1099

Immune Protection of Infants against Rotavirus Gastroenteritis by a Serotype 1 Reassortant of Bovine Rotavirus WC3 H F. Clark, F. E. Borian, and S. A. Plotkin

From the Joseph Stokes, Jr., Research Institute of the Children's Hospital of Philadelphia and the Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania

Rotavirus gastroenteritis is the major cause of infant diarrhea in all countries of the world and a significant cause of mortality in developing countries [1]. Therefore, an effective vaccine would be worthwhile and cost-effective [2]. Several strains of rotavirus have been proposed as candidates for a live orally administered vaccine against the infantile gastroenteritis caused by these agents. However, problems have been experienced with many of the strains proposed as vaccines. For example, RIT 4237 proved to be insufficiently protective in trials conducted in Africa [3, 4], whereas RRV and its reassortant derivatives induced feverand gastrointestinal symptoms in some vaccinees [5,6]. Also, protective efficacy ofRRV has been inconsistent [7-9]. The WC3 strain of bovine origin (serotype 6) developed in our laboratory has been safe and immunogenic [10]. In an initial double-blind efficacy trial involving 104 infants, a single dose ofWC3 vaccine wasassociated with a 76 % reduction in the incidence of rotavirus diarrhea and 100 % reduction in moderate to severe rotavirus diarrhea. This protection

Received 7 August 1989; revised 11 December 1989. Informed written censent was obtained from the parents of the study subjects. Vaccine trial protocols were approved by the Committee of Protection of Human Subjects, Institutional Review Board, Children's Hospital of Philadelphia, and the Human Subjects Review Committee of the Wistar Institute. Grant support: Institut Merieux, Lyon, France, and RR-QOO40 (National Institutes of Health). Reprints and Correspondence: Dr. H F. Clark, Division ofInfectious Diseases, Children's Hospital, 34th Street and Civic Center Blvd., Philadelphia, PA 19104. The Journal of Infectious Di!Ieases 1990;161:1099-1104 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6106-0008$01.00

was demonstrated during a winter outbreak caused by serotype 1 [11]. WC3 appears to protect by induction of heterotypic immunity. The serum antibody response to WC3 is predominantly directed to rotavirus serotype 6. About halfofWC3 vaccinees who exhibited a serum antibody response also developed serum antibody to serotype 3, but serum antibody responses to serotype 1 rotavirus were infrequent [10]. Thus the identity of the heterotypic immune response to WC3 is unclear, and recent unpublished observations cast doubt on the consistency of this heterotypic protection. It has been proposed that a serotype-specific vaccine may be necessary to induce consistent protection against the infecting wild virus serotypes [7, 8]. Therefore, it was of interest to determine whether the efficacy of WC3 rotavirus vaccine would be improved by incorporation into the WC3 virion of a serotype 1 antigen. We have developed a reassortant virus based on WC3 but containing gene segment 9 of serotype 1 which codes for the vp7 surface protein. The preparation, characterization, and phase I clinical safety and immunogenicity tests of this virus, called WI79-9, are described in detail elsewhere [12]. Here we present the initial evaluation of safety and efficacy of WI79-9 in a placebo-controlled double-blind efficacy trial in Philadelphia.

Study Design and Methods WI79-9 Vaccine. WI79-9 rotavirus contains gene segment 9 of human serotype 1 rotavirus strain WI79, which was isolated in our laboratory in 1984, and all other gene segments from serotype 6 strain WC3 rotavirus. The virus was administrated in a dose of 2.0 x 10' plaque-forming units in cell culture medium (Eagle's mini-

