JOURNAL OF VIROLOGY, OCt. 1991, p. 5374-5380 Vol. 65, No. 100022-538X/91/105374-07$02.00/0Copyright © 1991, American Society for Microbiology

Immune Response and Resistance to Rous Sarcoma Virus Challengeof Chickens Immunized with Cell-Associated Glycoproteins

Provided with a Recombinant Avian Leukosis VirusYAHIA CHEBLOUNE,* JAN RULKA,t FRANCOIS LOIC COSSET, SANDRINE VALSESIA,CORINNE RONFORT, CATHERINE LEGRAS, ANTOINE DRYNDA, JACEK KUZMAK,t

VICTOR MARC NIGON, AND GERARD VERDIER

Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique LA810, Centre National de laRecherche Scientifique UMR106, Universite Claude Bernard Lyon-I, Batiment 741, 43, Boulevard du

11 novembre 1918, 69622 Villeurbanne Cedex, France

Received 17 April 1991/Accepted 15 July 1991

The Rous-associated virus 1 env gene, which encodes the envelope gp85 and gp37 glycoproteins, was isolatedand inserted in place of the v-erbB oncogene into an avian erythroblastosis virus-based vector, carrying the neoresistance gene substituted for the v-erbA oncogene, to generate the pNEA recombinant vector. A helper-freevirus stock of the pNEA vector was produced on an avian transcomplementing cell line and used to infectprimary chicken embryo fibroblasts (CEFs) or quail QT6 cells. These infected cells, selected with G418(CEF/NEA and QT6/NEA, respectively) were found to be resistant to superinfections with subgroup Aretroviruses. The CEF/NEA preparations were used as a cell-associated antigen to inoculate adult chickens bythe intravenous route compared with direct inoculations of NEA recombinant helper-free viruses used as acell-free antigen. Chickens injected with the cell-associated antigen (CEF/NEA) exhibited an immune responsedemonstrated by induction of high titers of neutralizing antibodies and were found to be protected againsttumor production after Rous sarcoma virus A challenge. Conversely, no immune response and no protectionagainst Rous sarcoma virus A challenge were observed in chickens directly inoculated with cell-free NEArecombinant virus or in sham-inoculated chickens.

Infections of chickens with avian retroviruses are wide-spread, as evidenced by the synthesis of specific antibodiesin most chicken flocks (12, 14, 31). To keep flocks free fromavian sarcoma and leukosis viruses, eradication methods areapplied by eliminating all viremic and serologically positivebirds, essentially by eliminating hens that excrete virus intheir eggs (4, 36). Several approaches have been usedsimultaneously to provide resistance to avian retrovirusinfections. Studies have shown that chickens inoculated witheither pathogenic avian leukosis virus or low doses of Roussarcoma virus (RSV) produce an immune response andfewer tumors after RSV challenge with the same subgroup(16, 20, 23). Similarly, immunization of chickens has beenobtained by using purified envelope glycoproteins (gp85 andgp37) that induce both an immune response, as evidenced bydetection of virus-neutralizing antibodies (1), and resistanceto tumor production after subsequent RSV challenge (3).Finally, a recombinant avian leukosis virus containing struc-tural genes (gag-pol and env), originating from subgroup ARSV, and the long terminal repeats from Rous-associatedvirus type 0 (RAV-0), corresponding to a naturally attenu-ated nonpathogenic endogenous virus from subgroup E (30),was inoculated into chicken embryos (18 days of incubation)or hatched chicks (21, 33). Production of viral protein-specific antibodies was detected in most chicks at 5 weekspostinoculation of the recombinant avian leukosis virus (21).Moreover, significantly smaller tumors than those of sham-inoculated controls were observed when these treated birdswere RSV challenged (33).

* Corresponding author.t Present address: Institut VWtdrinaire, 24-100 Pulawy Al., Par-

tysantow 57, Poland.

We and other teams have recently reported the construc-tion of avian packaging cell lines (6, 32, 34) that enableproduction of replication-defective avian leukosis virus-based vectors as helper-free virus stocks (5, 7, 19, 26, 27).Therefore, these avian packaging cell lines would offer theopportunity to investigate avian vaccination approaches byusing retroviral vectors obtained as ecotropic helper-freevirus stocks. The purpose of this study was to examine boththe immune responses and resistance of chickens to RSVchallenge after immunization with either (i) a helper-freevirus stock of an avian erythroblastosis virus (AEV)-baseddefective vector carrying and expressing the env gene ofsubgroup A avian retroviruses which were used as cell-freeantigens and directly injected into animals or (ii) chickenembryo fibroblasts (CEFs) infected in vitro with the NEAvirus and then injected into chickens as cell-associated viralprotein antigens. Our results demonstrated a high-efficiencyimmune response induction, as well as resistance to tumorproduction after RSV challenge, for chickens immunizedwith cell-associated virus protein antigens. By contrast,neither an immune response nor resistance to tumor produc-tion was observed after treatment involving direct inocula-tion of a helper-free virus stock used as a cell-free antigen.

