Apr 1, 1986 - Robert Yolken (Johns Hopkins School of Medicine, Balti- more, Md.). ..... Arrobio, C. D. Brandt, W. J. Rodriguez, D. A. Sack, R. M.. Chanock, and ...
JOURNAL OF VIROLOGY, Nov. 1986, P. 491-496 0022-538X/86/110491-07$02.00/0 Copyright © 1986, American Society for Microbiology
Vol. 60, No. 2
Reassortant Rotaviruses Containing Structural Proteins vp3 and vp7 from Different Parents Induce Antibodies Protective against Each Parental Serotype PAUL A. OFFIT,12* H. FRED CLARK,' GERALDINE BLAVAT,"2 AND HARRY B. GREENBERG2'3 The Wistar Institute ofAnatomy and Biology and Division of Infectious Diseases, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 191041; Department of Medical Microbiology, Stanford University School of Medicine, Stanford, California 943052*; and Division of Gastroenterology, The Veterans Administration Medical Center, Palo Alto, California 943043 Received 1 April 1986/Accepted 25 July 1986 Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SAll and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SAll or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SAll. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SAll x NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections.
reassortant rotaviruses containing only gene segment 4 or 9 from the simian strain SAll or the antigenically distinct bovine strain NCDV, we were able to define the serotypic specificities of vp3 and vp7 in several mammalian rotavirus strains. We found that vp3 and vp7 were independently capable of stimulating a protective immune response against virulent rotavirus challenge.
Since their initial identification as a cause of human diarrhea in 1973, rotaviruses have been found to be the most important cause of acute gastroenteritis in infants and young children in both developed and developing countries (1, 14). The staggering worldwide impact of these viruses has excited interest in disease prevention by vaccine (15, 16, 33, 34). However, little is known about the molecular determinants of rotavirus responsible for inducing protection against challenge. A detailed knowledge of the rotavirus proteins which elicit protective immunity against challenge will contribute to the development of a successful vaccine. Rotaviruses contain inner and outer capsids enclosing a double-stranded RNA genome in 11 segments (3). There are two major outer capsid proteins, vp3 and vp7. vp7 is the protein product of gene segment 9 for most rotavirus strains; vp3 is the protein product of gene segment 4 (2, 12, 19). Gene segments 4 and 9 each segregate with the neutralization phenotype in reassortant rotaviruses (9, 23). In addition, monoclonal antibodies directed against either vp3 or vp7 of rhesus rotavirus neutralized viral infectivity in vitro (7) and passively protected suckling mice against rhesus rotavirus challenge (30). In this paper we describe the relative importance of vp3 and vp7 in eliciting a neutralizing immune response after homologous or heterologous host rotavirus infection. Using *
MATERIALS AND METHODS Animals. Conventionally bred CD-1 mice obtained from Charles River Breeding Laboratories, Inc., Wilmington, Mass., were housed in separate isolation units. Dams were bled on arrival by retro-orbital capillary plexus puncture, and sera were tested by radioimmunoassay as previously described (28); litters from seronegative dams were used in these studies. Cells and viruses. MA-104 cells were grown in antibioticfree BHK medium (18) or cell medium 199 supplemented with 10% fetal bovine serum. A seed stock of SAl1 virus was obtained from H. H. Malherbe (Gull Laboratories, Salt Lake City, Utah). The bovine rotavirus strain NCDV, adapted to growth in tissue culture at the University of Nebraska, was provided by Robert Yolken (Johns Hopkins School of Medicine, Baltimore, Md.). The bovine rotavirus strain UK, originally isolated by Gerald Woode, was provided by T. H. Flewett (World Health Organization Reference Center, Birmingham,
Corresponding author. 491
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OFFIT ET AL.
J.
TABLE 1. Gene segment assignments of a series of SAll x NCDV reassortant rotavriusesa Gene segment assignment Parent or reassortant
designation
1
2
3
4
5
6
7
8
9
10
11
NCDV SAll
N
N
N
N
N
N
N
N
N
N
N
S
S
S
S
S
S
S
S
S
S
S
N-1,5 N-3 N-7 N-10 N-li
N S S
S N S
S S
S S S S S
S S
S S S S S
N S S S S
S S S S S
S S N S S
S S S S S
S S S S S
S S S N S
S S S S N
S-2,11 S-7 N-2,4,9
N N S
S N N
N N S
N N N
N N S
N N S
N S S
N N S
N N N
N N S
S N S
S-4 N-9 N-4
N
N
N
S S
S S
S S
S S N
N S S
N S S
N S S
N S S
N N S
N S S
N S S
a Reassortant gene segment assignments were determined by comparison of gene segment migration in SDS-PAGE with that of SAll and NCDV parental strains. Reassortant viruses are designated S (SAll) or N (NCDV) based upon the least represented parental gene segments. Gene segment assignments from the least represented parental virus are indicated by a number following the letter designation. For example, S-4 contained SAll gene segment 4 and NCDV gene segments 1, 2, 3, 5, 6, 7, 8, 9, 10, and 11. The gene segment 4 and 9 assignments shown are in bold face.
