Immune and Histopathological Responses in Animals Vaccinated with Recombinant Vaccinia Viruses That Express Individual Genes

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1 JOURNAL OF VIROLOGY, Dec. 1987, p X/87/ $02.00/0 Copyright 1987, Americn Society for Microbiology Vol. 61, No. 12 Immune nd Histopthologicl Responses in Animls Vccinted with Recombinnt Vccini Viruses Tht Express Individul Genes of Humn Respirtory Syncytil Virus E. J. STOTT,t G. TAYLOR,t L. A. BALL,t K. ANDERSON,: K. K.-Y. YOUNG, A. M. Q. KING, AND G. W. WERTZt* Deprtment of Microbiology nd Immunology, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin Received 1 June 1987/Accepted 20 August 1987 Previous reports hve estblished tht vccini virus (VV) recombinnts expressing G, F, or N protein of respirtory syncytil (RS) virus protect smll nimls ginst intrnsl chllenge with live RS virus. This work demonstrtes tht vriety of prmeters ffect the protection induced by recombinnt viruses. The route of vccintion, the subtype of chllenge virus, nd the species used influenced the ntibody titers nd extent of protection. During these studies, observtions were lso mde on the subclss of ntibody generted, nd pulmonry histopthologicl chnges induced by chllenge fter vccintion were noted. The effect of route of inocultion on host response ws exmined by vccinting mice intrnslly, intrperitonelly, or by scrifiction with recombinnt VV expressing the RS virus G glycoprotein. Intrnsl vccintion induced 25-fold-higher titers of ntibody to RS virus in the lung thn the intrperitonel route did, but both routes resulted in complete suppression of virus repliction fter intrnsl chllenge 21 dys fter vccintion. Scrifiction ws less effective method of vccintion. The ntibody induced by recombinnt VV in mice ws mostly immunoglobulin G2 (IgG2) with some IgG2b. No ntibody to RS virus ws detected in the IgA, IgM, IgGl, or IgG3 subclss irrespective of the vccintion route. The G nd F glycoproteins were shown to elicit similr subclsses of ntibody. However, nimls vccinted with the G nd F vectors differed strikingly in their response to chllenge by heterologous virus. Mice or cotton rts vccinted with recombinnt VV crrying the G gene of RS virus were protected ginst chllenge only with homologous subtype A virus. Vccintion with recombinnt VV expressing the F glycoprotein induced protection ginst both homologous nd heterologous subtype B virus chllenge. The protection induced in mice ws greter thn tht detected in cotton rts, indicting tht the host my lso ffect immunity. Finlly, this report describes histologicl exmintion of mouse lungs fter vccintion nd chllenge. Vccinted mice tht were subsequently chllenged hd significntly greter lung lesion scores thn unvccinted chllenged mice. The lesions were primrily peribronchiolr nd perivsculr infiltrtions of polymorphonucler cells nd lymphocytes. Further work will estblish whether these pulmonry chnges re desirble immune response to virus invsion or potentil immunopthogenic hzrd. The results hve importnt implictions for plnning strtegy of vccintion ginst RS virus nd emphsize potentil dngers tht my ttend the use of recombinnt VV s vccines. Respirtory syncytil (RS) virus ws first isolted in 1956 nd over 30 yers lter remins the mjor cuse of hospitliztion for children in the first yer of life in the developed world. There re two mjor subtypes of humn RS virus, designted A nd B (1). A bovine RS virus (probbly relted to group B) is significnt cuse of respirtory disese nd mortlity in cttle in Europe nd the United Sttes. Although effective live or killed veterinry vccines re becoming vilble for the bovine RS virus (26, 33), there is no equivlent prophylxis for children. One of the first vccines tested in infnts, Formlin-inctivted virus, induced enhnced disese when vccinees were subsequently infected with RS virus (6, 10, 11, 13). This lrming observtion hd profound effect on reserch towrd sfe nd effective * Corresponding uthor. t Present ddress: Agriculturl nd Food Reserch Council, Institute for Animl Disese Reserch, Compton Lbortory, Compton, Berkshire RG16 ONN, United Kingdom. t Present ddress: Deprtment of Microbiology, The University of Albm t Birminghm, Birminghm, AL Present ddress: Agriculturl nd Food Reserch Council, Institute for Animl Disese Reserch, Pirbright