infection and (ii) the protective role of antibodies to VP3 and VP7 against challenge with virulent parental porcine rotaviruses.

Transcription

1 JOURNL OF VIROLOGY, Mr. 1988, p X/88/3744-5$2./ Copyright D 1988, mericn Society for Microbiology Vol. 62, No. 3 Infection Immunity of Piglets to Either VP3 or VP7 Outer Cpsid Protein Confers Resistnce to Chllenge ith Virulent Rotvirus Bering the Corresponding ntigen YSUTK HOSHINO,1* LIND J. S,2 MITZI M. SERENO,1 ROBERT M. CHNOCK,' ND LBERT Z. KPIKIN' Lbortory of Infectious Diseses, Ntionl Institute of llergy nd Infectious Diseses, Bethesd, Mrylnd 2892,1 nd Ohio griculturl Reserch nd Development Center, Ohio Stte University, Wooster, Ohio Received 23 July 1987/ccepted 6 November 1987 single-gene substitution ressortnt 11-1 s generted from to porcine rotviruses, OSU (serotype 5) nd Gottfried (serotype 4). This ressortnt derived 1 genes, including gene 4 ending VP3, from the OSU strin nd only gene 9, ending mjor neutrlition glyprotein (VP7), from the Gottfried strin nd s thus designted VP3:5; VP7:4. Orl dministrtion of this ressortnt to lostrum-deprived gnotobiotic neborn pigs induced high level of neutrliing ntibodies not only to Gottfried VP7 but lso to OSU VP3, thus demonstrting tht VP3 is s potent n immunogen s VP7 in inducing neutrliing ntibodies during experimentl orl infection. Gnotobiotic piglets infected previously ith the ressortnt ere mpletely resistnt to orl chllenge ith the virulent Gottfried strin (VP3:4; VP7:4), s indicted by filure of symptoms to develop nd lck of virus shedding. Similrly, prior infection ith the ressortnt induced lmost mplete protection ginst dirrhe nd significnt restriction of virus repliction fter orl chllenge ith the virulent OSU strin (VP3:5; VP7:5). Thus, it ppers tht (i) the immune system of the piglet responds eqully ell to to rotvirus outer cpsid proteins, VP3 nd VP7, during primry enteric rotvirus infection; (ii) ntibody to VP3 nd ntibody to VP7 re ech ssocited ith resistnce to dirrhe; nd (iii) infection ith ressortnt rotvirus bering VP3 nd VP7 neutrlition ntigens derived from to viruses of different serotype induces immunity to both prentl viruses. The relevnce of these findings to the development of effective ressortnt rotvirus vccines is discussed. Dirrhe is mplex disese of the gstrointestinl trct nd is of mjor importnce in humn nd veterinry medicine; rotvirus is mjor etiologic gent of this syndrome throughout the orld (4, 5, 1, 16, 27). Severl different pproches to immunoprophylxis re no being tken ith the im of protecting infnts nd young children ginst severe rotvirus dirrhe, nd these include the development nd evlution of (i) ttenuted humn rotviruses, including ld-dpted rotvirus mutnts; (ii) rotviruses of niml origin hich re restricted in humns; nd (iii) intr- nd interspecies ressortnt rotviruses (3, 9, 17-19, 21, 22, 28). Rotviruses hve to mjor surfce proteins, VP3 (gene 4 product) nd VP7 (gene 8 or 9 product), hich hve been shon to be independent neutrlition ntigens (12, 24). Recent studies hve shon tht VP3 is s potent in inducing neutrliing ntibodies s VP7 under nditions of prenterl or orl hyperimmunition (11, 12, 24, 25). More recently, mouse dms orlly hyperimmunied ith ressortnt rotvirus ntining the VP3 of one serotype nd the VP7 of nother serotype ere shon to nfer pssive protection to their progeny ginst chllenge ith either prentl rotvirus (25). In sine, the plcent is brrier to