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Immunogenicity of Recombinant Adenovirus-Respiratory Syncytial Virus Vaccines with Adenovirus Types 4, 5, and 7 Vectors in Dogs and a Chimpanzee Kuo-Hom Lee Hsu, Michael D. Lubeck, Alan R. Davis, Ramesh A. Bhat, Bernard H. Selling, Bheem M. Bhat, Satoshi Mizutani, Brian R. Murphy, Peter L. Collins, Robert M. Chanock, and Paul P. Hung

Division of Biotechnology and Microbiology, Wyeth-Ayerst Research, Philadelphia, Pennsylvania; Laboratory ofInfectious Diseases, National Institutes ofHealth, Bethesda, Maryland

Human respiratory syncytial virus (RSV) is the major cause of severe lower respiratory tract disease in infants and young children, It is estimated that 40%-50% of pediatric patients hospitalized with bronchiolitis and 25%hospitalized with pneumonia are infected with RSV [I, 2]. The peak incidence of severe disease occurs in 2-month-old infants [3]. Currently, there is no effective vaccine for RSV. A formalin-inactivated RSV vaccine tested in the midI960s failed to protect vaccinees from infection [4]. Moreover, this vaccine induced enhanced disease when vaccinees experienced natural infection by RSV [4]. Other experimental vaccines based on cold-adapted or temperature-sensitive RSV mutants are under development [5-8], but an attenuated RSV strain that is genetically stable and has the appropriate balance between attenuation and immunogenicity has yet to be identified. Modern approaches to new RSV vaccines are also under development, which include subunit vaccines [9-11] and live recombinant vaccines using adenovirus or vaccinia virus as vectors [12-15]. We are particularly interested in the development ofadenovirus-vectored vaccines for the following reasons: oral adenovirus types 4 and 7 (Ad4 and Ad7) vaccines have an established safety record [16, 17]; adenoviruses

Received 3 February 1992; revised 30 April 1992. Presented in part: Modern Approaches to New Vaccines Including Prevention of AIDS. September 1990. Cold Spring Harbor Laboratory. Cold Spring Harbor. NY. Reprints or correspondence: Dr. K.-H. Lee Hsu, Division of Biotechnology and Microbiology. Wyeth-Ayerst Research. P.O. Box 8299, Philadelphia, PA 19101. The Journal of Infectious Diseases

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© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6604-00 II $0 1.00

have been developed as high-level expression vectors [1820]; large segments of foreign DNA can be inserted after deletion of nonessential regions of the adenovirus genome [21, 22]; posttranslational processing of foreign proteins (i.e., glycosylation and phosphorylation) occurs in recombinant adenovirus-infected cells [20, 23, 24]; and vectors based on different adenovirus serotypes can be used for booster immunizations [25]. Both fusion (F) and attachment (G) glycoproteins ofRSV have been shown to be the major protective antigens. Preliminary studies of a recombinant adenovirus type 5 that expressed RSV F protein (Ad5F) showed promising results [I2]. Intranasal or enteric vaccination of cotton rats with Ad5F induced RSV-neutralizing antibodies and resulted in a significant reduction of RSV replication in lung tissue on RSV challenge [12]. In the present study, we wanted to compare immune responses induced by F or G protein individually and combined in the same recombinant. We constructed a series of recombinant adenoviruses that express F, G, or both F and G. The availability ofa series of heterotypic adenovirus recombinants allows optimizing immunization protocols by sequential booster vaccinations. Human adenoviruses exhibit a highly restricted host range. Although cotton rats and hamsters -support limited replication ofAd5 in the lungs [26-28], they are less susceptible to lung infection by Ad4 or Ad7 [26] (unpublished data). We have previously established a dog model for testing the immunogenicity of Ad4- and Ad7-vectored hepatitis B vaccines [25]. Infectious Ad7 was detected in lungs of dogs for 3-4 days after intratracheal inoculation ofthe virus, whereas only minimal levels of infectious Ad4 were detected in lungs at only I day after inoculation. Liver, kidney, heart, spleen, and blood were all negative for infectious Ad7 or Ad4. Inocu-

