected in theBAL of RSV infected mice, suggesting that the airway-hyper-responsiveness caused by the viral infection maybe due to in®ltration of these cells into the airway.RSV infection induces the expression of adhesion mol-ecules on the bronchial epithelium, particularly ICAM-1 [33±35], which contributes to airway in¯ammationby supporting adhesion and retention of these in®ltrat-ing in¯ammatory cells [36].

Comparison of lung IFN-g levels in control andpIFN-g treated mice following infection with RSVshowed that control infected mice showed barelydetectable levels of IFN-g proteins. This is consistent

Fig. 5. (A) Detection of IL-5 and IFN-g mRNA expression in the

lungs of BALB/c mice. Mice were treated as described in Fig. 2A,

IL-5 and IFN-g gene expression was checked by RT±PCR. lanes 1±4

and 11±14 are PBS and lipofectamine control respectively. Lanes 5±

10 are mice administered with pIFN-g construct. Control mice

(panel 2, lanes, 1±4 and 11±14) showed IL-5 speci®c ampli®cation.

pIFN-g treated mice showed IFN-g expression (panel 1, lanes 6±10).

Data are from two independent sets of experiments. (B) Expression

of IFN-g in BALF from treated and control mice following RSV

infection as estimated by ELISA. Values are mean2SD (n= 6). �

Indicates value signi®cant at p< 0.05 when compared with di�erent

controls.

M. Kumar et al. / Vaccine 18 (2000) 558±567 565

with the ®nding that compared some other viruses,such as in¯uenza; RSV infections induce less IFN-g[37]. Also vaccination of calves with formalin-inacti-vated BRSV lead to diminished IFN-g productionresulting in impaired viral clearance and enhanced dis-ease in FI-BRSV vaccinated calves [38]. However, noIFN-g transcripts were detected from lung tissue invarious control groups following RSV infection,despite detectable levels of IFN-g protein in BALF.This could be attributed to the minimal induction ofIFN-g by RSV infection and rapid turnover of theIFN-g mRNA. The levels of IFN-g in pIFN-g treatedand RSV infected group were still higher than the var-ious control groups infected with RSV.

Interferons also possess important immunomodula-tory functions. Because of the reciprocal regulation ofT helper cell subsets, it was anticipated that the pIFN-g gene transfer would promote a Th1-like instead of aTh2-like immune response expected against RSV. Thecytokine IL-5 is an important marker of Th2-likeimmunity induced by RSV and it plays a pivotal rolein eosinophils survival in the lung and asthma. Resultsfrom a RT±PCR analysis of IL-5 mRNA expressionindicated that pIFN-g gene transfer resulted in theexpression of IFN-g mRNA and inhibited IL-5 ex-pression in infected mice when compared to controls.Mice lacking IFN-g mRNA expression exhibited anupregulation in the levels of IL-5 mRNA.Furthermore, IL-5 protein could not be detected in theBAL ¯uid of infected mice, which expressed signi®cantamounts of IFN-g and had only a few eosinophils.Results from a RT±PCR analysis of IL-12 also showedan increased expression of IL-12 (p40) mRNA in someof the pIFN-g treated mice (data not shown).Together, these results suggest that pIFN-g inhibits theinduction of IL-5 mRNA and may play an importantrole in the induction of a Th1-like immunity againstRSV.

Epithelial cell damage and cellular in®ltration arehallmarks of RSV infection in humans and in variousanimal models. Prior treatment of mice with thepIFN-g signi®cantly reduced epithelial cell damage anddecreased interstitial and peri-bronchovascular in®ltra-tion of the cells in the lung upon RSV infection. Thecontrol RSV-infected mice predominantly showed amononuclear cell in®ltrate with very few eosinophils.No marked di�erence was observed between the unin-fected and pIFN-g treated and RSV infected mice,suggesting that pIFN-g gene transfer suppresses RSVinfection-induced in¯ammatory cell population in thelung. These ®ndings suggest that the pIFN-g per sedoes not induce any in¯ammatory changes in the air-way and the mice did not exhibit any external signs ofillness, which suggests that the vaccine may be safe inmice. The evidence that pIFN-g treated and RSVinfected mice exhibited a normal airway phenotype

suggests that the RSV replication is inhibited almostby about 83% of the control.

Recently it was reported [39] that murine IFN-g,when expressed by a recombinant respiratory syncytialvirus, attenuates virus replication in mice without com-promising immunogenicity. The attenuation was attrib-uted to the activity of high levels of IFN-g and micewere completely resistant to subsequent RSV chal-lenge. In contrast, our approach of over-expressingIFN-g through a gene transfer may provide protectionagainst a broader range of viral infection, includingthe most prevalent rhinovirus infection [24].