the trnsport of mternl ntibodies to the fetus. In this study, by using the homologous system of lostrum-deprived, neborn gnotobiotic pigs nd single VP7 gene substitution ressortnt porcine rotvirus, e hve studied (i) the immunogenicity of VP3 nd VP7 under nditions of single experimentl orl * Corresponding uthor. infection nd (ii) the protective role of ntibodies to VP3 nd VP7 ginst chllenge ith virulent prentl porcine rotviruses. The observtions mde during this study hve implictions for the development of effective ressortnt rotvirus vccines. MTERILS ND METHODS Viruses, cells, hyperimmune ntiser, nd PRN ssy. The folloing viruses ere used in this study: humn rotvirus DS-1 (serotype 2), porcine rotvirus Gottfried (serotype 4), porcine rotvirus SB-1 (serotypes 4 nd 5), nd porcine rotvirus OSU (serotype 5). ll rotviruses ere plque purified three times in this lbortory before use in this experiment. The OSU nd Gottfried strins used in the cross-protection studies ere gut-origin viruses hich hd been serilly pssged only in gnotobiotic piglets. n estblished cell line of fetl rhesus monkey kidney cells, M14 (14), s used for virus propgtion, production nd selection of ressortnt viruses, nd the plque reduction neutrlition (PRN) ssy. Hyperimmune ntiserum ginst ech of the viruses s prepred in guine pigs. Hyperimmune ntiserum s het inctivted t 56 C for 3 min before it s used in the neutrlition ssy. The PRN ssy s performed in plstic six-ell pltes (Costr, Cmbridge, Mss.) of M14 cell monolyers to mesure neutrliing ntibody, s previously described (15, 29). Genetic ressortment, selection of ressortnts, nd genotype nlysis of ressortnt rotvirus. Genetic ressortment s crried out s previously described (11). 744 Donloded from on Jnury 28, 219 by guest

2 ~~~~~~~~~~~~ VOL. 62, 1988 INFECTION IMMUNITY OF PIGLETS TO RESSORTNT ROTVIRUS 745 ds RN ere llected dily for 2 eeks postchllenge. Virus shedding s exmined by cell culture immunofluorescence osu test (1) nd in some cses by virus isoltion in M14 cell, Gott. o U o _ cultures. Blood smples ere obtined t the time of primry infection, 3 eeks lter t the time of chllenge, nd 2 eeks fter chllenge. -2,3 RESULTS -5 Genertion nd chrcterition of single-gene substitution OSU x Gottfried ressortnt fter mixed infection ~~-& of M14 cells ith the porcine OSU nd Gottfried rotviruses, e isolted single-gene substitution ressortnt, ^ , hich derived only its VP7 gene segment from the Gottfried prent, heres the remining 1 gene segments ere derived from the OSU prent (Fig. 1). Ressortnt 11-1 s neutrlied by hyperimmune ntiserum rised ginst OSU Gott. xgott' OSU either OSU or Gottfried (Tble 1). lso, hyperimmune Probe ntiserum to this ressortnt neutrlied the Gottfried nd FIG. 1. nlysis of genotype of porcine rotvirus OSU x Gottfried ressortnt Genomic RNs (ds RN) of OSU, Gottfried nturlly occurring intertypic porcine rotvirus, SB-1, ith OSU strins to the sme degree. It is of interest tht (Gott.), nd OSU x Gottfried ressortnt 11-1 (OSU x Gott. 11-1) VP3 of serotype 5 nd VP7 of serotype 4 s recently ere hybridied to single-strnded 32P-lbeled mrn 'trnscripts of identified (Tble 1) (11). OSU, Gottfried, nd OSU x Gottfried ressortnt Only gene 9 Resistnce in gnotobiotic piglets to both prentl viruses of the OSU x Gottfried ressortnt 11-1 is derived from Gottfried; induced by OSU x Gottfried ressortnt rotvirus the remining 1 genes re from OSU. Previous exposure of 4- to 5-dy-old