England). The cultivation and characterization of the Eb strain of murine rotavirus have been recently described (8). Plaque-purified stocks of the SAl1, NCDV, and UK viruses were prepared in MA-104 cells. A plaque-purified stock of Eb rotavirus was prepared in secondary African green monkey kidney cells (8). Reassortant rotaviruses were derived by coinfection of MA-104 cells with the SAl and NCDV strains each at a multiplicity of infection of 5.0 as previously described (5, 23, 24). After 48 h of incubation at 37°C, cultures infected with SAl and NCDV rotaviruses were harvested by freezing and thawing. Progeny virus was titrated by plaque assay as previously described (29). Individual progeny plaques were picked, passaged twice in MA-104 cells, and characterized as described below. Genotypic analysis of reassortant rotaviruses by SDSPAGE. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with slab gels 32 cm long and 0.75 mm thick (Hoefer Scientific Instruments). Gels were run for 16 h at 350 V per gel in Tris-glycine buffer prepared as described by Laemmli (17) except that the pH of the running buffer was adjusted to 9.0 with NaOH (22). Detection of RNA bands by silver nitrate staining was performed as previously described (29). Reassortant virus gene segment assignments were determined by comparison of gene segment migration in SDS-PAGE with that of parental strains (5, 23, 24). Immunization and handling of dams. Eight-week-old female CD-1 mice were orally inoculated with 6 x 106 PFU of SAl1, NCDV, UK, or SAl x NCDV reassortant rotaviruses and inoculated orally 3 and 6 weeks later with the same virus at an identical dose. On the day of the final booster inoculation mice were mated with CD-1 males in cages at a female-to-male ratio of 1:1. Eight-week-old female mice were inoculated with Eb virus according to one of the following protocols: (i) mice were inoculated orally with 105 50% tissue culture infective doses of Eb and inoculated
VIROL.
orally 4 weeks later with the same viral dose or (ii) mice were inoculated intraperitoneally with 105 50% tissue culture infective doses of Eb emulsified in a 50% suspension of complete Freund adjuvant and inoculated intraperitoneally 4 weeks later with the same viral dose in incomplete Freund adjuvant. Infection of infant mice. Five-day-old mice were orally inoculated with 106 PFU of SAl1 virus or 5 x 107 PFU of NCDV (approximately 10 times the dose for each virus at which 50% of inoculated animals developed diarrhea) (24, 27). We recently found that approximately 90% of 5-day-old mice orally inoculated with S x 107 PFU of UK virus developed diarrhea (unpublished data). Pups were inspected daily for diarrhea by gentle palpation of their abdomens. Collection of serum and milk. On the day of inoculation of suckling mice, sera were obtained by puncture of the retroorbital capillary plexus from dams immunized with SAil, NCDV, UK, or SAl1 x NCDV reassortants. Sera were obtained from mice immunized with Eb rotavirus 2 weeks after the booster inoculation. Milk was collected from dams 5 days after inoculation of suckling mice by a modification of the technique described by Feller and Boretos (4). Briefly, litters were removed approximately 16 h before milking. To secure adequate yields of milk, it was necessary to inoculate the lactating mice intraperitoneally with 0.1 U of oxytocin (Parke, Davis, & Co., Detroit, Mich.). Mice were then milked with the suction apparatus previously described (4). PRN assay. The plaque reduction neutralization (PRN) assay was a modification of the technique described by Matsuno et al. (21) and was performed as previously described (28). RESULTS Gene segment assignments of SAll x NCDV reassortant rotaviruses. Unambiguous gene segment assignments for all reassortant viruses were obtained by comparison of comigration of reassortant RNA with that of parental strains in SDS-PAGE (Table 1). Evocation of neutralizing antibodies directed against both the gene segment 4 and 9 protein products by oral inoculation of mice with rotavirus strain Eb. To determine the relative roles of the gene segment 4 and 9 protein products in evoking a neutralizing immune response after local or systemic administration of a homologous host rotavirus, we inoculated mice either orally or parenterally with the Eb strain of murine rotavirus and tested their sera by PRN (Table 2). Greenberg et al. (8) found that the Eb strain was serotypically indistinguishable from the SAl1 strain by PRN with hyperimmune antisera generated in guinea pigs. We found that hyperimmune antisera obtained by parenterally immunizing mice with Eb virus neutralized SAll virus as well as TABLE 2. PRN assay of sera from CD-1 mice immunized either orally or parenterally with murine rotavirus strain Eb Route of immunization
Serum sample
PRN titer against indicated virusa S-4 NCDV N-4 SAll
Parenteral
1
18,000
25,000
5,500
100
Oral
1 2
300 300
300 400
500 900