MATERIALS AND METHODS

Plasmids. Plasmid pRAV-1, containing the permuted ge-nome of RAV type 1 (RAV-1), was obtained from J. M.Bishop (University of California, San Francisco). PlasmidpAG50, containing the bacterial neomycin resistance gene,was obtained from A. G. Garapin (Institut Pasteur, Paris,France). Plasmids pTXN5' and pNL53 were derived from anAEV genome in which the v-erbA and v-erbB oncogenes

5374

on May 21, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

ALV IMMUNIZATION PROTECTS AGAINST RSV CHALLENGE 5375

were deleted and the v-erbA oncogene was replaced by thebacterial neomycin resistance gene, to generate pTXN5' (2).pNL53 was derived from pTXN5' by insertion of the bacte-rial lacZ gene in place of the v-erbB oncogene (7, 19).Chicken lines. Brown Leghorn chickens (C/E) closely

monitored for infection with ALSV were obtained from theStation de Pathologie Aviaire INRA Tours Nouzilly. Thisflock, maintained in isolation, was constantly monitored foravian sarcoma and leukosis virus infections. All chickensused in our experiments were obtained from this flock.

Construction of the pNEA vector. A 1.9-kb KpnI-AccIfragment containing the splice acceptor site and the env genewas isolated from plasmid pRAV-1 and then inserted be-tween the KpnI and AccI sites of the pUC19 polylinker togenerate the pUCenvA recombinant plasmid (see Fig. 1).

Partial digestion of the pUCenvA plasmid with EcoRI andHindIII allowed a 1.9-kb fragment containing the env genelinked to the splice acceptor site to be released and recov-ered. The EcoRI and Hindlll extremities of this fragmentwere converted to blunt ends with DNA polymerase 1(Klenow fragment). This fragment was then inserted intopTXN5' between the StuI and XbaI sites, which wereconverted to blunt ends. The resulting plasmid was calledpNEA (Fig. 1).

Cells and culture conditions. QT6 is a chemically derivedJapanese quail tumor cell line, kindly provided by C. Mos-covici (25). Haydee is a transcomplementing cell line derivedfrom QT6, in which we inserted a RAV-1 packaging mutantcontaining the expressing the gag and pol retroviral genes,as well as the selectable bacterial hygromycin resistancegene (6).CEFs were prepared from C/E Brown Leghorn 10-day-old

chicken embryos and grown as previously described (15).Both QT6 cells and CEFs were grown in F10 mediumsupplemented with 5% calf serum and 1% chicken serum.The Haydee cell line was grown in the same mediumcontaining 50 pg of hygromycin B (Boehringer Mannheim)per ml.

Recovery of NEA virions and virus titration. NEA plasmidDNA transfection into the Haydee cell line was performed asdescribed by Kawai and Nishizawa (18). Transfected cellswere grown in medium containing 200 ,ug of G418 (GIBCO-BRL) per ml and 50 ,ug of hygromycin B per ml. At 10 to 15days later, resistant colonies were submitted to the action oftrypsin to produce a polyclonal culture. Helper-free virusstocks were then harvested from subconfluent Neor cells infresh medium at 6 to 16 h later. Cell debris were removed bycentrifugation (10 min, 3,000 rpm, 4°C), and the harvestedsupernatant containing the virus stock was cleared again byfiltration through a 0.22-,um-pore-size membrane (Millipore).The resulting helper-free virus stock was either used directlyto infect a cell culture or stored at -70°C before use.NEA helper-free virus stocks recovered from the Haydee/

NEA cell line were titrated through induction of resistanceafter infection of either QT6 cells or CEFs with variousdilutions of virus stocks and selection of the infected cellswith G418. Neor clones were scored to determine the virustiter expressed as resistance-forming units per milliliter.

Preparation of immunogen products. G418-resistant CEFsresulting from NEA virus infection (called CEF/NEA) wereUV irradiated (4,000 R at 254 nm) and treated with trypsin,and dissociated cells were recovered in phosphate-bufferedsaline. Cells were then frozen and thawed before use.Uninfected CEFs, used as controls, were also UV irradiatedand received the same treatment as CEF/NEA.Immunization experiments. Three-month-old Brown

