Immune Response and Resistance to Rous ... - Journal of Virology

JOURNAL

OF

VIROLOGY, OCt. 1991, p. 5374-5380

Vol. 65, No. 10

0022-538X/91/105374-07$02.00/0 Copyright © 1991, American Society for Microbiology

Immune Response and Resistance to Rous Sarcoma Virus Challenge of Chickens Immunized with Cell-Associated Glycoproteins Provided with a Recombinant Avian Leukosis Virus YAHIA 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 la Recherche 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 isolated and inserted in place of the v-erbB oncogene into an avian erythroblastosis virus-based vector, carrying the neo resistance gene substituted for the v-erbA oncogene, to generate the pNEA recombinant vector. A helper-free virus stock of the pNEA vector was produced on an avian transcomplementing cell line and used to infect primary 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 A retroviruses. The CEF/NEA preparations were used as a cell-associated antigen to inoculate adult chickens by the intravenous route compared with direct inoculations of NEA recombinant helper-free viruses used as a cell-free antigen. Chickens injected with the cell-associated antigen (CEF/NEA) exhibited an immune response demonstrated by induction of high titers of neutralizing antibodies and were found to be protected against tumor production after Rous sarcoma virus A challenge. Conversely, no immune response and no protection against Rous sarcoma virus A challenge were observed in chickens directly inoculated with cell-free NEA recombinant virus or in sham-inoculated chickens.

Infections of chickens with avian retroviruses are widespread, as evidenced by the synthesis of specific antibodies in most chicken flocks (12, 14, 31). To keep flocks free from avian sarcoma and leukosis viruses, eradication methods are applied by eliminating all viremic and serologically positive birds, essentially by eliminating hens that excrete virus in their eggs (4, 36). Several approaches have been used simultaneously to provide resistance to avian retrovirus infections. Studies have shown that chickens inoculated with either pathogenic avian leukosis virus or low doses of Rous sarcoma virus (RSV) produce an immune response and fewer tumors after RSV challenge with the same subgroup (16, 20, 23). Similarly, immunization of chickens has been obtained by using purified envelope glycoproteins (gp85 and gp37) that induce both an immune response, as evidenced by detection of virus-neutralizing antibodies (1), and resistance to tumor production after subsequent RSV challenge (3). Finally, a recombinant avian leukosis virus containing structural genes (gag-pol and env), originating from subgroup A RSV, and the long terminal repeats from Rous-associated virus type 0 (RAV-0), corresponding to a naturally attenuated nonpathogenic endogenous virus from subgroup E (30), was inoculated into chicken embryos (18 days of incubation) or hatched chicks (21, 33). Production of viral proteinspecific antibodies was detected in most chicks at 5 weeks postinoculation of the recombinant avian leukosis virus (21). Moreover, significantly smaller tumors than those of shaminoculated controls were observed when these treated birds were RSV challenged (33). Corresponding author. t Present address: Institut

We and other teams have recently reported the construction of avian packaging cell lines (6, 32, 34) that enable production of replication-defective avian leukosis virusbased vectors as helper-free virus stocks (5, 7, 19, 26, 27). Therefore, these avian packaging cell lines would offer the opportunity to investigate avian vaccination approaches by using retroviral vectors obtained as ecotropic helper-free virus stocks. The purpose of this study was to examine both the immune responses and resistance of chickens to RSV challenge after immunization with either (i) a helper-free virus stock of an avian erythroblastosis virus (AEV)-based defective vector carrying and expressing the env gene of subgroup A avian retroviruses which were used as cell-free antigens and directly injected into animals or (ii) chicken embryo fibroblasts (CEFs) infected in vitro with the NEA virus and then injected into chickens as cell-associated viral protein antigens. Our results demonstrated a high-efficiency immune response induction, as well as resistance to tumor production after RSV challenge, for chickens immunized with cell-associated virus protein antigens. By contrast, neither an immune response nor resistance to tumor production was observed after treatment involving direct inoculation of a helper-free virus stock used as a cell-free antigen.