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The safety and protective efficacy of a serotype 1 reassortant of bovine rotavirus WC3, designated strain WI79-9, was evaluated in a double-blind placebo-controlled trial. Rotavirus reassortant WI79-9 contains a gene segment 9 coding for the surface structural protein vp7 of a human serotype 1 rotavirus, with all other gene segments derived from WC3 rotavirus, which had previously been shown to be safe and immunogenic in infants. Infants 2-11 months of age were given two doses of vaccine (107.3 plaque-forming units/dose) or of placebo 28 days apart. Adverse reactions to the vaccine were not detected. The incidence of serum plaque reduction neutralization antibody responses to two doses of vaccine was serotype 6, 97%; serotype 3, 68%; and serotype 1, 22%. Active surveillance during the subsequent rotavirus season revealed 8 cases of rotavirus gastroenteritis in 39 placebo control infants and no cases in 38 WI79-9 vaccine recipients (protection = 100%, P = .003). Six cases of rotavirus gastrolnteritis were caused by type 1 and two by type 3 virus. Although vaccination with WI79-9 affected only the incidence of rotavirus gastroenteritis, the vaccinated infants exhibited a significantly reduced incidence of total days of diarrhea, fever, and illness associated with gastroenteritis in general.

1100

Clark et al.

Table 1. Characteristics of infants enrolled in vaccine trial. Group Characteristic No. of infants No. (%) male Mean age at initial inoculation (months) Breast-fed (%) Day care (%) Siblings at home (%) Mean interval, first inoculation to midpoint rotavirus season (months)

WI79-9 38 21 (55)

5.6

± 2.6

Placebo 39 20 (51) 5.9 ± 2.9

39

28

42

26

55

54

3.4

±

1.0

3.5 ± 1.0

Results The vaccinated population. Of the 77 infants who completed the trial, 38 received vaccine and 39 received placebo. Characteristics of the cohorts are listed in table 1. The populations were closely matched in distribution of age, gender, and presence of siblings at home. There were slightly more vaccinated infants who were breast-fed or in day care. Postvaccine reactions. Clinical reactions detected within the first 7 days after each dose of either vaccine or placebo are listed in table 2. No differences between vaccine and placebo groups were noted with respect to observed diarrhea or vomiting; a slight excess of irritability in vaccinated infants did not approach statistical significance. Slightly more fevers were observed in vaccinees after the second dose only. However, all episodes of fever after the booster dose were associated with concomitant conditions not related to gastroenteritis: upper respiratory infections (three infants), otitis media (one), both conditions (four), or teething (two). Analysis of clinical observations made daily revealed no clustering of signs of diarrhea, vomiting, fever, or irritability on any particular day or group of days after either the primary or the booster vaccine dose. Although viral shedding in feces was not monitored during the 7-day postinfection period when shedding of WC3 and its reassortants had occasionally been observed [10], a plaque assay of fecal samples collected from 32 vaccinees at 14 days after infection revealed vaccine poliovirus in three and vaccine rotavirus in none. Immune response to vaccine. The serum PRN antibody response to two doses of vaccine or placebo was determined after the vaccine code was broken (table 3). All but one vaccinee (97 %) exhibited serum antibody responses to serotype 6 (stain WC3). The exception was 4.5 months old, male, and breast-fed, and before vaccine administration was seronegative to WC3 (type 6) but exhibited very high PRN titers to type 3 (1 :4680) and type 1 (1:2190). Only 22 % of vaccinated infants exhibited a serum antibody response to serotype 1 (strain WI79). A serum antibody response to serotype 3 (strain SAIl) was detected in 68 % of the vaccinees. In infants receiv-