Lbortory, Pirbright, Woking, Surrey GU24 ONF, United Kingdom control of RS virus bronchiolitis. Attention turned entirely towrd live ttenuted vccines which were used intrnslly or intrmusculrly (4, 5, 12, 31, 32). However, this pproch ws frustrted by genetic instbility or overttenution of the virus or interference by mternl ntibody with its repliction. Three theories hve been dvnced to explin enhnced disese. First, tht serum ntibody in the bsence of nsl ntibody ws immunopthogenic. Second, tht Formlin induced ltered rectivity to the killed virus. Finlly, tht the inctivted vccine cused delyed hypersensitivity. Although recent evidence (19) suggested tht formldehyde dmged the RS virus fusion protein ntigen so tht the vccine induced ntibody with reduced biologicl ctivity, this is by no mens estblished nd my not be the whole explntion of vccine-enhnced disese. There is, therefore, need to distinguish protective responses to RS virus from those which re potentilly pthogenic. Recombinnt vccini viruses (VV) crrying genes for RS virus proteins hve recently been shown to protect cotton rts nd mice ginst intrnsl chllenge with RS virus (9, 14, 20, 25, 30). Such recombinnt viruses re powerful tools with which the immune response to individul RS virus

2 3856 STOTT ET AL. ntigens my be dissected. For exmple, using recombinnt vectors, the surfce glycoproteins, G nd F, hve been shown to induce high titers of humorl ntibody which correlte with protection of lungs from live virus chllenge (2, 9, 20, 25, 30). In ddition, the internl nucleocpsid gene of RS virus hs been identified s significnt trget for the cell-medited immune response (3). These initil studies hve provided useful insight into the roles of individul virl proteins in the infection process. However, mny spects of the response to vccintion with these vectors require chrcteriztion. In n effort to understnd how expression of individul RS virus proteins might be providing protective effect ginst RS virus or prticipting in the potentition of disese, we nlyzed the following prmeters of vccintion with recombinnt vectors bering RS virus genes: (i) route of inocultion, (ii) clss of ntibody produced in lung nd serum s function of route of dministrtion, (iii) effect of species of test niml on protection level, nd (iv) cross subgroup protection in reltion to the surfce glycoproteins. In ddition to the bove studies we lso crried out histopthologicl nlyses of lung sections fter vccintion nd chllenge. These studies were imed t determining whether prticulr RS virus protein might be involved in potentiting disese. MATERIALS AND METHODS Viruses. The Austrlin A2 strin (16) belongs to RS virus subtype A, nd the 8/60 strin (8) belongs to subtype B (1). Both viruses were grown nd titrted on HEp-2 cells. The overly for plque ssy consisted of Egle miniml essentil medium supplemented with 10% fetl clf serum, 20,ug of bromodeoxyuridine per ml, 25 mm HEPES (N-2-hydroxyethylpiperzine-N'-2-ethnesulfonic cid) (ph 7.6), nd 0.25% grose. Pltes were incubted for 7 dys t 37 C. The construction nd chrcteristics of the recombinnt VV vg301, contining the lrge glycoprotein (G) gene, vf325 nd vf317, contining the fusion protein (F) gene, nd vn125 nd vn333, contining the nucleoprotein (N) gene, hve been described previously (2, 14, 30). Briefly, cdnas of the RS virus genes were inserted into the EcoRI site of the VV thymidine kinse gene, downstrem from the initition site for trnscription of the VV 7.5K promoter. A recombinnt vector contining the gene for the nonstructurl protein 1B ws mde by similr methods. Detils of its construction will be reported elsewhere (K. Anderson, L. A. Bll, nd G. Wertz, mnuscript in preprtion). Recombinnt VV were grown nd ssyed on HEp-2 cells. Infected cell lystes were sonicted for 2 min nd centrifuged t 1,000 x g for 5 min, nd the superntnt ws lyered over 35% sucrose in 10 mm Tris hydrochloride, ph 8.8. After centrifuging t 18,000 rpm in n SW27 rotor for 80 min t 4 C, the pelleted virus ws resuspended in 1 ml of 10 mm Tris hydrochloride, ph 8.8. This mteril ws stored t -70 C nd diluted in 10 mm Tris hydrochloride (ph 8.8) before inocultion into nimls. Animls. Mle BALB/c ByJ mice free from infection with Sendi virus, pneumoni virus of mice, nd Mycoplsm pulmonis were obtined from Jckson Lbortory (Br Hrbor, Mine) t 4 