Leghorn chicks were divided into four groups of 9 to 11animals (see Table 1). The chicks were inoculated intrave-nously with 300 RI of a virus-free preparation of CEFscontaining 3 x 106 cells (group 1), 300 ,l of a CEF/NEApreparation corresponding to 3 x 106 cells (group 2), or 1 mlof a helper-free NEA virus stock containing 1.5 x 105resistance-forming units (group 3); group 4 birds received noinoculation and were used as controls. At 10 days later, asecond inoculation, similar to the first one, was given togroups 1, 2, and 3.At 2 weeks after the first injection, chicks were bled by

venipuncture every week and sera were prepared fromclotted blood samples allowed to stand at room temperatureovernight. Sera were inactivated for 30 min at 56°C and useddirectly in a serum neutralization test or stored at -70°Cbefore use. All timings for events reported in the tables andfigures refer to the first immunization injection.Serum neutralization tests. A mixed virus stock containing

a replication-defective AEV-based vector called NL53, carry-ing and expressing the bacterial neomycin resistance andlacZ genes (26, 27) and associated with the helper RAV-1,was used for serum neutralization tests. The advantage ofvector NL53 was mainly rapid detection by focus reductionassay of the bacterial P-galactosidase encoded by the lacZgene (7, 19).From 200 to 500 blue colony-forming units in 100 ,u were

incubated for 1 h in ice, with 100 ,ul of serial twofold dilutionsof sera. Mixtures were then plated onto monolayers of QT6cells in 16-mm (well diameter) tissue culture plates. At 48 hlater, infected cells were fixed in 4% paraformaldehyde,washed twice with phosphate-buffered saline, and incubatedfor periods ranging from 2 h to overnight at 37°C in a solutioncontaining 2 mM MgCl2, 3% dimethyl sulfoxide, 5 mMpotassium hexacyanoferrate III [(K3FeCN)6, 3H20], 5 mMpotassium hexacyanoferrate II [K4Fe(CN)6, H20], and 1 mgof 5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside perml. Blue-stained cells or colonies were scored, and theneutralization dose giving 50% reduction of blue cells (ND50)was determined for every neutralizing serum. Sera preparedfrom control (noninoculated) animals were used to deter-mine the reference value of blue cells obtained with theNL53/RAV-1 virus stock.RSV challenge. An RSV Schmidt-Ruppin strain A stock

was produced on a CEF culture. The titer of the virus stockwas determined both in vitro and in vivo, with infected CEFcultures, by scoring both transforming foci using a colony-forming assay in soft agar, as previously described (24), andtumor production in animals after wing web inoculation ofvarious virus stock dilutions. Animals from groups 1 to 4were RSV challenged with 100 pAl of a virus stock containing103 tumor-forming units by the subcutaneous route via wingweb injection at 5 weeks after the first immunizing inocula-tion. From 10 days after RSV challenge, the chicks were

examined every day to detect tumor induction, and tumorsizes were measured.

RESULTS

Production of a helper-free vector expressing the env gene.The Haydee cell line, expressing the gag and pol avianretroviral genes from a packaging mutant derived from theRAV-1 genome, was transfected with pNEA plasmid DNA,which corresponds to a packaging-competent vector carry-ing and expressing both the neomycin resistance and envAgenes. Details of construction are given in Materials andMethods, as well as in Fig. 1. Transfected cells were then

VOL. 65, 1991

on May 21, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

J. VIROL.5376 CHEBLOUNE ET AL.

5'LTR

pRAV1 I [-ggSD

BcoR! Kpaf

AcoRJ/lIdf digestionsiGenow polymerasetreatment

1.9 kb env.Afragment

po

/prIl AccI

SA

EcoR

, Acdf

L puc 19 S\ 5A7

Kim! 5YuJ

5'LTR A gag J A e 3'LTR

pTX5' II AL~~ SSD I

XbaJ digestionKienow polymerasetreatmentSizUi digestion

Ligation

5'LTR Agag a 3'LTR

pNE A_ -ESD SA

FIG. 1. Construction of the pNEA vector. The 1.9-kb fragment containing the env gene was isolated from the pRAV-1 genome after KpnIand AccI digestions. This fragment was then inserted between the KpnI and Accl sites of the pUC19 plasmid. The env gene was released fromthe resulting plasmid after partial EcoRI digestion, followed by HindIll digestion. The EcoRI and HindlIl extremities of this 1.9-kb fragmentwere blunt ended by Klenow polymerase treatment, and then the resulting fragment was inserted between the blunt-ended XbaI and StuIextremities of the TXN5' vector to generate the pNEA vector. Abbreviations: SA, splice acceptor site; SD, splice donor site; Ac, AccI; E,EcoRI; Hd, HindlIl; K, KpnI; Sp, SphI; Xb, Xbal; Xh, XhoI; LTR, long terminal repeat.

selected with G418, and Neor colonies were grown as a

polyclonal culture to subconfluence. Virus stocks recoveredfrom producer cells were titrated on QT6 cells and CEFs,giving a titer of i05 to 1.5 x 105 neomycin resistance-formingunits per ml. These viruses were used to infect fresh CEFs,which were selected with G418, and gave CEF/NEA prepa-

rations (see Materials and Methods). To verify that the virusstock contained no replication-competent viruses, the super-natant recovered from resultant infected Neor cells (CEF/NEA) was used for massive infection of fresh QT6 cells andCEFs. After G418 selection, no resistant QT6 cell or CEFcolonies were observed. This demonstrated that the super-

natant was unable to transmit G418 resistance to normalcells, providing evidence of the helper-free character of theused virus stock.