lostrum-deprived gnotobiotic piglets to the Gottfried strin did not prevent virus shedding or dirrhe fter chllenge 3 eeks lter ith RN-RN hybridition ssys ere performed to determine the prentl origin of the genes present in ressortnt pprent decrese in durtion of virus shedding (Tble 2, the virulent heterotypic OSU strin; hoever, there s n rotviruses, s described previously (6). Genertion nd group 1). Similrly, piglets in group 2 initilly exposed to the genotype nlysis of single VP3 gene substitution OSU x virulent OSU nd chllenged 3 eeks lter ith the virulent DS-1 ressortnt 46-1, hich derived only its VP3 gene Gottfried strin shed virus, lthough ith n pprent decrese in durtion, nd developed dirrhe. Thus, significnt segment from its porcine OSU prent nd 1 gene segments from its humn DS-1 prent, s described previously (11). heterotypic protection s not observed. In erlier studies, Experimentl nimls nd cross-protection studies. Gnotobiotic piglets used in this study ere obtined, housed, nd mpletely protected gnotobiotic piglets from dirrhe or it s shon tht prior infection ith OSU or Gottfried mintined s previously described (1). Individuls from to virus excretion hen chllenged 3 eeks lter ith the litters (2 piglets) of lostrum-deprived gnotobiotic piglets homotypic virulent virus (2; dt not shon). Chllenge of ere treted orlly ith 2 ml ech of one of the folloing previously uninfected 25-dy-old gnotobiotic piglets ith preprtions: Gottfried virus s 5% suspension of intestinl ntents from gnotobiotic piglet hich hd titer of 2 dirrhe of shorter durtion thn tht observed in 4- to either OSU or Gottfried virus (groups 5 nd 6) induced x 16 fluorescent-focus units (group 1), OSU virus s 4% 5-dy-old piglets (5.5 dys versus 9. dys for OSU nd 7. suspension of intestinl ntents from gnotobiotic piglet dys versus 12. dys for Gottfried); hoever, the medin hich hd titer of 2.5 x 16 fluorescent-focus units (group durtion of virus shedding s similr or identicl. 2), or OSU x Gottfried ressortnt 11-1 s 1:2 dilution of dministrtion of single-gene substitution OSU x Gottfried ressortnt 11-1 to 4- to 5-dy-old gnotobiotic piglets infected M14 cell lyste ntining 5 x 16 PFU (groups 3 nd 4). To groups (groups 5 nd 6) of piglets ere induced dirrhe, nd the infected piglets shed virus (Tble designted ntrols nd ere not dministered virus t ge 4 2, groups 3 nd 4). Hoever, hen these piglets ere to 5 dys. ll piglets, including the ntrols, ere chllenged chllenged 3 eeks lter ith virulent OSU, significnt orlly 3 eeks lter ith 2 ml of either the OSU or the shoed Gottfried virus preprtion described bove. Piglets ere exmined dily for evidence of dirrhe, nd rectl sbs dirrhe s not observed (to of four piglets trnsient mild dirrhe) nd there s delyed onset of virus shedding of short durtion (group 3). When nimls Donloded from on Jnury 28, 219 by guest TBLE 1. Porcine rotvirus ntigenic chrcterition of single-gene substitution OSU x Gottfried ressortnt 11-1 by PRN ssy VP3 Origin of genes Reciprocl of 6% PRN ntibody titer' of hyperimmune guine pig ntiserum to indicted rotvirus (serotype): VP7 ll other Gottfried OSU x Gottfried SB-1 OSU proteins (4) 11-1 (4 nd 5) (4 nd 5) (5) Gottfried Gottfried Gottfried Gottfried 4,96 81,92 2,48 <8 OSU x Gottfried 11-1 OSU Gottfried OSU 81,92 '81,92 4,96 2,48 SB-1 SB-1 SB-1 SB-1 4,96 81,92 4,96 4,96 OSU OSU OSU OSU <8 4,96 1,24 81,92 Vlues derived from one test. Homologous titers re in boldfce type.

