JOURNAL OF VIROLOGY, June 1990, p. 2687-2691

Vol. 64, No. 6

0022-538X/90/062687-05$02.00/0 Copyright © 1990, American Society for Microbiology

Serum-Neutralizing Antibody to VP4 and VP7 Proteins in Infants following Vaccination with WC3 Bovine Rotavirus WARD,'*

DOUGLAS R. KNOWLTON,1 HARRY B. GREENBERG,2 GILBERT M. SCHIFF,' AND DAVID I. BERNSTEIN1 The James N. Gamble Institute of Medical Research, 2141 Auburn Avenue, Cincinnati, Ohio 45219,1 and Division of Gastroenterology, Veterans Administration Medical Center, Palo Alto, California 943042 RICHARD L.

Received 10 October 1989/Accepted 7 March 1990

Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypicaHly distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], 80% of the neutralizing antibody was found to have been made to the VP4 protein. These results have important implications in vaccine development in which reassortant rotaviruses containing VP4 of animal strains and VP7 of human strains have been constructed as possible vaccine candidates (3, 13, 14). In a report by Flores et al. (4), it was suggested that human-rhesus rotavirus reassortants stimulated greater neutralizing antibody to the rhesus rotavirus VP4 protein than to the human rotavirus VP7 protein in orally inoculated infants. Therefore, it was of immediate interest to determine whether VP4 is consistently the dom-

Inoculation of animals or humans with rotaviruses results in the production of neutralizing antibody which is believed to be at least partially responsible for protection against rotavirus disease. Rotaviruses contain two outer capsid proteins, VP4 and VP7, which have been shown to elicit the production of neutralizing antibody (5, 6, 10, 11, 15, 24). The relative importance of each protein in protection against rotavirus disease is not known. Passive protection studies with mice indicate that antibody to either protein can be protective when delivered orally (12, 16, 17). Similarly, when piglets were immunized with a reassortant strain containing the VP4 protein of one virulent porcine strain and the VP7 protein of a second porcine strain belonging to a different serotype, they were protected against both porcine rotaviruses (8). Thus, neutralizing antibody to either VP4 or VP7 appeared to be protective in these animal studies. The relative immunogenicities of the two rotavirus neutralization proteins have not been firmly established. It was originally reported that most neutralizing antibody was made to VP7 after hyperimmunization of animals (5, 6, 11). More recent studies indicate that high titers of neutralizing antibody to both VP4 and VP7 can be generated in hyperimmunized (8, 10, 15, 16) as well as in orally inoculated (8, 16) animals. On the basis of differences in immune responses in mice, it was suggested that VP4 may be more immunogenic after intestinal infection than after parenteral hyperimmunization (20). Direct evidence that VP4 can be more immunogenic than VP7 during intestinal infection was shown in human studies. Svensson et al. (23) found, by using radioimmunoprecipitation, that serum antibody to VP4 appeared to be made following oral inoculation of infants with either of two live experimental vaccine strains, but no antibody to VP7 was detected. Ward et al. (27) showed that after administration of *

inant immunogen for induction of neutralizing antibody following intestinal infection of humans with rotavirus. Our laboratory conducted a rotavirus vaccine trial during the 1988-1989 rotavirus season to determine the efficacy of the WC3 strain of bovine rotavirus in the protection of infants 2 to 12 months old against rotavirus disease. All but 3 of the 103 infants orally administered this live virus vaccine seroconverted to the WC3 strain (D. I. Bernstein, V. E. Smith, D. S. Sander, K. A. Pax, G. M. Schiff, and R. L. Ward, submitted for publication). Infants who had the largest rises in serum-neutralizing antibody to the vaccine virus and no evidence of previous rotavirus exposure were examined to determine the relative immunogenicities of the WC3 VP4 and VP7 proteins. As shown in this report, the VP7 protein was clearly the dominant immunogen. MATERIALS AND METHODS Virus strains. The properties of the WC3 bovine rotavirus and the passage of history of the vaccine have been described previously (1, 2). The final titer of the vaccine lot used in this study was 107-5 PFU per ml. Rhesus rotavirus (RRV) was originally from N. Schmidt (22). The EDIM virus (EW strain of mouse rotavirus) was from J. Wolf (28) and was adapted to grow in cell culture by Greenberg et al. (7).