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mum essential medium [EMEM] with no protein supplement) containing 20 % cherry syrup. Placebo was EMEM alone with 20 % cherry syrup. Study design. The study was initiated in September 1987 and was performed in three private pediatric practices in the suburbs of Philadelphia. After a brief physical examination, healthy normal infants 2-11 months old were given WI79-9 vaccine or placebo orally (volume 2.5 ml). Vaccineor placebo was assigned to numbered vials according to a table of random numbers, and all investigators were blinded to code. Infants receiving diphtheria-pertussis-tetanus or oral polio vaccine within 7 days were excluded. Breast-feeding was withheld for 1 h before and after immunization. Commercial infant formula (30 ml) was fed immediately before administration of vaccine to buffer stomach acidity. Infants who refused formula received an antacid containing 40 mg of magnesium hydroxide and 45 mg of aluminum hydroxide/ml (Maalox; Rorer, Fort Washington, PA), 1 ml/kg of body weight. A second dose of vaccine was given 28 days after the first dose. Blood samples were collected by finger stick before the first WI79-9 dose, 28 days after the second dose, and after the end of the rotavirus season (July 1988). Stool samples for fecal antibody study were collected at the time of and 14 days after each oral inoculation. To assess possible adverse reactions to vaccine, parents were called daily for 7 days after administration of each dose. Parents were also instructed to take rectal temperatures twice daily and to fill out a postcard form recording temperature, number of consistency of stools, and incidence of vomiting or other signs of illness during the 7-day postinoculation period. Active surveillance for rotavirus disease during the study period was maintained by instructing parents to report any episode of gastroenteritis immediately to the study nurse, weekly phone calls to the parents of each study infant, and daily monitoring of the telephone logs of pediatricians serving the study population. Parents were instructed to supply a stool sample to the study nurse at the inception of any episode of diarrhea and to maintain a symptom log for the duration. Disease severity was also monitored by daily phone calls to parents during the course of the illness. A numerical score of clinical severity of gastroenteritis was established by two blinded clinicians independently evaluating symptom records according to a grading system previously described [11]. Virus diagnosis. Stools were examined for the presence of rotavirus by a commercial latex agglutination test (Slidex; BioMerieux [13], Charbonnieres les Bains, France) provided by the manufacturer, and by polyacrylamide gel electrophoresis for detection of rotavirus genome segments [14]. Representative rotavirus-positive samples of each observed electropherotype were submitted to the Viral Gastroenteritis Unit, Centers for Disease Control (Atlanta), for the determination of serotype. SAIl virus was obtained from Dr. H. Malherbe, San Antonio, TX; all other reference viruses were isolated in our laboratory. Serum antibody determination. Serum antibody titers were assessed by the plaque reduction neutralization (PRN) test as previously described [15]. Test viruses used were type 1 (WI79), type 2 (SC2), type 3 (SAl1), type 4 (CC4), type 9 (WI61), and type 6 (WC3). Statistical analysis. Discrete variables were analyzed by X2 test using Fisher's exact test and Yates's correction where appropriate.

1ID 1990;161 (June)

Serotype 1 Reassortant of Rotavirus WC3

JID 1990;161 (June)

Table 2. Postinoculation reactions of infants given two doses of WI79-9 vaccine (n

=

38) or placebo (n

llOl

DISTRIBUTION OF ROTAVIRUS INFECT10NS BYELECTROPHEROTYPE 11181

= 39).

VACCINE TRIAL wu..

No. of infants experiencing reaction after Dose 2

Dose 1

J.

Reaction Diarrhea Vomiting Fever (~38.1 0c) Irritability

WI79-9

Placebo

WI79-9

Placebo

4 2 4 15

5 3 6 11

5 3 10 14

3 1 2 9

III

I.

ltI

...0

10

II:

11/

ID ~

~

z

5

-

V

........t ~

X

~ X

i---l

No. (%) with PRN antibody response WI79-9 (n = 38)

6 (WC3) 3 (SAIl) 1 (WI79) Any

37 25 8/37 37

(97) (68) (22) (97)

Placebo (n = 39) 2 2 2 2

(5)* (5) (5) (5)

NOTE. PRN = plaque reduction neutralization. • Infants no. 68 and 75 (table 6) experienced clinical rotavirus disease before postplacebo bleeding; onset was 21 days after placebo dose 2 for infant 68 and 16 days after placebo dose I for infant 75.