weeks of ge. Cotton rts (Sigmodon hispidus) 8 to 12 weeks old were supplied by W. Clyde from colony mintined by the University of North Crolin. All experiments were bsed on groups of t lest five nimls. Vccintion nd chllenge. Unnesthetized mice were vccinted with 100,ul by intrperitonel injection or with 10 j.l by scrifiction of the til with bifurcted needle. J. VIROL. Intrnsl vccine in 50,u1 ws instilled into the noses of mice nesthetized with 2 mg of sodium pentobrbitl injected intrperitonelly. Cotton rts were nesthetized by inhltion of methoxyflurne (Abbott Lbortories, North Chicgo, Ill.) before intrderml injection of 100,u1 of vccine. Animls were chllenged under nesthesi s described bove by intrnsl instilltion of 50 p.l (mice) or 100,ul (cotton rts) of phosphte-buffered sline contining to 105 PFU of RS virus. Antibody ssy. Live mice were bled from the til vein. After nimls were killed with n overdose of sodium pentobrbitl, blood ws collected from the xill or hert. Mouse lungs were wshed by tying 20-guge cnnul into the trche nd inflting nd deflting the lungs twice with 1 ml of phosphte-buffered sline. Antibody to RS virus in ser nd lung wshings ws ssyed by enzyme-linked immunosorbent ssy (ELISA). Microtiter pltes (Flcon; Becton Dickinson Lbwre, Oxnrd, Clif.) were coted with RS virus-infected HEp-2 cell lyste or uninfected cell lyste s control nd dried overnight t 37 C. After incubtion with blocking buffer (phosphte-buffered sline contining 0.05% Tween 20 nd 5% pig serum) for 30 min t 22 C, the pltes were wshed thoroughly in phosphtebuffered sline contining 0.05% Tween 20. Dilutions of ser or lung wshings were dded nd incubted for 60 min t 22 C. After further wshing, bound ntibody ws detected by dding got nti-mouse immunoglobulin G (IgG) serum coupled to horserdish peroxidse (Kirkegrd nd Perry Lbortories Inc., Githersburg, Md.) or horserdish peroxidse-conjugted rbbit nti-mouse IgM, IgA, IgGl, IgG2, IgG2b, or IgG3 (Miles Scientific, Div. Miles Lbortories, Inc., Nperville, Ill.). After further wshing, 3,3',5,5'- tetrmethylbenzidine nd hydrogen peroxide in 0.1 M citrte buffer (ph 5.0) were dded s substrtes. The rection ws stopped by the ddition of 0.1 M sulfuric cid, nd the opticl density (OD) ws red t 450 nm. ELISA titers were clculted fter subtrction of OD with control ntigen from OD with virus-infected ntigen nd plotting this specific OD ginst smple dilution. By regression nlysis of the liner prt of this curve, the endpoint ws deduced t n OD 1.5 times the bckground. Virus ssy. Lungs were weighed nd ground in Hnks blnced slt solution contining M sucrose, 4.8 mm glutmte, 30 mm mgnesium chloride, 25 mm HEPES buffer, 200 U of penicillin per ml, nd 200,ug of streptomycin per ml to give 10% suspension. After centrifugtion t 10,000 x g for 2 min, the superntnt fluid ws ssyed in HEp-2 cells s described bove. Histology. Lungs were inflted with nd then immersed in fixtive consisting of 10% formldehyde in sturted mercuric chloride. Sections of lung were stined with hemtoxylin nd eosin nd exmined for bnorml pthology (lesions). Coded sections of lung were scored for lesions by n rbitrry scle of 0 to 3, with 1 representing infiltrtion involving one or two bronchioles or blood vessels nd 3 representing lesions involving most of the bronchioles nd blood vessels. A minimum of three lobes were exmined nd scored for ech mouse so tht the men lesion score for ech group ws bsed on t lest 15 observtions. RESULTS Route of vccintion. To determine the effect of route of dministrtion of vccine on ntibody levels stimulted in serum nd lung nd on protection, we inoculted groups of mice with 2 x 106 PFU of vg301 by one of the following

3 VOL. 61, 1987 RESPONSES TO VACCINIA VIRUSES CARRYING RS VIRUS GENES ).0 c Ri 0~~~~ Dys fter vccintion FIG. 1. Effect of vccintion route on ELISA ntibody response to RS virus. Symbols: , intrnsl; 0-4, intrperitonel; *----, scrifiction. Arrow indictes intrnsl chllenge with RS virus. routes: (i) intrperitonel, (ii) intrnsl, or (iii) scrifiction of the til. Seven dys lter, the men serum ntibody titer to RS virus ws 2.9 log1o in mice vccinted intrperitonelly (Fig. 1). Antibody ws not detected in nimls vccinted intrnslly or by scrifiction until 14 dys. Three weeks fter vccintion mice were chllenged intrnslly with RS virus. At this time the titer