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Recombinant adenovirus type 4, 5, and 7 expressing the fusion glycoprotein (F) gene, the attachment glycoprotein (G) gene, or both F and G genes ofrespiratory syncytial virus (RSV) was constructed. Intratracheal immunization of dogs with Ad7F induced moderate titers of RSVneutralizing antibodies. After booster immunization with Ad4F, the dogs developed high titers of RSV-specific antibody. Subsequently, three two-dose vaccination regimens, Ad4F/Ad5F, Ad7G/Ad4G, and Ad7FG/Ad4FG, were compared with Ad7F/Ad4F for immunogenicity and protective efficacy.The results indicated that Ad4F/ Ad5F was equal or greater in immunogenicity to Ad7F / Ad4F, but Ad7G/Ad4G and Ad7FG/Ad4FG were less effectivethan Ad7F / Ad4F in inducing RSV-neutralizing antibody. All vaccination regimens completely protected the lungs of dogs from RSV infection. A chimpanzee was sequentially immunized orally with Ad7F, Ad4F, and Ad5F. A low-level antibody response to RSV was induced after the primary immunization, but no significant increases were observed after booster immunizations.

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lation of dogs with recombinant Ad7 and Ad4 vaccines expressing hepatitis B surface antigen induced substantial adenovirus type-specific antibody responses as well as responses to hepatitis B surface antigen [25]. We have now further developed the dog as a challenge model for adenovirus-vectored RSV vaccines and tested four different vaccination regimens for immunogenicity and protective efficacy. Chimpanzees are susceptible to enteric infection by recombinant human Ad7 and Ad4 [29]. Chimpanzees are also highly susceptible to infection by RSV [30] and thus represent the optimal challenge model for oral-enteric immunization with recombinant adenovirus-RSV vaccines. Here we report the results of sequential immunization of I chimpanzee with Ad7F, Ad4F, and Ad5F.

Viruses and cells. Wyeth-Ayerst's (Marietta, PA) vaccine strains of Ad4 (CL68378) and Ad7 (55142), as well as an Ad5 strain selected for use on the basis of previous safety testing in adults [31], were used for the construction of recombinant viruses. Adenoviruses were grown and titrated by plaque formation on A549 human lung carcinoma cells and then purified by centrifugation on discontinuous (25% and 60%)and continuous (33%-60%) metrizamide gradients. Metrizamide was then removed from virus preparations by dialysis against PBS. For controls in dog experiments, inactivated virus was prepared by exposure of the virus to UV light for 2 h at a distance of 8 em. For capsule production, purified virus was lyophilized and packed into gelatin capsules; the capsules were then enteric coated with cellulose acetate phthalate as previously described [30]. RSV strain A2 was grown and titrated by plaque formation on the HEp-2 human epidermoid carcinoma cell line. Construction ofrecombinant viruses. cDNA clones ofRSV F and G genes were prepared previously [32, 33]. Construction of recombinant viruses followed previously described methods [19]. In brief, recombinant plasmids were constructed that contained a fragment of the adenovirus genome comprising all or most of the E3 region. The cloned DNA was modified by deleting a portion of the E3 region (1.9-3.56 kbp, which includes all E3 coding sequences except the carboxy portion of the 14.7kDa protein), and a segment of DNA consisting of a serotypespecific tripartite leader followed by the F and/or G gene was then inserted in the deletion site. Recombinant viruses were then generated by homologous recombination. Each recombinant virus was subjected to three cycles of plaque purification, after which expression of the F and/or G protein in infected cells was confirmed in tissue culture. Genetic stability was determined by restriction enzyme analysis. ELISA. Serum RSV antibody ELISA titers were determined using 96-well plates (Corning Glass Works, Corning, NY) coated with bovine anti-RSV (Wellcome Diagnostics, Dartford, UK) and RSV strain A2. Serial twofold dilutions of each serum sample were evaluated. Dog and chimpanzee RSV antibodies were detected by using horseradish peroxidase-conjugated goat anti-dog and anti-human IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), respectively. The optical density (OD) at