MATERIALS AND METHODS Plasmids. Plasmid pRAV-1, containing the permuted genome of RAV type 1 (RAV-1), was obtained from J. M. Bishop (University of California, San Francisco). Plasmid pAG50, containing the bacterial neomycin resistance gene, was obtained from A. G. Garapin (Institut Pasteur, Paris, France). Plasmids pTXN5' and pNL53 were derived from an AEV genome in which the v-erbA and v-erbB oncogenes

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ALV IMMUNIZATION PROTECTS AGAINST RSV CHALLENGE

were deleted and the v-erbA oncogene was replaced by the bacterial neomycin resistance gene, to generate pTXN5' (2). pNL53 was derived from pTXN5' by insertion of the bacterial 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 the Station de Pathologie Aviaire INRA Tours Nouzilly. This flock, maintained in isolation, was constantly monitored for avian sarcoma and leukosis virus infections. All chickens used in our experiments were obtained from this flock. Construction of the pNEA vector. A 1.9-kb KpnI-AccI fragment containing the splice acceptor site and the env gene was isolated from plasmid pRAV-1 and then inserted between the KpnI and AccI sites of the pUC19 polylinker to generate the pUCenvA recombinant plasmid (see Fig. 1). Partial digestion of the pUCenvA plasmid with EcoRI and HindIII allowed a 1.9-kb fragment containing the env gene linked to the splice acceptor site to be released and recovered. The EcoRI and Hindlll extremities of this fragment were converted to blunt ends with DNA polymerase 1 (Klenow fragment). This fragment was then inserted into pTXN5' between the StuI and XbaI sites, which were converted to blunt ends. The resulting plasmid was called pNEA (Fig. 1). Cells and culture conditions. QT6 is a chemically derived Japanese quail tumor cell line, kindly provided by C. Moscovici (25). Haydee is a transcomplementing cell line derived from QT6, in which we inserted a RAV-1 packaging mutant containing the expressing the gag and pol retroviral genes, as well as the selectable bacterial hygromycin resistance gene (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 medium supplemented with 5% calf serum and 1% chicken serum. The Haydee cell line was grown in the same medium containing 50 pg of hygromycin B (Boehringer Mannheim) per ml. Recovery of NEA virions and virus titration. NEA plasmid DNA transfection into the Haydee cell line was performed as described by Kawai and Nishizawa (18). Transfected cells were grown in medium containing 200 ,ug of G418 (GIBCOBRL) per ml and 50 ,ug of hygromycin B per ml. At 10 to 15 days later, resistant colonies were submitted to the action of trypsin to produce a polyclonal culture. Helper-free virus stocks were then harvested from subconfluent Neor cells in fresh medium at 6 to 16 h later. Cell debris were removed by centrifugation (10 min, 3,000 rpm, 4°C), and the harvested supernatant containing the virus stock was cleared again by filtration through a 0.22-,um-pore-size membrane (Millipore). The resulting helper-free virus stock was either used directly to 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 resistance after infection of either QT6 cells or CEFs with various dilutions of virus stocks and selection of the infected cells with G418. Neor clones were scored to determine the virus titer expressed as resistance-forming units per milliliter. Preparation of immunogen products. G418-resistant CEFs resulting from NEA virus infection (called CEF/NEA) were UV irradiated (4,000 R at 254 nm) and treated with trypsin, and dissociated cells were recovered in phosphate-buffered saline. Cells were then frozen and thawed before use. Uninfected CEFs, used as controls, were also UV irradiated and received the same treatment as CEF/NEA. Immunization experiments. Three-month-old Brown

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Leghorn chicks were divided into four groups of 9 to 11 animals (see Table 1). The chicks were inoculated intravenously with 300 RI of a virus-free preparation of CEFs containing 3 x 106 cells (group 1), 300 ,l of a CEF/NEA preparation corresponding to 3 x 106 cells (group 2), or 1 ml of a helper-free NEA virus stock containing 1.5 x 105 resistance-forming units (group 3); group 4 birds received no inoculation and were used as controls. At 10 days later, a second inoculation, similar to the first one, was given to groups 1, 2, and 3. At 2 weeks after the first injection, chicks were bled by venipuncture every week and sera were prepared from clotted blood samples allowed to stand at room temperature overnight. Sera were inactivated for 30 min at 56°C and used directly in a serum neutralization test or stored at -70°C before use. All timings for events reported in the tables and figures refer to the first immunization injection. Serum neutralization tests. A mixed virus stock containing a replication-defective AEV-based vector called NL53, carrying and expressing the bacterial neomycin resistance and lacZ genes (26, 27) and associated with the helper RAV-1, was used for serum neutralization tests. The advantage of vector NL53 was mainly rapid detection by focus reduction assay of the bacterial P-galactosidase encoded by the lacZ gene (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 dilutions of sera. Mixtures were then plated onto monolayers of QT6 cells in 16-mm (well diameter) tissue culture plates. At 48 h later, infected cells were fixed in 4% paraformaldehyde, washed twice with phosphate-buffered saline, and incubated for periods ranging from 2 h to overnight at 37°C in a solution containing 2 mM MgCl2, 3% dimethyl sulfoxide, 5 mM potassium hexacyanoferrate III [(K3FeCN)6, 3H20], 5 mM potassium hexacyanoferrate II [K4Fe(CN)6, H20], and 1 mg of 5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside per ml. Blue-stained cells or colonies were scored, and the neutralization dose giving 50% reduction of blue cells (ND50) was determined for every neutralizing serum. Sera prepared from control (noninoculated) animals were used to determine the reference value of blue cells obtained with the NL53/RAV-1 virus stock. RSV challenge. An RSV Schmidt-Ruppin strain A stock was produced on a CEF culture. The titer of the virus stock was determined both in vitro and in vivo, with infected CEF cultures, by scoring both transforming foci using a colonyforming assay in soft agar, as previously described (24), and tumor production in animals after wing web inoculation of various virus stock dilutions. Animals from groups 1 to 4 were RSV challenged with 100 pAl of a virus stock containing 103 tumor-forming units by the subcutaneous route via wing web injection at 5 weeks after the first immunizing inoculation. From 10 days after RSV challenge, the chicks were examined every day to detect tumor induction, and tumor sizes were measured. RESULTS Production of a helper-free vector expressing the env gene. The Haydee cell line, expressing the gag and pol avian retroviral genes from a packaging mutant derived from the RAV-1 genome, was transfected with pNEA plasmid DNA, which corresponds to a packaging-competent vector carrying and expressing both the neomycin resistance and envA genes. Details of construction are given in Materials and Methods, as well as in Fig. 1. Transfected cells were then

