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Field Trial of Rhesus Rotavirus or Human-Rhesus Rotavirus Reassortant Vaccine ofVP7 Serotype 3 or 1 Specificity in Infants H. P. Madore,* C. Christy, M. Pichichero, C. Long, P. Pincus, D. Vosefsky, A. Z. Kapikian, R. Dolin, and the Elmwood, Panorama, and Westfall Pediatric Groups t

Departments ofAdult and Pediatric Infectious Diseases, University of Rochester School of Medicine and Dentistry, New York; National Institute ofAllergy and Infectious Diseases, National Institutes ofHealth. Bethesda, Maryland

Orally administered live rhesus monkey rotavirus vaccine (RRV, VP7 serotype 3) and humanrhesus reassortant rotavirus vaccine (DxRRV, VP7 serotype 1) were evaluated in a placebo-controlled field trial of 223 infants 2-4 months old. Both vaccines were mildly reactogenic but were generally well tolerated in the 10 days after vaccination. RRV and DxRRV were immunogenic, inducing serum antibody responses in 78% and 71% of the vaccinees, respectively. Efficacy of RRV vaccine was 66% (P = .01) and of DxRRV vaccine 77% (P = .002) against rotavirus-associated illness in the first season after vaccination. EfficacyofRRV vaccine against rotavirus-associated illness over three rotavirus seasons was 51.2% (P = .045) and of DxRRV vaccine was 67.3% (P = .006). RRV vaccine provided heterotypic protection of 58.5% (P = .041) and DxRRV vaccine provided homotypic protection of 72.8% (P = .005) over three seasons against the predominant serotype 1 rotavirus. Extensive efforts are underway to develop a live attenuated vaccine to prevent rotavirus gastroenteritis in infants and children. The rhesus monkey rotavirus strain MMUl8006 (RRV, VP7 serotype 3) was found to be immunogenic and mildly reactogenic in several trials [1-6]. The RRV vaccine was protective against homotypic serotype 3 rotavirus illness [7, 8], but efficacy against heterotypic rotavirus strains was variable [6, 9-12]. To obtain homotypic protection against the four prevalent rotavirus serotypes, a "modified Jennerian" approach has been taken, and humanrhesus reassortant vaccines have been prepared that contain the serotype-specific gene for the major neutralization protein, VP7, from human rotavirus and the remaining 10 genes from the rhesus monkey rotavirus [13-15]. The reassortant vaccines have been shown to be safe and immunogenic [16-18]. In 1987, we initiated a placebo-controlled field trial with 223 2 to 4-month-old infants comparing the rhesus monkey

Received 5 November 1991; revised 3 March 1992. , Presented in part: Society for Pediatric Research, Washington, DC, May 1989 (abstract 1035); 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, October 1990 (abstract 1227); 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, October 1991 (abstract 1289). Informed consent was obtained from parents of each subject. The studies were approved by the University of Rochester's Committee on Investigations involving Human Subjects and the National Institute of Allergy and Infectious Disease Clinical Research Study Subpanel on Human Studies. Financial support: National Institutes of Health (AI-52577, RR-00044). Reprints or correspondence: Dr. H. Paul Madore, University ofRochester Medical Center, Box 689.601 Elmwood Ave., Rochester, NY 14642. * Present affiliation: Praxis Biologics, Rochester, New York. t Group members listed after text. The Journal of Infectious Diseases 1992;166:235-43 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6602-0002$01.00

RRV and the human-rhesus reassortant (DxRRV, VP7 serotype I) vaccines. As serotype I was the prevalent rotavirus in the Rochester area, the relative efficacy ofRRV and DxRRV vaccines could be assessed. The safety, immunogenicity, and long-term protection provided by the vaccines over three rotavirus seasons was evaluated.

Methods Study population. Infants (2-4 months old) from three pediatric group practices in the Rochester area (predominantly suburban middle-class patients) were enrolled from 15 September 1987 to 22 February 1988. All vaccinees were healthy full-term infants; those who had missed scheduled well-child care were excluded. Each infant had received the first diptheria-tetanus toxoid-pertussis and oral polio vaccines, and further immunizations were withheld for 4 weeks before and 3 weeks after rotavirus vaccination. Information on attendance at day care facilities was not obtained. Vaccines. The RRV vaccine is derived from the natural simian virus with a VP7 ofspecificitysimilar by neutralization assay to human VP7 serotype 3, and a "simian" VP4 [19]. The DxRR V vaccine is a human-rhesus reassortant with the human VP7 (D strain) of serotype 1 specificity and the remaining 10 genes, including the VP4,-from the rhesus monkey rotavirus [14, 15]. Suspensions of these vaccines (lot RR V-1, DxRR V-1) were prepared by Flow Laboratories (Mel.can, VA) and yielded a titer of undiluted RR V and DxRR V vaccine of --106 pfu/rnl.. In our laboratory. each vaccine was diluted 1:100 in soy formula (Isomil; Ross Laboratories, North Chicago, IL), aliquoted in dram vials, and stored at -70°C until use. The TCID so titers for the 1:100 dilution of the RR V and DxRRV vaccines after thawing were 5 X I0 4/mL and 5 X lOs/mL, respectively, IOO-fold less than the TCIDso titers of the undiluted vaccines. These TCID so titers were higher than the expected 104 pfu/rnl, for the