of serum ntibody in scrified mice ws t lest fivefold lower thn tht in the other two vccinted groups (Fig. 1; Tble 1). Antibody titers in lung wshings were highest (102.2) in mice vccinted intrnslly nd lowest (<100 5) in scrified mice. Five dys fter chllenge, the men titer of RS virus in lungs of unvccinted mice ws 4.8 log1o PFU/g. In contrst, no virus ws recovered from mice vccinted intrperitonelly or intrnslly. Virus ws recovered from two of five mice vccinted by scrifiction giving men titer of 1.6 log1o PFU/g. Seven dys fter chllenge, ntibody titers in serum were 3- to 50-fold greter thn t dy 21. During the sme period, titers of ntibody in the lung rose by 1,000-fold or more in mice vccinted intrperitonelly or by scrifiction, but by only 5-fold in mice vccinted intrnslly. Antibody clss. The isotype of ntibody to RS virus ws determined by ELISA before (dy 21) nd fter (dy 28) chllenge (Tble 2). Antibody in both serum nd lung wshings ws predominntly IgG2 nd IgG2b. At dy 21, the route of dministrtion hd little or no effect on distribution of ntibody isotype in serum. In the lung, however, mesurble IgG2 nd IgG2b ntibody ws detected only fter intrnsl vccintion. At 28 dys postvccintion, significnt titers of ntibody were present in serum of nimls vccinted by ll these methods, lthough titers in nimls vccinted by intrperitonel inocultion were significntly higher for both the IgG2 nd IgG2b clsses. No ntibody to RS virus ws detected in the IgA, IgM, IgGl, or IgG3 subclsses. The dt on ntibody isotype generted bove used recombinnt expressing the RS virus glycoprotein (G) s the ntigenic stimulus. The G protein is n unusul virl membrne protein. G is estimted to hve 50 to 60% of its 84,000 moleculr weight contributed by crbohydrte, the mjority of which is tunicmycin resistnt, thereby suggesting tht it is 0- rther thn N-linked. It ws of interest, therefore, to determine whether the ntibody isotype distribution would differ if recombinnt expressing the second RS virus surfce protein F, the fusion protein, ws used for vccintion. The F protein hs only five crbohydrte ttchment sites, nd ll re tunicmycin sensitive, indicting tht they re N-linked. The ntibody induced by vccintion in mice with vf325 ws predominntly of the IgG2 type with some IgG2b (dt not shown). This pttern is nlogous to tht observed fter vccintion with the G-expressing vector. Also, s with the G vccintion, no ntibody of the IgA, IgM, IgGl, or IgG3 subclss ws detected. Effect of heterologous chllenge. There re t lest two mjor subtypes (A nd B) of humn RS virus, nd nlyses with monoclonl nd polyclonl ntibodies indicte tht the mjor difference between these subtypes resides in the G glycoprotein. We therefore investigted the degree of cross protection tht the glycoproteins G or F might elicit ginst the two mjor subtypes of RS virus. Mice were vccinted intrperitonelly with recombinnts expressing either the G gene or the F gene of the subgroup A virus (A2 strin) nd chllenged 3 weeks lter with either A2 strin or 8/60 strin (B subtype) RS virus (Tble 3). At the time of chllenge ntibody titers to A2 virus were 3.9 to 4.0 log1o. Although mice given vf325 vccine hd similr titers ginst 8/60 virus (4.1 log1o), ntibody titers of nimls given vg301 vccine were 40-fold lower ginst 8/60 (Tble 3). Despite the fct tht the 8/60 B-type RS virus only grows to low titers in mice, i.e., 2.9 log1o PFU/g of lung (see below for comprtive growth in cotton rts), differences in the response to chllenge were detected which reflected the observed differences in ntibody levels. Mice vccinted with vg301 or TABLE 1. Effect of route of vccintion on ntibody production nd protection Dy 21 ntibody titer Dy 28 ntibody titer Vccine Route of t chllengeb Dy 26 virus titer fter chllenged vccintion fter chllenge' Serum Lung Serum Lung vg301 Intrperitonel 3.8 ± ± 0.5 < ± 0.4 vg301 Intrnsl 3.9 ± ± 0.2 < ± ± 0.2 vg301 Scrifiction 3.1 ± 0.2 < ± ± ± 0.5 None <1.5 < ± ± 0.3 <0.5 All vccines were inoculted t dose of 2 x 106 PFU. b Antibody titers were mesured by ELISA nd re expressed s men loglo ± stndrd error. Mice were chllenged intrnslly with PFU of RS virus 21 dys fter vccintion. c Virus titers re expressed s men log1o PFU per grm ± stndrd error. Mice were killed for virus titrtion 5 dys fter chllenge. d Antibody ws ssyed 7 dys fter chllenge.