ing placebo, a rise in serum antibody was seen only in two who experiencedclinical attacks of rotavirus gastroenteritis beforethe postplacebosampling (within 56 daysof the original placebo inoculation). The presenceof serum antibody to serotypes 1 or 3 before vaccination was associated with a reduced incidence of serum antibody responses to those serotypes. Type 1 antibody responses occurred in 7 (24%) of 29 type l-seronegative but only 1 (12.5%) of 8 type l-seropositive infants. Type 3 antibodyoccurredin 20 (83%) of 24 type 3-seronegativebut only 5 (36%) of 14 type 3-seropositive infants. The inhibition of serotype 3 PRN antibody responses in serotype3-seropositive infants was significant (P = .005). There wasa trend toward a higher incidenceof heterotypic PRN serumantibody responses in older infants. The incidence of antibody responses to type 1 in infants5-11 months of age was6 (28.6%) of21 compared with 2 (12.5%)of 16 in infants 2-4 monthsold at vaccination. The frequency of antibody responses to type 3 (strain SAll) was 18 (85.7%) of 21 in infants 5-11 months old compared with only 7 (41.2%) of 17 in the younger vaccinees. FecallgAantibodyresponse. Fecal samplescollectedbefore vaccineadministration and 14 daysafter the initial dose were evaluated for anti-rotavirus 19A content by Dr. Richard Ward, Cincinnati. Assays were performed on 9 infants given vaccineand 13givenplacebo. No fecal rotavirus 19A response was detected.

1.

1.

I.

E-I". .1wptyM

r---

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I.

.....

X.

I

I

I

I

-

... ·

J,K,X

V

. .. . ·· .. .. . · J

..

X

X

I

I

I

I

K

D

I•

I

X

10

I,D

12

1.

1 2

3

h3

1.

WEEKaf 1988

FigUl;e 1. Distribution of individual episodes of rotavirus infant gastroentiritis during the study period, according to week of the year, serotype, and electropherotype.

Surveillance datafor rotavirus gastroenteritis. Rotavirus surveillance was performed in parallel for the vaccine study population and, to determine whether the study group challenged wascharacteristic of the larger community, amonginfants admitted with gastroenteritis to Children's Hospital of Philadelphia. The electropherotype associated with each infectionwasdetermined, and the serotype wasdetermined for representatives of each electropherotype. The epidemic pattern is illustrated in figure 1. Sevenelectropherotypes were detected in the Children'sHospital population. Viruses were type 1 or 3 except for one type 2. Types 1 and 3 were equally prevalent; indeed, three infants were simultaneously infected with type 1 and type 3 strains. The onset of rotavirus disease appeared to be slightly delayedin the suburban vaccine study population, and study cases appeared after the cessation of cases among infants at Children's Hospital. However, two of the study cases appearing after the 10th week of 1988 were mild and undoubtedly would not have been noted without active surveillance. Vaccine-associated protection. All rotavirus diseasenoted in the vaccine study occurred in the placebo cohort (table 4). Two episodes were caused by type 3 (both graded moderate to severe) and six were caused by type 1 virus (4 moderate to severe, 2 mild). Thus,the rotavirus attackrate in the placebo group was 21% and the protection rate for vaccinewas 100% (P = .003). Wealso ascertained the prevalenceof asymptomaticinfections in the study population by identifying infants with an increase in' serum antibody to serotypes 1 and 3 at the end of the rotavirus season (table 4). Twelve asymptomatic infections were detected in vaccinated infants but only four in

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Type

12

11tE CHILDREIl'8I1OS1'1TAL OF PHILADELPHIA

X

Table 3. Immune response of infants to WI79-9.

X.

~

Clark et aI.

1102

Table 4. Rotavirus infections in WI79-9 vaccinees and placebo recipients. No. (%) with infection Vaccinees

Type of infection Symptomatic Type 1 Type 3 Total Asymptomatic Types 1 and 3 Type 1 Type 3 Total Total rotavirus infections

Placebos 6 2 8 (21)

0 0 0

4

2 7 3 12 (34) 12 (34)

0 0

4 (10.5) 12 (32)

season.