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I: 100 dilution of the RRV and DxRRV vaccines due to variability of both the TCID so and plaque-forming unit (pfu) assays, but the doses of the diluted vaccines are equivalent and are expressed as 104 pfu. Subjects received a vial of either vaccine or identically appearing placebo previously coded by a table of random numbers. Vaccine administration. The infants drank 30 mL of soy formula buffered with 400 mg of sodium bicarbonate to reduce stomach acidity, followed immediately by I mL of vaccine or placebo (soy formula) administered in a tuberculin syringe with the needle removed..All feedings were withheld for I h before and after vaccination. Reactogenicity. To assess reactions to the vaccine, parents filled out a daily report for 10 days after vaccination, which included evening rectal temperature, symptoms of gastrointestinal illness, and behavioral changes. Each instance of rectal temperature ;;::.38°C or acute illness during this period was evaluated by a member of the study team, and stool or rectal swab and nasopharyngeal specimens were taken for culture of bacteria and viruses. Gastrointestinal illness was defined as the occurrence of diarrhea (more than twice the usual number of stools or any watery stools) or vomiting or both. Immunogenicity. Blood samples for measurement of antibody response to rotavirus were taken immediately before vaccination, 21 days later, and after the first and second rotavirus seasons. Sera were not collected after the third season. Serum neutralizing antibody response to rotavirus was determined by a microneutralization assay on 96-well plates as previously described [9]. Serum antibody rises of fourfold or greater were considered significant. Serum pairs were tested against 250-500 TCID so of human rotavirus Wa (VP7 serotype I, provided by Y. Hoshino, National Institutes of Health), 100-250 TCID so of DxRRV vaccine (VP7 serotype I), or 100-250 TCID so ofRRV vaccine (VP7 serotype 3) in the microneutralization assays. Serum IgA to rotavirus was measured by ELISA modified from a previously described procedure [20-22]. Briefly, polystyrene 96-well plates (3799; Costar, Cambridge, MA) were coated with hyperimmune guinea pig antirotavirus (SA-II) capture antibody diluted at 1/5000 in PBS for 6 h at 20°C, followed by one wash with PBS-0.05% Tween 20 (PBS-T). Plates were subsequently washed five times with PBS-T between steps. RRV-infected or uninfected MA I04 cells extracted with I, 1,2-trichloroI,2,2,-trifluoroethane (Genetron; Baker Chemical, Phillipsburg, NJ) and diluted 1/10 in PBS containing 0.5% gelatin and 0.5 M NaCI (dilution buffer) were added and incubated overnight at 4°C. Plates were washed and human serum pairs starting at a 1/50 dilution with subsequent twofold dilutions in PBS containing 10%fetal bovine serum, 0.5% gelatin, and 0.5 M NaCI were added and incubated for 2 h at 37°C, followed by a wash. Indicator anti-IgA antibody linked to alkaline phosphatase (2491; Tago, Burlingame, CA) at 1/2500 in dilution buffer was added and incubated for 2 h at 37°C, followed by a wash. Plates were incubated in I mg/ml. p-nitrophenyl phosphate (104 tablets; Sigma, St. Louis) in 10% diethanolamine buffer for 30 min at 20°C, the reaction stopped with 3 M sodium hydroxide, and plates read at 405 nm (Model EL309; Biotek Instruments, Winooski, VT). End point was the last dilution with a positive-to-

Jlf) 1992; 166 (August)