4 3858 STOTT ET AL. J. VIROL. TABLE 2. Immunoglobulin subclss of ntibody to RS virus0 Antibody titers on dy 21 Antibody titers on dy 28 Vccine Route of Serum Lung Serum Lung vccintion IgG2 IgG2b IgG2 IgG2b IgG2 IgG2b IgG2 IgG2b vg301 Intrperitonel 1.7 ± 0.5 <1.0 <0.3 < ± ± ± ± 0.5 vg301 Intrnsl 1.7 ± ± ± ± ± ± ± ± 0.2 vg301 Scrifiction 1.3 ± ± 0.6 <0.3 < ± ± ± ± 0.3 None <1.0 <1.0 <0.3 <0.3 <1.0 <1.0 <0.3 <0.3 Antibody titers were mesured by ELISA nd re expressed s men logi0 ± stndrd error. vf325 hd no detectble virus in the lungs 5 dys fter chllenge with the homologous A2 virus. In contrst, only the lungs of mice vccinted with vf325 were free of virus fter chllenge with the heterologous 8/60 (B-type) virus. There ws no significnt difference between men 8/60 virus titers in the lungs of unvccinted mice nd those given vg301 vccine (Tble 3). Effect of species. There re two mjor smll niml model systems for humn RS virus, the BALB/c mouse nd the cotton rt (S. hispidus). To compre the results obtined in mice with those from n lterntive niml model of different species, we exmined the effectiveness of vccintion in cotton rts ginst A nd B subtype viruses with tht described bove in mice. Cotton rts were vccinted with recombinnts contining the G or F gene nd chllenged with A2 or 8/60 virus (Tble 4). In nimls given the vg301 vccine, no virus ws detected in the lungs of two of five rts chllenged with the A2 virus nd the men virus titer ws significntly reduced (from 5.6 log1o to 3.5 log1o). There ws no protection ginst the 8/60 B-type chllenge virus, nd the reduction of 0.4 log1o in virus titer ws not sttisticlly significnt. No virus ws recovered from the lungs of six of seven of the rts vccinted with vf317 nd chllenged with A2 nd four of five rts chllenged with 8/60 virus. The men virus titer ws reduced by 3.8 or 2.8 log1o fter chllenge with A2 or 8/60 virus, respectively. The results obtined in cotton rts re consistent with those obtined in mice. However, the reduction in virus titer in the lungs of the cotton rts ws more vrible. This might be due to the fct tht wheres the mice re inbred, the rts re not, or tht the interderml inocultion of the cotton rts is less reproducible (Tbles 1, 3, nd 4). Effect of vccintion on lung lesions. The effect of vccintion nd chllenge on the histology of the lung ws lso exmined. In the bsence of RS virus chllenge, vccintion by scrifiction or intrperitonel injection hd no significnt effect on the lungs when compred with those of untreted mice (Tble 5; P = 0.45 or 0.93, respectively). In contrst, 21 dys fter intrnsl vccintion the lesion score ws 0.74 compred with 0.01 in unvccinted nimls, highly significnt difference (P = ). The lesions in the intrnslly vccinted mice t this time showed peribronchiolr nd perivsculr ccumultions of lymphocytes nd mcrophges. In some res, the bronchiolr epithelil cells were proliferting, nd in others the epithelil cells were elongted nd flttened. The epithelil lining ws infiltrted with mononucler cells, nd there were few mononucler cells nd desqumted epithelil cells in the lumen. However, the bronchiolr exudte ws miniml, nd this my be due to the lung lvge performed before fixtion. In some res of the lung prenchym there ws thickening of the lveolr wlls owing to mononucler cell infiltrtion, nd the lveoli contined lrge fomy mcrophges. After chllenge, the lesion score of the intrnslly vccinted mice did not chnge; however, the lesions, lthough similr to those described bove, showed greter predominnce of lymphocytes in the peribronchiolr nd perivsculr cell ccumultions nd fewer res of epithelil cell prolifertion. The epithelil cells were still enlrged, but there ws less infiltrtion with mononucler cells. After RS virus chllenge, the lesion scores of unvccinted mice nd of those vccinted intrperitonelly or by scrifiction were significntly greter thn those of unchllenged mice (Tble 5; P = to 0.024). Furthermore, lesions in mice vccinted intrperitonelly or by scrifiction were significntly greter thn those due to RS virus lone (P = 0.05 nd 0.029, respectively). The lesions in unvccinted mice hve been described in detil elsewhere (29) nd re composed of slight peribronchiolr