Interference test on NEA_infected cells. Neor CEFs ex-

pressing the NEA vector (i.e., CEF/NEA obtained as re-

ported above), CEFs infected with RAV-1, or CEFs used as

controls were infected with 103 focus-forming units ofRSV-A and grown under soft agar. Two weeks later, no

transforming focus was observed in either CEF/NE' orCEF/RAV-1 cells, while transforming foci were obtainedwith the RSV-A-infected CEFs used as controls. This dem-onstrated that the env gene carried by the pNEA vector wascorrectly expressed and presented on membrane cells, mak-ing cells resistant to superinfection by an exogenous virus ofthe same subgroup. A similar result was obtained when thevector NL53 carrying the lacZ gene and pseudotyped withthe RAV-1 helper (see Materials and Methods) was used tosuperinfect CEF/NEA cells; i.e., no ,-galactosidase-positivecells were detected as blue cells or colonies after 5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside staining of in-fected cells.

Preparation of antigens. Neor CEFs infected by NEA virus(i.e., CEF/NEA) and noninfected CEFs used as controlswere UV irradiated (4,000 R at 254 nm) and treated withtrypsin to recover cells for antigen preparation, as describedin Materials and Methods. To test UV irradiation efficiency,two dishes containing UV-irradiated cells were not submit-ted to trypsin action and were maintained in fresh medium.

on May 21, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

ALV IMMUNIZATION PROTECTS AGAINST RSV CHALLENGE 5377

TABLE 1. Antibody response and protection againstRSV challenge'

No. Antigen No. with an antibody % withbirds inoculated response tumors

1 10 CEFs 0 1002 9 CEF/NEA 9 03 9 NEA 0 1004 11 None 0 100

a Chickens were inoculated intravenously with 3 x 10' CEFs per bird(group 1), 3 x 106 CEF/NEA cells per bird (group 2), or 1.5 x 105resistance-forming units of a helper-free NEA virus stock (group 3). Group 4corresponded to noninoculated birds. Antibody response was measured byserum neutralization test 2 weeks after the first immunization treatment.Chickens were challenged with 103 tumor-forming units per bird from an RSVSchmidt-Ruppin strain A stock by the subcutaneous route 5 weeks after thebeginning of the experiment, and percentages of tumor production weredetermined 3 weeks later.

At 24 h later, only 0.1 to 1% of the irradiated cells were alive;however, these cells did not divide and died 3 to 5 days later.This observation suggests that the UV irradiation conditionsused led to inactivated DNAs.

Chick immunization and immunological response. To in-vestigate the biological properties of the different antigenpreparations, 3-month-old Brown Leghorn chicks were di-vided into four groups of 9 to 11 animals, and each group wascomparatively treated as reported in Materials and Methodsand Table 1. At time zero, birds were bled by venipunctureto test sera by the serum neutralization method beforeinoculation. Antigen preparations were then inoculated asdescribed in Materials and Methods. Ten days later, chickswere inoculated again under the same conditions. At day 15and every week thereafter, chicks were bled and sera wereprepared to test the presence of viral protein-specific anti-bodies in serum neutralization assays with vector NL53carrying and expressing the bacterial lacZ gene. Blue colo-ny-forming units were scored on QT6 cultures infected withvector NL53 and previously incubated with the collectedsera. The reduction of blue colony numbers in sera of treatedbirds compared with those of controls indicated induction ofviral protein-specific antibodies. The results of the serumneutralization test for each group of birds are reported inTables 1 and 2. They illustrate that all sera collected 2 weeksafter the first immunization treatment from birds inoculated

TABLE 3. Dilutions of sera from group 2 birds allowing either100 or 50% NL53 virus neutralizationa

Serum dilution allowing:Chick no.

100% neutralization 50% neutralization

1 <128 8,0002 <128 4,0003 <64 7,0004 <4 9005 <256 >8,000 (14,000)6 <64 6,0007 <32 9,0008 <128 >8,000 (19,000)9 <32 7,000

"The average value of NL virus particles submitted to the neutralizationtest corresponded to 234 blue colony-forming units. The protocol used forTable 2 was used to estimate the highest serum dilutions to obtain the 100%level of NL53 virus neutralization. Dilution values leading to 50% NL53 virusneutralization were determined from curves as reported in the text.

with CEF/NEA antigen (group 2) revealed the presence of ahigh level of specific antibodies directed against subgroup Aenv glycoproteins. Conversely, sera from birds either di-rectly inoculated with the NEA virus stock or sham inocu-lated with a noninfected CEF preparation were negative, aswere those from noninoculated birds.Serum neutralization test values obtained with sera col-

lected 35 days after immunization from group 2 birds wereused to plot neutralization curves ofNL virus against serumdilutions as previously described (13). From these curves,the ND50 for each serum was determined (Table 3). Theseresults demonstrate that in sera of all of the birds except one(bird 4 [Table 2]), the ND50 corresponded to high serumdilution values, two of them being higher than 1/8,000 per10-1. Moreover, to determine the kinetics of neutralizingantibody production, ND50 values were determined everyweek (i.e., 15, 21, 28, and 35 days after immunization) forevery serum from all of the birds of group 2 (Fig. 2). Theaverage curve showed a linear increase in neutralizingantibody production until the week 4, and the rate ofproduction decreased during the last week.