3 746 HOSHINO ET L. J. VIROL. TBLE 2. Immunity to chllenge ith prentl porcine rotviruses OSU nd Gottfried induced by the ressortnt rotvirus 11-1 (Gottfried VP7, OSU ll other genes) in gnotobiotic piglets Primry infection (t ge 4 to 5 dys) Chllenge (3 eeks fter primry infection) Gnotobiotic Durtion (medin Onset (medin dy) Durtion (medin Onset (medin dy) piglet group dys) of: of: Rotvirus dys) of: of: (no.) Rotvirus dministered Virus Dirrhe Virus dministered Virus Dirrhe Virus Dirrhe shedding shedding shedding shedding 1 (4) Gottfried OSU (4) OSU Gottfried (4) OSU x Gottfried OSU 1.5 none (4) OSU x Gottfried Gottfried none none none none 5 (2) none Nb N N N OSU (2)C none N N N N Gottfried To of four piglets shoed trnsient mild dirrhe. b N, Not pplicble. c One piglet died on dy 7 (virus shedding nd dirrhe occurred from dy 1 to dy 6). previously infected ith the OSU x Gottfried 11-1 ressortnt ere chllenged ith virulent Gottfried, neither virus shedding nor dirrhe s observed (group 4). Neutrliing ntibody response of gnotobiotic piglets initilly treted ith OSU x Gottfried ressortnt 11-1 rotvirus nd chllenged 3 eeks lter ith either virulent OSU or Gottfried. Exposure of 4- to 5-dy-old piglets to single-gene substitution OSU x Gottfried ressortnt 11-1 induced significnt ntibody response to both Gottfried nd OSU viruses (Tble 3, groups 3 nd 4). Of note is the finding tht the neutrliing ntibody titer induced by OSU VP3 is higher thn tht induced by the VP7 of Gottfried. The level of serum neutrliing ntibodies to OSU VP3 s determined ith OSU virus (VP3:5; VP7:5) s higher thn hen single-gene substitution OSU x DS-1 ressortnt 46-1, hich derived only the VP3 gene from its OSU prent nd the remining 1 genes from humn DS-1 virus (VP3:5; VP7:2), s used for ssy. Ser obtined 16 dys fter OSU virus chllenge of group 3 piglets shoed fourfold or greter increse in serum ntibody to homologous virus but not to heterologous Gottfried virus. Chllenge of group 4 piglets ith Gottfried virus induced n increse in neutrliing ntibodies hich pproched fourfold, lthough significnt increse of neutrliing ntibody to heterologous OSU virus s not observed. lso, the serologic response of group 2 piglets suggested tht neutrliing ntibodies to VP3 of serotype 5 develop sloly nd require 37 dys or more to rech mximum titers. DISCUSSION Recent studies hve estblished tht the neutrlition specificities on VP3, gene product 4, nd VP7, gene 8 or 9 product, segregte independently in nture nd tht VP3 is s potent n immunogen s VP7 in inducing neutrliing ntibodies under nditions of prenterl or orl hyperimmunition. lso, it hs been shon tht mouse dms hyperimmunied orlly ith ressortnt ntining VP3 nd VP7 from to distinct rotvirus serotypes protect their pups pssively ginst dirrhe induced by either prentl rotvirus (25). Criticl questions remining unnsered ere (i) ho do the rotvirus outer cpsid proteins VP3 nd VP7 behve ntigeniclly in the host during single enteric infection, (ii) to ht extent re ntibodies induced ginst VP3 nd VP7 involved in resistnce ginst rotvirus disese, nd (iii) ho effective is single enteric infection by ressortnt rotvirus in providing resistnce to both prentl viruses? We ddressed these questions in this study by using homologous system of lostrum-deprived, neborn gnotobiotic pigs free of mternl ntibodies nd single VP7 gene substitution porcine rotvirus ressortnt. First, e nlyed the immunogenicity in guine pigs of the single-gene substitution OSU x Gottfried rotvirus ressortnt 11-1, hich derived 1 gene