nd perivsculr ccumultions of lymphocytes with polymorphonucler leukocytes nd enlrgement of the bronchiolr epithelil cells nd occsionl res where the lveolr wlls re thickened. The lesions in the vccinted mice were essentilly the sme but with incresed peribronchiolr nd perivsculr ccumultions nd greter infiltrtion of the lveoli with lymphocytes nd mcrophges. As described bove, mice given recombinnt vg301 hd incresed lesion scores fter chllenge (Tble 5). To estblish whether VV recombinnts contining other RS virus genes induced the sme effect, the lungs of mice given TABLE 3. Effect of heterologous chllenge Vccine Route of Dose Dy 21 ntibody titer virus vciin10pf)chllenge vccintion(10~~~ PFU) A2 8/60tieinlgs virusb Dye 26 viunsr vg301 Intrperitonel ± 0.4 A2 <1.7 8/ ± 0.4 vf325 Intrperitonel ± ± 0.4 A2 <1.7 8/60 <1.7 None <1.5 <1.5 A2 4.4 ± 0.2 8/ ± 0.1 Antibody titers were mesured by ELISA nd re expressed s men loglo ± stndrd error. b Mice were chllenged intrnslly with PFU of either the A2 or 8/60 strin of RS virus 21 dys fter vccintion. c Virus titers expressed s men logl0 PFU per grm ± stndrd error. Mice were killed for virus titrtion 5 dys fter chllenge.

5 VOL. 61, 1987 RESPONSES TO VACCINIA VIRUSES CARRYING RS VIRUS GENES 3859 TABLE 4. Protection of cotton rts by recombinnt VV Vccine Route of Dose Dy 21 Chllenge No. protected/ Virus titer Difference (P) vccintion (107 PFU) ntibody titer"' (PFU) no. chllenged in lung' vg301 Intrderml ± 0.6 A2 (5 x 104) 2/5 3.5 ± /60 (1 x 105) 0/5 4.2 ± vf317 Intrderml ± 0.5 A2 (5 x 104 ) 6/7 1.8 ± 0.4 < /60 (1 x 105) 4/5 1.8 ± None <1.5 A2 (5 x 104) 0/7 5.6 ± 0.2 8/60 (1 x 105) 0/ Antibody titers were mesured by ELISA ginst A2 virus nd re expressed s men logl0 I Virus titers re expressed s men loglo PFU per grm ± stndrd error. C Probbility of differences from unvccinted mice were clculted by Student's t test. + stndrd error. vf325, vn125, or vlb were exmined 7 dys fter RS virus chllenge (Tble 6). Both recombinnts vf325 nd vn125 induced significntly incresed lesion scores 7 dys fter chllenge compred with unvccinted mice (P = nd 0.017, respectively). The lesions were similr to those described bove for mice vccinted intrperitonelly with vg301. However, the lesion score of mice given vlb ws not significntly greter thn tht in unvccinted mice (P = 0.81). This ltter observtion demonstrted tht the vccini vector itself is not responsible for the increse in scores in vccinted nimls. DISCUSSION Recombinnt VV expressing foreign genes hve been proposed s potentil vccines. Before this pproch cn be fesible, extensive studies of the biologicl properties of these viruses in vrious niml models must be undertken. Vccintions with recombinnt viruses expressing the G, F, or N protein of RS virus bolish or significntly reduce the titer of virus in the lung fter intrnsl infection (14, 25, 30) nd thus provide system in which recombinnt-induced immunity to this respirtory pthogen my be nlyzed. One of the most importnt vribles ffecting ntibody level nd extent of protection in mice ws the route of vccintion. Intrnsl vccintion induced 25-fold-higher titers of ntibody in the lung thn either of the prenterl routes. Despite this difference, fter chllenge there ws no detectble virus in the lungs of nimls vccinted by either intrnsl or intrperitonel routes. Prt of the explntion for this immunity is probbly tht the ppernce in the lung of RS virus ntigen, with or without limited repliction, stimulted the locliztion of lrge numbers of sensitized lymphocytes nd consequent locl production of lrge mounts of protective ntibody. Support for this hypothesis is provided by the histologicl finding of peribronchiolr infiltrtion of lymphocytes (Tble 5; discussed below) nd the lrge increses in ntibody in the lung 7 dys fter chllenge (Tble 1). The high levels of ntibody re sufficient to explin the protection observed since it is known tht pssive trnsfer of monoclonl ntibodies to G glycoprotein protects mice (28). It is unlikely tht cytotoxic T cells plyed significnt role in the protection induced by this recombinnt becuse vg301 does not stimulte cytotoxic T-cell memory (3). The reltively poor response to vccintion by