Induction of protection against RSV challenge. Five weeksafter the first immunization, all chickens were challengedwith 103 tumor-forming units of RSV Schmidt-Ruppin strainA via wing web injection. The results in Table 1 demonstrate

TABLE 2. Accurate determination of antibody responses of birds in group 2a

No. of serum ,B-Galactosidase-positive cell score after serum neutralization tests using the following serum dilution:tested 1/16 1/64 1/256 1/512 1/1,024 1/2,048 1/4,0% 1/8,192

1 0 0 0 7 21 47 85 1292 0 2 21 45 83 116 144 1723 0 3 11 33 57 95 106 1234 8 30 47 83 155 216 NTb NT5 0 0 0 3 5 13 26 586 0 0 1 4 9 33 101 2117 0 0 11 18 27 56 84 1488 0 0 0 0 2 10 36 649 0 3 11 23 54 83 118 147

Control 300 300 NT NT NT NT NT NT

a Sera of the different birds (1 to 9) in group 2 (see Table 1 and Materials and Methods) were collected 5 weeks after immunization assays, diulted, and thenmixed with NL53 viruses as described in Materials and Methods. After infection of QT6 cells, blue cells, expressing ,B-galactosidase from the lac gene carriedby the NL vector, were scored. The control values are the average values obtained with sera of sham-inoculated birds (group 1; Table 1).

b NT, not tested.

VOL. 65, 1991

on May 21, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

5378 CHEBLOUNE ET AL.

ND 50 /

/

9000-/8000-

7000

6000

5000

4000

3000

2000

1000

DAYS

7 14 21 28 35

FIG. 2. Kinetics of neutralizing antibody production in sera ofgroup 2 birds. Each week, the average ND50, expressed as the serum

dilution leading to 50% NL53 virus neutralization, was calculatedfrom the individual values of the tested birds. Ordinate, average

ND50; abscissa, time after immunization treatment.

that all of the chickens immunized with a CEF/NEA prepa-

ration (group 2) were resistant to tumor production, while allof the other chickens (groups 1, 3, and 4) developed tumors,which progressively increased in size in most cases (Fig. 3).These observations are in agreement with those on theproduction of neutralizing antibodies. Indeed, all chickens ofgroup 2 produced specific antibodies directed against sub-

size of tumor

50

40

30

20

10

O -4.l ~~~~~~~T--D- days7 14 21 28 35

FIG. 3. Kinetics of tumor size after RSV challenge. Birds of all

four groups were RSV challenged as described in Materials and

Methods. After 10 days, the birds were observed every day and

tumor sizes were measured. Ordinate, tumor sizes in millimeters;

abscissa, time. Symbols: 0, group 1 (sham-inoculated) birds; 0,

group 2 (CEF/NEA_inoculated) birds; A, group 3 (NEA virus-inoculated) birds; *, group 4 (noninoculated) birds (used as con-

trols).

group A env glycoproteins and showed protection againsttumor development, whereas no antibody production wasobserved in all sera of chickens of the other groups, whichwere not protected. It should be noted that tumor progres-sions were practically similar for groups 1 and 4 comparedwith group 3, in which greater tumor volumes were observedduring the same period (Fig. 3).

DISCUSSION

In this work, we investigated the possibility of obtainingan immune response and resistance of chickens to RSVchallenge following immunization treatment involvingrecombinant AEV-based vectors. The transcomplementingvector pNEA was transfected into the Haydee cell line toproduce a helper-free virus stock, NEA. Neor cells resultingfrom infection with NEA virus and G418 selection becametotally resistant to superinfection with either a mixed stockcontaining both NL53 and RAV-1 virus particles (subgroupA) or RSV Schmidt-Ruppin strain A. We conclude that theenv gene carried by the pNEA vector was correctly ex-pressed, allowing interference with other retroviruses fromsubgroup A. Inoculation of adult chickens with either NEAvirus or a cell-associated antigen produced by expression ofthe NEA vector in recipient cells demonstrated that only thesecond type of immunizing preparation produced an efficientimmune response. Indeed, a high level of neutralizing anti-bodies was observed in the sera of most of the birds fromgroup 2 (Table 3). This humoral response was detectable 2weeks after the first immunization treatment and increasedprogressively over the following weeks (Fig. 2), until week 5to 6. In contrast, no antibody response was observed in theseroneutralization test from sera of group 3 birds inoculatedwith the NEA virus. Several explanations may be given forthe last observation. (i) env gene glycoproteins associatedwith NEA viral particles may be less efficient than theantigens present in cell membranes because of insufficientamounts for induction of an immune response. (ii) Afterintravenous inoculation, the infectious ability of NEA viralparticles was rapidly abolished, leading to a low level ofinfected cells in the inoculated birds. (iii) We assume thatcells of inoculated birds infected with the NEA vector andexpressing the env glycoproteins were rapidly excludedbefore they could be used to stimulate an effective immuneresponse.