segments from the OSU virus (serotype 5) nd only the VP7 gene segment (gene 9) from the Gottfried virus (serotype 4). When hyperimmunied prenterlly ith ressortnt 11-1, guine pigs produced high levels of neutrliing ntibodies not only to Gottfried virus but lso to OSU virus (Tble 1). Since Gottfried is ntigeniclly distinct from OSU, neutrlition of OSU virus by OSU x Gottfried ressortnt 11-1 ntiserum is due to OSU VP3 ntibodies, nd neutrlition of Gottfried virus by the sme ntiserum is due to Gottfried VP7 ntibodies. This nfirms tht VP3 is s potent in stimulting neutrliing ntibodies s VP7 fter prenterl hyperimmunition (11, 12, 24, 25). Next, e nlyed serum smples obtined from lostrum-deprived, neborn gnotobiotic pigs infected orlly ith the single-gene substitution ressortnt 11-1 nd observed tht VP3 s s potent n immunogen s VP7 in inducing neutrliing ntibodies fter enteric infection. Tht is, the immune system of the host regnies both VP3 nd VP7 eqully s judged by induction of equl levels of neutrliing ntibodies by the to rotvirus outer cpsid proteins (Tble 3). It is of interest tht the VP3 of ressortnt rotvirus such s 11-1 my be more effective thn its prentl rotvirus in inducing neutrliing ntibodies not only under nditions of hyperimmunition reported previously (11, 12, 24, 25) but lso under nditions of single orl immunition (this study). In ddition, such ntibodies ere ssocited ith resistnce to infection, disese, or disese nd infection cused by the virulent prentl rotviruses used to derive the ressortnt. Neutrliing ntibodies induced by the Gottfried VP7 of the OSU x Gottfried ressortnt 11-1 ere ssocited ith resistnce of piglets to chllenge ith virulent Gottfried virus, s indicted by bsence of dirrhe nd virus shedding. Similrly, ntibodies to OSU VP3 of the ressortnt 11-1 ere ssocited ith protection of piglets from dirrhe (to of four shoed trnsient, mild dirrhe) upon chllenge ith virulent OSU virus; virus shedding s lso delyed in onset nd s decresed in durtion (Tble 2). These observtions indicte tht single orl dministrtion of ressortnt Donloded from on Jnury 28, 219 by guest

4 VOL. 62, 1988 INFECTION IMMUNITY OF PIGLETS TO RESSORTNT ROTVIRUS CL t'3.9. -h. b (I 3D :! C n. C X. X ) ] o, o Co e... ~ CD o o oo o -~t-j f! s CD W o *- CD oo 8 oo I. P. 1-- o_ oo c ) C : W! : IIV <b o C> X ii.~ P.- Q Q o> oo o _ ~~~~~~ ~ g -" go o " o-. 8 >o " oo tofoo " "- Q " " -,. C D Ci t-. -- C : c C C) s L.. t :; > X<c I- v <: = Zs L..- '2,L c Q P. 3 go I. Cl to 2C, : U) l < CD to r C, e go l- ( C,L cn _. C.. C, F C) - rotvirus possessing VP3 nd VP7 derived from to distinct rotvirus serotypes induced sufficient neutrliing ntibodies nd ssocited resistnce to protect the host ginst dirrhe upon chllenge ith either of the virulent prentl rotviruses. The durtion of virus shedding by gnotobiotic piglets initilly fed OSU or Gottfried virus nd then chllenged 3 eeks lter ith heterotypic Gottfried or OSU virus s significntly shorter (4. dys in group 1 nd 3.5 dys in group 2) thn tht of previously unimmunied 25-dy-old nimls fed OSU or Gottfried virus (1. dys in group 5 nd 7. dys in group 6). Becuse OSU virus nd Gottfried virus re serotypiclly distinct on the bsis of their VP3 nd VP7 specificities (Tbles 1 nd 3), the observed difference in the durtion of virus shedding suggests tht cell-medited immunity my lso ply role in resistnce to hererotypic rotvirus infection, disese, nd revery. Recently, London et l. (2) hve shon tht (i) single intrduodenl dministrtion of reovirus type 1, nother enteric virus of the