negative ratio ;;::.2 and optical density (OD) ;;::. 0.1. A serum pair with a fourfold or greater rise was included as a positive control for each assay. Adult serum pairs from a previous rotavirus trial [2] tested by this assay and by use with the assay described by Midthun et al. [22] for rotavirus-specific IgA had 13 significant rises in both assays, 19 pairs had no rises in both assays, and I pair had a significant rise in this assay. Clinical illness. In the Rochester area, the rotavirus season is generally January-June, with peak activity in March. Infants were followed from the time of enrollment through the following 3 years. The first year included time of enrollment (15 September 1987-22 February 1988) through 8 August 1988. The second year included 9 August 1988-8 August 1989. The third year was 9 August 1989-8 August 1990. Parents notified their pediatricians and the study nurse when their child developed a gastrointestinal illness. A clinical symptom sheet was filled out by the parent or nurse, and stool or rectal swabs were taken during illness for culture of viruses and bacteria. To assure compliance, maintain interest, and minimize missing of diarrheal illness, we alternated telephone, letter, or postcard reminders monthly during the rotavirus season. Each episode of gastrointestinal illness was also evaluated by the World Health Organization clinical indicators of severity: presence of fever (;;::.38°C), vomiting, dehydration, more than six stools in 24 h, and duration of illness ~24 h. We compared the number of vaccine and placebo recipients with gastrointestinal illnesses and rotavirus-associated illnesses lasting >24 h using one or more of the above criteria. Laboratory methods. Stool and rectal swab specimens were placed in veal infusion broth, stored at 4°C, and processed within 24 h. An aliquot was assayed for rotavirus antigen by ELISA (Pathfinder; Kallestad Laboratories, Austin, TX) according to manufacturer's directions. A second aliquot for rotavirus isolation was cultured in African green monkey kidney cell cultures and cell extracts were assayed by ELISA for rotavirus antigen as previously described [9]. The remainder of the specimen was stored at -70°C. Rotaviruses were serotyped with serotype-specific neutralizing monoclonal antibody as previously described [23]. Nasopharyngeal swabs for virus isolation were placed in veal infusion broth, transported at 4°C, and inoculated into HEp-2 cells, human foreskin fibroblasts, and cynomologous monkey kidney cells. Stool and rectal swab specimens were placed in Cary-Blair transport medium, transported at 4°C, and tested for Shigella, Salmonella, and Campylobacter species by standard microbiologic assays. Statistical analysis. x 2 analysis with Yates's correction for continuity, Fisher's exact test (two-tailed), or Students t test (two-tailed) were used where appropriate; 95%confidence intervals (CI) were determined as described by Kleinbaum et al. [24].

Results Study population. Of the 223 infants enrolled and vaccinated, 76 received RRV vaccine, 74 received DxRRV vaccine, and 73 received placebo; 40 RRV vaccinees, 30 DxRRV vaccinees, and 38 placebo recipients were breastfed at the time of vaccination. Mean ages for each group at

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Table 1. Clinical reactions of recipients of live rhesus monkey rotavirus (RRV; VP7, serotype 3), human-rhesus reassortant vaccine (DxRRV; VP7, serotype I), or placebo. No.Ztotal (%) with Vaccine group

Loose stools

Watery stools

Vomiting

Irritability

Placebo RRV DxRRV

39/63 (62) 58/73 (79)* 47/67 (70)

22/63 (35) 29/74 (39) 30/66 (45)

18/63 (28) 22/75 (29) 23/66 (35)

42/66 (64) 44/75 (57) 38/68 (56)

*p

=

.039.

x2 analysis.

RR V vs. placebo group.

vaccination were 3.8 ± 0.8,3.7 ± 0.7, and 3.8 ± 0.8 months, respectively. Reactogenicity. DxRRV and RR V vaccines were well tolerated by the infants. In the 1O-day period after vaccination, no significant differences were observed between DxRRV vaccinees and placebo recipients in number of loose or watery stools, number ofvomiting episodes, occurrence ofirritability (table 1), or occurrence of more than three stools a day during the 10 days after vaccination (data not shown). A significantly greater number of RRV vaccinees had loose stools compared with placebo recipients (P = .039), but frequencies were similar with regard to watery stools, vomiting, and irritability (table 1). A significantly greater number ofRRV vaccinees had more than three stools a day on day 7 compared with placebo recipients (P = .03, data not shown). TheRRV and DxRRV vaccinations were associated with mild febrile reactions. Fever peaked on day 4 after vaccination in RRV vaccinees and on day 5 after vaccination in DxRRV vaccinees; fever in placebo recipients was distributed more evenly over the 10-day period (figure 1). The mild febrile reactions associated with RR V differed significantly from those of placebo recipients on days 4 and 5 (figure 1).

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The maximum temperature for the RRV vaccinees during this period was 39.5°C. The frequency of mild febrile reactions associated with DxRRV was significantly greater than that of placebo recipients on day 5 (figure 1), with a maximum recorded temperature of 38. 7°C. The duration offever in RRV and DxRRV vaccinees was generally 1-2 days. Immunogenicity. Serum neutralizing antibody, rotavirus-specific IgA, or any serologic response (table 2) occurred in most vaccinees by 21 days after vaccination. Serologic responses to one vaccine were in most instances accompanied by serologic response to the other vaccine, but 7 of the RRV vaccinees had serologic responses only to DxRRV or Wa and 4 of the DxRRV vaccinees had serologic responses only to RR V. The serologic responses of placebo recipients and RRV vaccinees to rotavirus Wa serotype 1 by neutralization (9.6% and 19.1%, respectively) may represent intercurrent rotavirus infection during the vaccination period. The serum antibody rises in these subjects were not accompanied by detectable gastrointestinal illness, indicating that asymptomatic rotavirus infections occurred during the vaccination period. Of 12 RRV vaccinees and 5 placebo recipients with serologic responses to human rotavirus Wa,5 of the 9 tested RR V vaccinees and 0 of the 3 tested placebo recipients had accompanying serologic responses of rotavirus-specific IgA. Geometric mean titers in the prevaccination sera were