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selected with G418, and Neor colonies were grown as a polyclonal culture to subconfluence. Virus stocks recovered from producer cells were titrated on QT6 cells and CEFs, giving a titer of i05 to 1.5 x 105 neomycin resistance-forming units per ml. These viruses were used to infect fresh CEFs, which were selected with G418, and gave CEF/NEA preparations (see Materials and Methods). To verify that the virus stock contained no replication-competent viruses, the supernatant recovered from resultant infected Neor cells (CEF/ NEA) was used for massive infection of fresh QT6 cells and CEFs. After G418 selection, no resistant QT6 cell or CEF colonies were observed. This demonstrated that the supernatant was unable to transmit G418 resistance to normal cells, providing evidence of the helper-free character of the used virus stock. Interference test on NEA_infected cells. Neor CEFs expressing the NEA vector (i.e., CEF/NEA obtained as reported above), CEFs infected with RAV-1, or CEFs used as controls were infected with 103 focus-forming units of RSV-A and grown under soft agar. Two weeks later, no

transforming focus was observed in either CEF/NE' or CEF/RAV-1 cells, while transforming foci were obtained with the RSV-A-infected CEFs used as controls. This demonstrated that the env gene carried by the pNEA vector was correctly expressed and presented on membrane cells, making cells resistant to superinfection by an exogenous virus of the same subgroup. A similar result was obtained when the vector NL53 carrying the lacZ gene and pseudotyped with the RAV-1 helper (see Materials and Methods) was used to superinfect CEF/NEA cells; i.e., no ,-galactosidase-positive cells were detected as blue cells or colonies after 5-bromo4-chloro-3-indolyl-3-D-galactopyranoside staining of infected cells. Preparation of antigens. Neor CEFs infected by NEA virus (i.e., CEF/NEA) and noninfected CEFs used as controls were UV irradiated (4,000 R at 254 nm) and treated with trypsin to recover cells for antigen preparation, as described in Materials and Methods. To test UV irradiation efficiency, two dishes containing UV-irradiated cells were not submitted to trypsin action and were maintained in fresh medium.

VOL. 65, 1991

ALV IMMUNIZATION PROTECTS AGAINST RSV CHALLENGE

TABLE 1. Antibody response and protection against RSV challenge' No.

Antigen

birds

inoculated

10 9 9 11

CEFs CEF/NEA NEA None

1 2 3 4

TABLE 3. Dilutions of sera from group 2 birds allowing either 100 or 50% NL53 virus neutralizationa

No. with an antibody response

% with tumors

0 9 0

100 0 100 100

0

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Serum dilution allowing:

Chick no.

1 2 3 4 5 6 7 8 9

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 105 resistance-forming units of a helper-free NEA virus stock (group 3). Group 4 corresponded to noninoculated birds. Antibody response was measured by serum neutralization test 2 weeks after the first immunization treatment. Chickens were challenged with 103 tumor-forming units per bird from an RSV Schmidt-Ruppin strain A stock by the subcutaneous route 5 weeks after the beginning of the experiment, and percentages of tumor production were determined 3 weeks later.

100% neutralization

50% neutralization