In summary, these results taken together demon-strate that a pIFN-g gene transfer inhibits RSV repli-cation, alters the lung cytokine pattern from a Th2-dominant to Th1-dominant milieu, and protects thelung from RSV-induced epithelial damage and cellularin®ltration. The in vivo expression of IFN-g proteinfollowing gene transfer renders signi®cant protectionof BALB/c mice against RSV infection. Because of theanti-viral property of IFN-g, an IFN-g DNA vaccinemay be useful against respiratory viral infections.

Acknowledgements

SSM is a recipient of the American LungAssociation of Florida Career Development Award.This study was supported by the funds from theUniversity of South Florida Creative Scholarship, andthe Grant-in-Aid from the Aging Institute awarded toSSM, the Joy McCann Culverhouse Endowment tothe Airway Disease Research Center.

References

[1] Chanock RM, Parott RH. Acute respiratory disease in infancy

and childhood: present understanding and prospects for preven-

tion. Pediatrics 1981;36:21±39.

[2] Brandt CD, Kim HW, Arrobio JO, et al. Epidemiology of res-

piratory syncytial virus infection in Washington, DC. III.

Composite analysis of eleven consecutive yearly epidemics. Am

J Epidemiol 1990;98:355±64.

[3] Kim HW, Arrobio JO, Brandt CD, et al. Epidemiology of res-

piratory syncytial virus infection in Washington, DC. I.

Importance of the virus in di�erent respiratory tract disease syn-

dromes and temporal distribution of infection. Am J Epidemiol

1973;98:216±25.

[4] McIntosh K, Chanock RM. Respiratory Syncytia Virus. In:

Fields BN, Knipe DM, editors. Virology, 2, 1990. p. 1045.

[5] Murray AR, Dowell SF. Respiratory syncytial virus: not just

for kids. Hospital Practice 1997;15:87±104.

[6] Center for Disease Control and Prevention. Respiratory syncy-

tial virus activity 1996-1997 season. MMWR 1996;45:1053.

[7] Hall CB, McBride JT. Respiratory syncytial virus-from chimps

with colds to conundrums and cures. New Engl J Med

1991;325:57±8.

M. Kumar et al. / Vaccine 18 (2000) 558±567566

[8] Warren KS. New scienti®c opportunities and old obstacles in

vaccine development. Proc Natl Acad Sci USA 1986;83:9275±7.

[9] Walsh J. Establishing health priorities in the developing world.

New York: United Nations Development Programme, 1988.

[10] Hall CB, Walsh EE, Long CE, Schnabel KC. Immunity to and

frequency of reinfection with respiratory syncytial virus. J Infect

Dis 1991;163:693±8.

[11] Chanock RM, Parrott RH, Connors M, Collins PL, Murphy

BR. Serious respiratory tract disease caused by respiratory syn-

cytial virus: prospects for improved therapy and immunization.

Pediatrics 1992;90:137±43.

[12] Hall CB. Prospects for a respiratory syncytial virus vaccine.

Science 1994;265:1393±4.

[13] Mok JYK, Simpson H. Symptoms, atopy and bronchial reactiv-

ity after lower respiratory infection in children. Arch Dis Child

1984;59:299±305.

[14] Weiss ST, Tager IB, Munoz A, Speizer FE. The relationship of

respiratory infection in early childhood to the occurrence of

increased levels of bronchial responsiveness and atopy. Am Rev

Res Dis 1985;131:573±8.

[15] Sly PD, Hibbert ME. Childhood asthma following hospitaliz-

ation with acute viral bronchiolitis in infancy. Pediatr Pulmonol

1989;7:153±8.

[16] Schwarze J, Hamelmann E, Bradley KL, Takeda K, Gelfand

EW. Respiratory syncytial virus infection results in airway

hyperresponsiveness and enhanced airway sensitization to aller-

gen. J Clin Invest 1997;100:226±33.

[17] Mosmann TR, Co�man RI. Th1 and Th2 cells: di�erent pat-

terns of lymphokine secretion lead to di�erent functional prop-

erties. Annu Rev Immunol 1989;7:145±73.

[18] Roman M, Calhoun WJ, Hinton KL, Avendano LF, Simon V,

Escobar AM, Gaggero A, Diaz PV. Respiratory syncytial virus

infection in infants is associated with predominant Th-2-like re-

sponse. Am J Resp Crit Care Med 1997;156:190±5.

[19] Connors M, Kulkarni AB, Firestone CY, Holmes KL, Morse

HC, Sotnikov AV, Murphy BR. Pulmonary histopathology

induced by respiratory syncytial virus (RSV) challenge of for-

malin-inactivated RSV-immunized BALB/c mice is abrogated

by depletion of CD4+ T cells. J Virol 1992;66:7444±51.

[20] Connors M, Geise NA, Kulkarni AB, Firestone CY, Morse

HC, Sotnikov AV, Murphy BR. Enhanced pulmonary histo-

pathology induced by respiratory syncytial virus (RSV) chal-

lenge of formalin±inactivated RSV-immunized BALB/c mice is

abrogated by depletion of interleukin-4 (IL-4) and IL-10. J

Virol 1994;68:5321±5.