415 nm was developed by the substrate systems using ABTS (2,2'-azino-di[3-ethylbenzthiazoline sulfonate]). Titers are expressed as the reciprocal of the lowest serum dilution at which the OD was fivefold above the background. Expression of F and/or G proteins in cell culture was quantitated by using anti-F or anti-G mouse monoclonal antibodies (MAb 858-1 [lgG2a] and MAb 858-2 [IgG 1], respectively; Chemicon International, Temecula, CA). Recombinant adenovirusRSV-infected A549 cells (MOl = 10, infected for 2 days) were harvested and resuspended in PBS. Serial twofold dilutions of cell suspension were applied to ELISA plates and air dried at room temperature. After fixing with 50%acetone and 50%methanol and blocking with 5% BSA in PBS for 30 min, MAb anti-F or anti-G was added, and plates were incubated at room temperature for another 60 min. The plates were then washed with PBS, and bound antibody was probed with horseradish peroxidaseconjugated goat anti-mouse IgG (Chemicon). Standard curves were generated by coating the plate with purified mouse IgG I and IgG2a (Chemicon). OD readings at 415 nm from virus-infected cells were plotted against sample dilution. By regression analysis of the linear part of this curve, the concentrations of F and G proteins were deduced from the IgG2a and IgG I standard curves, respectively, on the basis of the assumption that one antibody molecule binds to one molecule ofRSV For G protein (i.e., 1 Ilg ofMAb 858-1 binds to 0.47 Ilg ofRSV F protein and 1 Ilg of MAb 858-2 binds to 0.6 Ilg of RSV G protein). Dogs. Normal healthy beagles -- 5 months old were purchased from Marshall Farms (North Rose, NY). Virus was diluted with PBS to 5 mL and injected intratracheally. Samples of blood were drawn weekly or dogs were sacrificed at various intervals by overdosing with nembutal intravenously. This method is efficient and humane. Tissues of interest were resected and fixed with 10% formalin for histopathology or homogenized in PBS for virus quantitation as previously described [25]. Chimpanzee. Sera from 20 chimpanzees housed at Bioqual (Rockville, MD) were screened for neutralizing antibodies to RSV and human adenoviruses. Chimpanzee 1443 was selected because it alone was seronegative for RSV-neutralizing antibodies and for antibodies to Ad4, Ad5, and Ad7. After the chimpanzee was anesthetized, virus capsules were administered orally via a gastric tube, and blood samples were taken biweekly or as indicated. Stool samples were collected as scheduled for virus isolation. Detection of adenovirus is detailed elsewhere [29]. Microneutralization assay. Serum samples were heat inactivated at 56°C for 30 min before testing. Serial twofold dilutions (beginning with 1:4) were incubated with 10 TCID so ofadenovirus and then inoculated onto A549 cell monolayers seeded in 96-well microtest tissue culture plates (Becton Dickinson, Lincoln Park, NJ) that were incubated for 5-7 days at 37°C in 5% CO 2 , All samples were analyzed in duplicate. Virus and cell controls were included, which served as markers for determining the end points of test sera. Titers are expressed as the reciprocal of the lowest serum dilution at which 100% protection was observed. RSV-neutralizing antibody titers were determined by the same method except that RSV and HEp-2 cells were used and incubation was 4-5 days. The chimpanzee sera were also tested with a complement-enhanced 60% plaque neutralization test as

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Materials and Methods

JID 1992; 166 (October)

Adenovirus-Vectored RSV Vaccine

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Table 1. Expression of RSV antigens in A549 cells infected with recombinant adenovirus. RSV protein Adenovirus or adenovirus-RSV recombinant

Attachment (G)