Corresponding author. 2687

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Vaccine administration. This was a double-blind, placebocontrolled study. Two hundred six healthy infants 2 to 12 months old were enrolled according to procedures approved by The Christ Hospital Institutional Review Board. After consumption of 25 to 50 ml of infant formula or 5 to 10 ml of Maalox Antacid (Rorer Pharmaceuticals, Fort Washington, Pa.), vaccine (1 ml) plus diluent (1 ml) or placebo (2 ml of diluent) was orally administered according to a code supplied by Institut Merieux, Lyon, France. Equal numbers of subjects (i.e., 103) received vaccine or placebo. Nothing except infant formula was consumed for at least 1 h after vaccination. Blood collection and determination of neutralizing antibody titers. Blood specimens (0.5 ml) were collected immediately before vaccination and 25 to 31 days after administration of vaccine to measure the rise in antibody titers to the WC3 strain. Neutralizing antibody responses to WC3 and the reassortant strains were determined by a fluorescent focus reduction assay. For this, stock preparations of WC3 and reassortants were activated by incubation with 20 ,ug of trypsin per ml for 1 h at room temperature, then diluted to approximately 50,000 focus-forming units per ml with Dulbecco modified minimal essential medium (DMEM). For neutralization, an equal volume (12.5 [lI) of serum (heat inactivated at 56°C for 30 min and serially diluted in the same medium) was mixed with virus and incubated (37°C, 1 h). Confluent monolayers of MA-104 cells (4 days old) grown in 96-well microdilution plates were washed twice with DMEM, and the serum-virus mixture was diluted 10-fold in the same medium before 0.1 ml was added to each well. The plates were then centrifuged (1,000 x g, 1 h, room temperature) to promote virus adsorption to cells. Unadsorbed virus was aspirated from the wells, and the cells were washed once with DMEM before being overlaid with 0.2 ml of DMEM containing antibiotics and 4 pLg of trypsin per ml. After 15 h of incubation at 37°C, medium was aspirated from the wells and cells were fixed with 80% acetone at -20°C. After storage for -10 min at -20°C, acetone was aspirated and monolayers were allowed to dry completely. Heatinactivated hyperimmune guinea pig serum to Wa virus was diluted 1:800 in phosphate-buffered saline (PBS) containing 5% nonfat dry milk, and 0.1 ml of the solution was added to each well of the microdilution plates. After 1 h at 37°C, the guinea pig serum was aspirated and the wells were washed once with PBS. Fluorescein-conjugated goat anti-guinea pig immunoglobin G (Cappel Laboratories, Downington, Pa.; 0.1 ml of a 1:200 dilution in PBS plus milk per well) was added. The plates were incubated (1 h, 37°C), and the conjugated antibody was removed by aspiration. The wells were washed twice with PBS, the cell monolayers were dried, and the fluorescein-tagged cells were visualized with a UV microscope. The reciprocal of the serum dilution that reduced recoverable focus-forming units by 60% was considered its titer. Criteria for selection of serum specimens. Although 103 subjects were administered vaccine and 100 of these were infected as determined by a rise of -threefold in serumneutralizing antibody to WC3, only 32 subjects had titers of .160 (D. I. Bernstein, et al., submitted). One of these subjects, along with 7 other vaccinees, had been previously infected as determined by the presence of serum rotavirus immunoglobulin A (25) and rises in heterotypic neutralizing antibody titers following WC3 vaccination (D. I. Bernstein et al., submitted). All subjects identified as previously infected were .7 months old at the time of vaccination. To maximize the possibility of observing differences in antibody

titers to the VP4 and VP7 proteins of WC3 in previously uninfected subjects, only serum specimens with neutralizing antibody titers of .160 were used for analysis. Thus, 31 specimens were included. None of the subjects from whom these specimens were obtained had detectable serum-neutralizing antibody to WC3 prior to vaccination (i.e., all had titers of

Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus.

Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neu...
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