[21] Graham BS, Henderson GS, Tang YW, Lu X, Neuzil KM,

Colley DG. Priming immunization determines T helper cytokine

mRNA expression patterns in lungs of mice challenged with res-

piratory syncytial virus. J Immunol 1993;151:2032±40.

[22] Pene J, Rousset F, Briere F, Chretien I, Bonnefoy JY, Spits H,

Yokota T, Arai N, Arai KI, Banchereay J, de Vries J. IgE pro-

duction by human lymphocytes is induced by interleukin±4 and

suppressed by interferons gamma, alpha and prostaglandin E2.

Proc Natl Acad Sci USA 1988;85:6880±4.

[23] Li XM, Chopra RK, Chou TY, Scho®eld BH, Wills-Karp M,

Huang SK. Mucosal IFN-g gene transfer inhibits pulmonary

allergic responses in mice. J Immunol 1996;157:3216±9.

[24] Sethi SK, Bianco A, Allen JT, Knight RA, Spiteri MA.

Interferon-gamma down-regulates the rhinovirus-induced ex-

pression of intercellular adhesion molecule-1 (ICAM-1) on

human airway epithelial cells. Clin Exp Immunol 1997;110:362±

9.

[25] Ledley FD. Nonviral gene therapy: the promise of genes as

pharmaceutical products. Hum Gene Ther 1995;6:1129±44.

[26] Graham BS, Perkins MD, Wright PF, Karzon DT. Primary res-

piratory syncytial virus infection in mice. J Med Virol

1988;26:153±62.

[27] Taylor G, Stott EJ, Hughes M, Collins AP. Respiratory syncy-

tial virus infection in mice. Infect Immun 1984;43:649±55.

[28] Tautz D, Renz M. An optimized freeze-squeeze method for the

recovery of DNA fragments from agarose gels. Anal Biochem

1983;132:14±9.

[29] Muller U, Steinho� U, Reis LFL, Hemmi S, Pavlovic J,

Zinkernagel RM, Aguet M. Functional role of type I and type

II interferons in antiviral defense. Science 1994;264:1918±21.

[30] Will A, Hemmann U, Horn F, Rollingho� M, Gessner A.

Intracellular murine IFN-g mediates virus resistance, expression

of oligoadenylate synthetase and activation of STAT transcrip-

tion factors. J Immunol 1996;157:4576±83.

[31] Taylor G, Stott EJ, Hayle AJ. Cytotoxic lymphocytes from the

lung of mice infected with respiratory syncytial virus. J Gen

Virol 1985;66:2533±8.

[32] Openshaw PJM. Flow cytometric analysis of pulmonary lym-

phocytes from mice infected with respiratory syncytial virus.

Clin Exp Immunol 1989;75:324±8.

[33] Arnold R, Werchau H, Konig W. Expression of adhesion mol-

ecules (ICAM-1 LFA-3) on human epithelial cells (A549) after

respiratory syncytial virus infection. Int Arch Allergy Immunol

1995;107:392±3.

[34] Matsuzaki Z, Okamoto Y, Sarashina N, Ito E, Togawa K,

Saito I. Induction of intercellular adhesion molecule-1 in human

nasal epithelial cells during respiratory syncytial virus infection.

Immunology 1996;88:565±8.

[35] Arnold R, Konig W. ICAM-1 expression and low-molecular-

weight G-protein activation of human bronchial epithelial cells

(A549) infected with RSV. J Leuk Biol 1996;60:766±71.

[36] Stark JM, Godding V, Sedgwick JB, Busse WW. Respiratory

syncytial virus infection enhances neutrophil and eosinophil ad-

hesion to cultured respiratory epithelial cells. Roles of CD18

and intercellular adhesion molecule-1. J Immunol

1996;156:4774±82.

[37] Chonomaitre T, Roberts NJ, Douglas RG, Hall CB, Simons

RL. Interferon production by human mononuclear leukocytes:

di�erences between respiratory syncytial virus and in¯uenza

virus. Infect Immun 1981;32:300±3.

[38] Woolums AR, Singer RS, Boyle GA, Gershwin LJ. Interferon

gamma production during bovine respiratory syncytial virus

(BRSV) infection is diminished in calves vaccinated with forma-

lin-inactivated BRSV. Vaccine 1999;17:1293±7.

[39] Bukreyev A, Whitehead SS, Bukreyeva N, Murphy BR, Collins

PL. Interferon g expressed by a recombinant respiratory syncy-

tial virus attenuates virus replication in mice without compro-

mising immunogenicity. Proc Natl Acad Sci USA 1999;96:

2367±72.

M. Kumar et al. / Vaccine 18 (2000) 558±567 567