fmily Reoviride, is cpble of generting detectble levels of reovirus-specific cytotoxicity in Peyer's ptch lymphocytes upon in vitro restimultion nd (ii) the reovirus-specific cytotoxic T lymphocytes thus generted re not specific for the immuniing reovirus serotype. Virus-specific, cell-medited immunity s determined by delyed-type hypersensitivity response hs been reported to develop in dult mice fed murine rotvirus EDIM 7 dys fter primry orl infection; hoever, serotype specificity of the phenomenon s not determined (26). mmon neutrlition epitope(s), if ny, on VP3 nd VP7 my ply certin role in this phenomenon. lthough single-gene substitution OSU x Gottfried ressortnt 11-1 (VP3:5; VP7:4) nd nturlly occurring intertypic porcine rotvirus SB-1 (VP3:5; VP7:4) re identicl by reciprocl PRN ssy ith hyperimmune guine pig serum (Tble 1), subtle difference in ntigenic mposition beteen these to viruses s reveled hen postinfection piglet ser ere exmined by PRN ssy ginst these to viruses; titers ginst homologous ressortnt 11-1 virus ere lys higher thn those ginst SB-1 virus (Tble 3). This suggests difference beteen the to viruses in neutrlition epitopes on VP3 nd VP7. Midthun et l. (23) hve shon tht gene 4 of the OSU virus is similr but not identicl to gene 4 of the SB-1 virus by RN-RN hybridition ssy. Our observtions tht (i) fter single orl exposure to rotvirus, the host immune system responds eqully to to rotvirus outer cpsid proteins, VP3 nd VP7, (ii) ntibodies to VP3 nd VP7 thus induced re independently ssocited ith resistnce to chllenge ith virulent rotvirus, nd (iii) ressortnt rotvirus tht hs the VP3 of one serotype nd the VP7 of nother serotype cn elicit sufficient neutrliing ntibodies nd ssocited resistnce to both serotypes to prevent dirrhe upon chllenge ith either prentl virus suggest tht ressortnt rotvirus vccines my be useful in immunoprophylxis. For exmple, n ttenuted mutnt of ressortnt 11-1 might prove useful s live vccine in sine. lso, single-gene substitution humn x humn rotvirus ressortnts generted in this lbortory (for exmple, W x DS-1 [VP3:2; VP7:1], P x DS-1 [VP3:2; VP7:3], ST3 x DS-1 [VP3:2; VP7:4], W x ST3 [VP3:1; VP7:4], or W x P [VP3:1; VP7:3]) s ell s single-gene substitution ressortnts beteen nturlly ttenuted (7, 8, 13) nd virulent humn rotviruses hich derive genome segment 4 from ttenuted neontl rotviruses (for exmple, ST3 x P or M37 x P) my prove useful in prevention of humn rotvirus disese. Donloded from on Jnury 28, 219 by guest

5 748 HOSHINO ET L. CKNOWLEDGMENTS This ork s supported in prt by Public Helth Service grnt I from the Ntionl Institute of llergy nd Infectious Diseses to L.J.S. We thnk Lul Ttum, nnie L. Pittmn, nd Peggy Weilnu for excellent technicl ssistnce, nd Susn Hrding nd Debr Brunelle for editoril ssistnce in the preprtion of this mnuscript. LITERTURE CITED 1. Bohl, E. H., L. J. Sif, K. W. Theil,. G. gnes, nd R. F. Cross Porcine prrotvirus: detection, differentition from rotvirus, nd pthogenesis in gnotobiotic pigs. J. Clin. Microbiol. 15: Bohl, E. H., K. W. Theil, nd L. J. Sif Isoltion nd serotyping of porcine rotviruses nd ntigenic mprison ith other rotviruses. J. Clin. Microbiol. 19: Clrk, H. F., P.. Offit, K. T. Doln,. Te, K. Goglin, E. M. Tist, nd S.. Plotkin Response of dult humn volunteers to orl dministrtion of bovine nd bovine/humn ressortnt rotviruses. Vccine 4: Cukor, G., nd N. R. Blcklo Humn virl gstroenteritis. Microbiol. Rev. 48: Estes, M. K., E. L. Plmer, nd J. F. 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