Resistance against tumor production after RSV challengein all of the animals from group 2 illustrates the efficiency ofsuch a modality of immunization. Indeed, no tumor produc-tion was observed in this group, although animal 4 (Tables 2and 3) appeared to have fewer neutralizing antibodies in itsserum. This animal produced less than 10% neutralizingantibodies compared with animals 5 and 8. We can concludethat in most cases, humoral responses induced by a cell-associated antigen produced a sufficient rate of neutralizingantibodies, ranging from 3 to 10 times higher than necessaryto induce resistance to RSV challenge. In the other groups(1, 3, and 4), all of the birds had developed tumors whichappeared 2 weeks after RSV challenge and then tumorvolume increased progressively during the next weeks (Fig.3). No significant difference was observed between thetumor sizes of group 1 (sham-inoculated) and group 4 (non-inoculated) birds after RSV challenge, while increased vol-umes of tumors were measured in group 3 birds (Fig. 3). Thereason for such large tumor sizes in animals directly inocu-lated with NEA virus compared with those in the other twogroups (1 and 4) is unknown. In previous works, it has been

J. VIROL.

on May 21, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

ALV IMMUNIZATION PROTECTS AGAINST RSV CHALLENGE 5379

reported that chickens either expressing endogenous viralgene ev2-producing RAV-0 virus or infected with the RAV-0virus as embryos (on day 6 of incubation) became immuno-logically tolerant to further exogenous retrovirus infectionsby limiting immune responses (8-11, 22). In the BrownLeghorn line we used, we showed that four endogenousstructures (evBL) are present and that some of them areexpressed (29). Taking into account the above-mentionedhypothesis, we suppose that evBL expression can also limitthe immune response in birds inoculated with either aCEF/NEA preparation or NEA virus. It would be interestingto know whether birds from group 2, which produce a hightiter of neutralizing antibodies, possess expressed endoge-nous structures.

Several previous approaches have been attempted toconfer resistance to RSV challenge on chickens. In the mostrecent studies, using either purified glycoproteins encodedby the env gene, isolated by sedimentation (3), or an atten-uated recombinant avian leukosis virus (21, 33), researchersdemonstrated that the conditions used stimulated an immuneresponse capable of partially protecting birds against tumorproduction after RSV challenge. Our results demonstratedthat cell-associated antigens are able to induce an efficientimmune response correlated with highly efficient resistanceto tumor production. In a mammalian retrovirus system, theability of a cell-associated antigen to induce an immuneresponse and protect animals against retrovirus infectionwas tested by using infection with a replication-competentretrovirus of either lymphoblastoid cells (17, 28) or anestablished bone marrow (BL-3) cell line (35). However, allof the experiments done under these conditions producedboth partial induction of an immune response and limitedprotection against retrovirus infection. These results couldbe explained by the occurrence of several viral antigensexpressed from such infected cells, with some of them, forexample, gag-encoded proteins, being more abundant thanenv-encoded glycoproteins. Consequently, the presence ofother antigens could partially prevent the expected immuneresponse against the envelope glycoproteins and therebylead to a decrease in the neutralizing antibody level, which iscorrelated with reduced protection against retrovirus infec-tion.

In conclusion, CEFs infected with a recombinant AEV-based vector produced as a helper-free virus stock, carryingand expressing the env gene of subgroup A, corresponded toan efficient immunogen preparation. Immunized chickensshowed production of neutralizing antibodies and were pro-tected against RSV challenge. These results demonstratedthat expression of the env gene in CEFs produced a proteinwith its natural folded structure. Experiments are in progressto (i) determine whether the route of immunization caninfluence the efficiency of tumor resistance, as previouslyreported (1), and (ii) test our vaccination procedure onyounger target chickens (hatched birds and 18-day-old em-bryos), as well as the subgroup specificity of protection.Moreover, in another work, we have successfully usedsimilar approaches to induce protection against Newcastledisease virus (unpublished data).

ACKNOWLEDGMENTS

We thank J. M. Bishop, A. G. Garapin, and C. Moscovici forproviding plasmids pRAV-1 and pAG50 and the QT6 cell line,respectively. We thank G. Dambrine and all members of the Stationde Pathologie Aviaire INRA Tours Nouzilly for providing aviansarcoma and leukosis virus-free animals. We thank A. Dorier(I.U.T. Lyon I) for technical assistance with the animals.