scrifiction my be reflection of the difficulty of determining the exct dose bsorbed. Although 2 x 106 PFU were pplied to the skin, only smll proportion my hve initited infection. A similr mrked dependence of immune response on route of vccintion hs been reported by Smll nd collegues (24), who showed tht intrderml vccintion of mice with recombinnt VV contining the H-2 influenz virus hemgglutinin gene protected only the lower respirtory trct, wheres intrnsl vccintion protected both nose nd lung from infection. A further considertion highlighted by our experiments is tht of sfety. We hve lredy shown tht wild-type VV given intrnslly is lethl to mice (25), but it is now cler tht lthough mice remined cliniclly norml fter intrnsl vccintion with recombinnt vg301, significnt lesions were present in their lungs over 3 weeks fter vccintion (Tble 5). Thus, lthough intrnsl vccintion my seem logicl route for protection ginst respirtory infection, it my be neither necessry nor desirble if the primry objective is to protect the lower respirtory trct. Our observtions tht ntibody responses to both vg301 nd vf325 were predominntly in the IgG2 subclss with some IgG2b were consistent with the finding tht the response of mice to infection by vriety of other respirtory nd enteric viruses is restricted to the IgG2 subclss with some ctivity in the IgG2b subclss but reltively little in IgGl or IgG3 (7). Furthermore, we re confident tht the sensitivity of the ssys used here ws dequte to hve detected the other ntibody clsses since similr ssys on mice fter infection with M. pulmonis demonstrted specific Vccine TABLE 5. Effect of route of vccintion on lung lesions before nd fter chllenge Route of Lung lesion scoreb vccintion Before chllenge p1c After chllenge pl p2d vg301 Intrperitonel 0.01 ± vg301 Intrnsl 0.74 ± ± vg301 Scrifiction ± None 0.01 ± ± The dose of vccine ws 2 x 106 PFU. bmen ± stndrd devition 21 dys fter vccintion (before chllenge) or 28 dys fter vccintion (fter chllenge). C pl, Probbility of differences between vccinted groups nd control clculted by Student's t test. d p2, Probbility of differences between chllenged nd unchllenged mice clculted by Student's t test.

6 3860 STOTT ET AL. J. VIROL. TABLE 6. Effect of vccine on lung lesions Vccine Route of Dose Chllenge Antibody Lesion score Difference (p)b vccintion (107 PFU) Clnetiter vf325 Intrperitonel 2 A2 4.0 ± ± vn125 Intrperitonel 1 A2 3.4 ± ± vlb Intrperitonel 2 A2 < ± None A2 < ± 0.05 None None < ± 0.10 Antibody titer ws mesured by ELISA 28 dys fter vccintion nd is expressed s men ± stndrd error. b Probbility of differences between vccinted nd unvccinted groups ws clculted by Student's t test. ntibody in immunoglobulin subclsses IgGl, IgG2, IgA, nd IgM in both ser nd lung wshes (27). Murine ntibody responses to soluble proteins nd crbohydrtes re generlly restricted to the IgGl nd IgG3 subclsses, respectively (17, 21, 23). The fct tht no ntibody to RS virus G glycoprotein ws found in the IgG3 subclss suggests tht lthough pproximtely 60% of its mss is crbohydrte (2), this moiety does not mke significnt contribution to its ntigenicity, t lest in mice. The IgGl nd IgG2 subclsses differ in their bility to ctivte mouse complement (15) nd possibly in their bility to effect ntibody-dependent cell cytotoxicity. Thus, the IgG2 restriction of the response to recombinnt VV my be significnt spect of the protection they fford in mice nd hs importnt implictions for vccine strtegy. There re t lest two subgroups of RS viruses which re distinguished by monoclonl nd polyclonl ntibodies to F nd G proteins (1, 18, 28). We therefore chllenged mice vccinted with recombinnts contining the F or G gene from A2 virus, subtype A virus, with representtive strins of A nd B subtypes. Although vccintion with the F protein vectors protected ginst both subtype A nd B virus, the G protein vectors only reduced repliction of the homologous virus. This observtion is consistent with results from the pssive trnsfer of monoclonl ntibodies. Monoclonl ntibodies ginst F protein which protect lso cross-rect with both A nd B subtype viruses. In contrst, protective ntibodies specific for G of subtype A fil to rect with