This work was supported by research grants from the CentreNational de la Recherche Scientifique, the Institut National de laRecherche Agronomique, the Commission of the European Com-munities, the Ministere de la Recherche et de l'EnseignementSuperieur and Etablissements Rh6ne-Merieux.

REFERENCES1. Bauer, H., M. Hayami, and J. C. Stehfen-Gervinus. 1979.

Influence of different routes of anti-tumor immunization: alter-native induction of tumor immunity and tumor enhancement. J.Immunol. 122:806-812.

2. Benchaibi, M., F. Mallet, P. Thoraval, P. Savatier, J. H. Xiao, G.Verdier, J. Samarut, and V. M. Nigon. 1989. Avian retroviralvectors derived from avian leukemia viruses: role of the trans-actional context of the inserted gene on the efficiency of thevectors. Virology 169:15-26.

3. Bennet, D. D., and S. E. Wright. 1987. Immunization withenvelope glycoprotein of an avian RNA tumor virus protectsagainst sarcoma virus tumor induction: role of subgroup. VirusRes. 8:73-77.

4. Calnek, B. W. 1968. Lymphoid leukosis virus: a survey ofcommercial breeding flocks for genetic resistance and incidenceof embryo infection. Avian Dis. 12:105-111.

5. Chebloune, Y., F. L. Cosset, C. Legras, A. Drynda, C. Faure,V. M. Nigon, and G. Verdier. 1990. Construction of AEV-basedsuicide vectors expressing transferred gene under control of anonretroviral promoter, p. 129-134. Proceedings of the 4thWorld Congress on Genetics Applied to Livestock Production,XVI. Edinburgh, 23-27 July 1990.

6. Cosset, F.-L., C. Legras, Y. Chebloune, P. Savatier, P. Thoraval,J. L. Thomas, J. Samarut, V. M. Nigon, and G. Verdier. 1990. Anew avian leukosis virus-based packaging cell line that uses twoseparate transcomplementing helper genomes. J. Virol. 64:1070-1078.

7. Cosset, F.-L., C. Legras, J. L. Thomas, R. M. Molina, Y.Chebloune, C. Faure, V. M. Nigon, and G. Verdier. 1991.Improvement of avian leukosis virus (ALV)-based retrovirusvectors by using different cis-acting sequences from ALVs. J.Virol. 65:3388-3394.

8. Crittenden, L. B., and A. M. Fadly. 1985. Responses of chickenslacking or expressing endogenous avian leukosis virus genes toinfection with exogenous virus. Poultry Sci. 64:454-463.

9. Crittenden, L. B., A. M. Fadly, and E. J. Smith. 1982. Effect ofendogenous leukosis virus on response to infection with avianleukosis and reticuloendotheliosis viruses. Avian Dis. 26:279-294.

10. Crittenden, L. B., S. McMahon, M. S. Halpern, and A. M.Fadly. 1987. Embryonic infection with the endogenous avianleukosis virus Rous-associated virus-0 alters responses to exog-enous avian leukosis virus infection. J. Virol. 61:722-725.

11. Crittenden, L. B., E. J. Smith, and A. M. Fadly. 1984. Influenceof endogenous viral (ev) gene expression and strain of exoge-nous avian leukosis virus (ALV) on mortality and ALV infec-tion and shedding in chickens. Avian Dis. 28:1037-1056.

12. DeBoer, G. F., J. Van Vloten, J. E. Groenendal, and H. J. L.Maas. 1978. On epizootiology and control of lymphoid leukosisin chickens. Comp. Immunol. Microbiol. Infect. Dis. 1:93-106.

13. Dougherty, R. M., J. A. Stewart, and H. R. Morgan. 1960.Quantitative studies of the relationships between infecting doseof Rous sarcoma virus, antiviral immune response and tumorgrowth in chickens. Virology 11:349-370.

14. Fadly, A. M., and W. Okazaki. 1982. Studies of avian leukosisvirus infection from a commercial breeder flock. Poultry Sci.61:1055-1060.

15. Gandrillon, O., P. Jurdic, M. Benchaibi, J. H. Xia, J. Ghysdael,and J. Samarut. 1987. Expression of v-erb A oncogene inchicken embryo fibroblasts stimulates their proliferation in vitroand enhances tumor growth in vivo. Cell 49:687-697.

16. Hall, M. R., L. F. Qualtiere, and P. Meyers. 1979. Cellular andhumoral immune reactivity to tumor-associated antigens inchickens infected with Rous sarcoma virus. J. Immunol. 123:1097-1105.

17. Jarret, W., 0. Jarret, L. Mackey, H. Laird, C. Hood, and D.

VOL. 65, 1991

on May 21, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

5380 CHEBLOUNE ET AL.

Hay. 1975. Vaccination against feline leukemia virus using a cellmembrane antigen system. Int. J. Cancer. 16:134-141.

18. Kawai, S., and M. Nishizawa. 1984. New procedure for DNAtransfection with polycation and dimethyl sulfoxide. Mol. Cell.Biol. 4:1172-1174.

19. Legras, C., F. L. Cosset, Y. Chebloune, R. M. Molina, J. L.Thomas, C. Faure, V. M. Nigon, and G. Verdier. Submitted forpublication.

20. Maas, H. J. L., G. F. DeBoer, and J. E. Groenendal. 1982. Agerelated resistance to avian leukosis virus: III. Infectious virus,neutralizing antibody and tumors in chickens inoculated atvarious ages. Avian Pathol. 11:309-327.

21. McBride, H. A. T., and R. M. Shuman. 1988. Immune responseof chickens inoculated with a recombinant avian leukosis virus.Avian Dis. 32:96-102.

22. McMahon, S. B., D. Ewert, R. Carroli, and M. S. Halpern. 1986.Expression of endogenous retroviral envelope glycoprotein as adeterminant of immunity to Rous sarcoma. Virology 155:737-741.

23. Meyers, P., H. M. Sigel, and H. T. Holden. 1972. Crossprotection in vivo against avian sarcoma virus subgroups A, Band C induced by Rous-associated viruses. J. Natl. Cancer Inst.49:173-181.

24. Moscovici, C., L. Gazzolo, and M. G. Moscovici. 1978. Avianacute leukemia viruses: transforming ability in vitro. Microbio-logica 1:1-13.

25. Moscovici, C., M. G. Moscovici, H. Jiminez, M. M. C. Lai, M. J.Hayman, and P. K. Vogt. 1977. Contains tissue culture cell linesderived from chemically induced tumors of Japanese quail. Cell11:95-103.

26. Nigon, V. M., Y. Chebloune, and G. Verdier. 1990. Constructionand use of ALV replication defective vectors for germ lineinfection, p. 97-106. Proceedings of the 4th World Congress onGenetics Applied to Livestock Production, XVI. Edinburgh,23-27 July 1990.

27. Nigon, V. M., G. Verdier, J. Samarut, C. Bagnis, M. Benchaibi,Y. Chebloune, F. L. Cosset, C. Faure, 0. Gandrillon, P. Jurdic,

C. Legras, F. Mallet, A. Reyss-brion, P. Savatier, and P.Thoraval. 1987. Contribution to the construction of retroviralvector and helper cell lines for gene transfer in chickens.Biotechnol. Action Prog. Progr. Rep. 2:1037-1044.

28. Pedersen, N. C., G. H. Thielen, and L. L. Werner. 1978. Safetyand efficacy studies of live and killed feline leukemia virusvaccines. Am. J. Vet. Res. 40:1120-1126.

29. Ronfort, C., M. Afanassieff, Y. Chebloune, G. Dambrine, V. M.Nigon, and G. Verdier. Identification and structure of endoge-nous proviral sequences in a brown leghorn chicken strain.Poultry Sci., in press.

30. Salter, D. W., and L. B. Crittenden. 1989. Artificial insertion ofa dominant gene for resistance to avian leukosis virus into thegerm line of the chicken. Theor. Appl. Genet. 77:457-461.

31. Sandelin, K., and T. Estola. 1974. Occurrence of differentsubgroups of avian leukosis virus in Finnish poultry. AvianPathol. 3:159-168.

32. Savatier, P., C. Bagnis, P. Thoraval, D. Poncet, M. Belakebi, F.Mallet, C. Legras, F. L. Cosset, J. L. Thomas, V. Chebloune, C.Faure, G. Verdier, J. Samarut, and V. M. Nigon. 1989. Gener-ation of a helper cell line for packaging avian leukosis virus-based vectors. J. Virol. 63:513-522.

33. Shuman, R. M., and H. A. T. McBride. 1988. Resistance ofchickens to Rous sarcoma virus challenge following immuniza-tion with a recombinant avian leukosis virus. Avian Dis. 32:96-102.

34. Stoker, A. W., and M. J. Bissell. 1988. Development of aviansarcoma and leukosis virus-based vector-packaging cell lines. J.Virol. 62:1008-1015.

35. Thielen, G. H., J. M. Miller, J. Higgins, R. N. Ruppaner, and W.Garrett. 1982. Vaccination against bovine leukemia virus infec-tion. Curr. Top. Vet. Med. Anim. Sci. 15:547-559.

36. Zander, D. V., R. G. Raymond, C. F. McClary, and K. Goodwin.1975. Eradication of subgroup A and B lymphoid leukosis virusfrom commercial poultry breeding flocks. Avian Dis. 19:408-423.

J. VIROL.

on May 21, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from