viruses of subtype B (28). Few monoclonl ntibodies to G cross-rect between the subtypes, indicting tht there re few shred epitopes (1, 18, 28). This is probbly the explntion for the low ntibody rection to 8/60 virus in mice given vg301. Thus, in considering the components desirble in n effective vccine it is pprent tht vccintion with F protects ginst both heterologous nd homologous virus in mice but tht G ntigens of both subtypes will be necessry for mximum efficcy. In the originl reports of recombinnt VV contining G nd F genes, differences in the level of immunity induced were pprent (9, 20, 25, 29). It ws uncler whether these discrepncies were due to differences between the recombinnts used, the routes of vccintion, or the species involved. The levels of protection we observed in cotton rts given vg301 nd vf317 re similr to those reported by Olmsted nd collegues (20) using different recombinnts crrying F nd G genes, indicting tht the potency of the different recombinnts is similr in terms of the protection they induce. It is cler from the present investigtion tht intrderml vccintion is less efficient thn other routes (Tble 1). However, even by this route the reduction in RS virus in the lung ws 10-fold greter in mice (103.2 PFU) thn in cotton rts (102.1 PFU), lthough the dose of vg301 given to the rts ws 45-fold higher. Greter efficcy in mice my be relted to the mouse dpttion of the WR strin of vccini which ws the prent of the recombinnt viruses. A further difference between the mouse nd cotton rt model systems is highlighted by the work reported here. In mice, vccintion with recombinnt VV crrying either the G or F gene resulted in no detectble virus in the lungs fter chllenge with homologous virus. However, recombinnts crrying the F gene protected cotton rts more effectively thn those contining the G gene (Tble 4). This ltter observtion ws lso mde by Olmsted et l. (20). In view of these differences between the species it is importnt to test these recombinnt VV in lrger nimls, such s chimpnzees, or to test protection ginst the bovine RS virus in its nturl host, cttle. Histologicl exmintion of the lungs of nimls vccinted with the recombinnt VV nd chllenged with RS virus hs not previously been reported nd is especilly relevnt for RS virus for which prior vccintion hs been reported to cuse enhnced disese upon chllenge. We hve reported tht intrnsl inocultion of mice with wildtype WR strin VV cused severe clinicl disese but tht no overt disese followed intrnsl vccintion with thymidine kinse-negtive recombinnt VV (25). It ws, therefore, importnt to note extensive histopthologicl chnges in lungs of mice 21 dys fter intrnsl infection with vg301 (Tble 5). Since this infiltrtion ws not seen in mice vccinted by prenterl routes it ws probbly due to locl repliction of virus in the respirtory trct. Of even greter concern ws the significnt increse in lesion score fter vccintion with vg301, vf325, or vn125 followed by intrnsl chllenge with RS virus (Tbles 5 nd 6). Since the VV ws purified nd no such enhnced lesions were observed fter vccintion with recombinnt contining the 1B gene of RS virus nd subsequent chllenge (Tble 6) it is unlikely tht they were cused by the VV vectors or hostderived components ssocited with them. Similr incresed infiltrtion occurred fter vccintion of cotton rts with Formlin-inctivted RS virus nd subsequent intrnsl chllenge (22). However, fter Formlin-inctivted virus the infiltrtion ws predominntly neutrophils, wheres in the present work lymphocytes predominted. In view of the relevnce of these observtions to the sfety of RS virus vccines, it is now importnt to estblish whether these pulmonry responses re prt of norml nd desirble immune response to virus invsion or whether they present potentil immunopthogenic hzrd. Work is therefore in progress to identify the type nd function of the cells infiltrting the lungs. The present work illustrted the vlue of recombinnt VV s tools with which to investigte immunity to RS virus. However, dngers hve been highlighted which demnd cution in considering these viruses s potentil vccines in nimls or in humns.

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