Table S. Symptoms in 8 placebo recipients with rotavirus disease. Symptom

Total days

Mean no. days/episode

Diarrhea Vomiting Fever illness

44 12 17 48

5.5 1.5 2.1 6.0

NOTE. Mean clinical severity score = 12.4. Four of eight infants (50%) with acute otitis media.

the placebo group. The total number of infections was therefore identical in vaccine and placebo cohorts (32%-34%). The severity of the rotavirus disease encountered in the placebo infants wasjudged by an enumeration of the total days of expression of individual symptoms (table 5). The mean durations of diarrhea and illness were >5 days; in a population of only 39 infants, 48 days of rotavirus illness were encountered in a single winter season. Immune response by serotypeofinfantswith rotavirusdis-

ease. The eight placebo infants with rotavirus gastroenteritis were evaluated for the type-specificity of their serum PRN antibody responses to the five most common human rotavirus serotypes and to bovine serotype 6 (table 6). All exhibited broadly cross-reactive active antibody response. The two infants infected with serotype 3 viruses developed maximum PRN titers to type 3. Infants infected with type 1 variously exhibited highest-titered responses to type 1 (two infants), type 4 (three), or type 3 (one). Nonrotavirus gastroenteritis. There was no evidence of WI79-9 vaccine-associated protection against nonrotavirus gastroenteritis. Fifteen of 38 vaccinated infants experienced 18 episodes (attack rate = 39 %) and 11 of 39 placebo recipients experienced 12 episodes (attack rate = 28%). Although there was actually a higher rate of nonrotavirus gastroenteritis in vaccinees than in placebo infants, the total days of gastroenteritis (rotavirus-associated plus nonrotavirus) in vaccinated and placebo cohorts were compiled to determine the possible overall impact of an effective rotavirus vaccine (table 7). The number of total episodes of gastroenteritis was virtually identical in the WI79-9-vaccinated and placebo populations. However, because of the absence of rotavirus disease in the vaccinated infants, there was a lower frequency of moderate to severe disease and fewer days of distinct symptoms of gastroenteritis in the vaccinated cohort. There were significantly fewer total days each of diarrhea, fever, and gastroenteritis-associated illness (P = < .05) in the vaccinated group.

Discussion The incorporation of the gene 9 of human type 1 rotavirus, which codes for serotype l-specific surface protein vp7, into WC3 (reassortant WI79-9) gave it enhanced capacity to induce a type 1 antibody response. In this small study, infants orally immunized with WI79-9 before the rotavirus season exhibited complete protection against rotavirus disease during a year when natural disease was caused by either type

Table 6. Immune response to symptomatic rotavirus infection in placebo recipients.

Infant 4 28 39

46 50 56 68 75

Seropositivity before infection*

Infectious virus serotype

None None None 4 (only) None None None 3 (only)

1 1 3 1 1 I 1 3

PRN serum antibody response to type

+ + + + + + + +

2

3

4

9

+ +

+ + + + + + + +

+

+ + + + + + + +

+ + +

+ + +

+ + +

6

+

+ +

NOTE. PRN = plaque reduction neutralization. Bold type indicates highest-titered response. • Based on PRN antibody titer ~IOO 28 days after the second dose of placebo, except infants 68 and 75 (day 0 serum used because natural infection occurred before sampling after second placebo dose).

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NOTE. Symptomatic infections were confirmed by the presence of rotavirus in stool specimens; asymptomatic by increases in serum antibody at the end of rotavirus

1ID 1990;161 (June)

Serotype 1 Reassortant of Rotavirus WC3

JID 1990;161 (June)

1103

Table 7. Total rotavirus and nonrotavirus gastroenteritis in enrolled infants. WI79-9 Vaccinees (n = 38)

Infants affected Total episodes Episodes of moderate to severe disease Days of Diarrhea Vomiting Fever Illness

Placebo recipients (n = 39)

Rotavirus gastroenteritis

Nonrotavirus gastroenteritis

Total

Rotavirus gastroenteritis

Nonrotavirus gastroenteritis

Total

Protection, total (%)

0 0

15 18

15 18

8 8

11 12

19 20

19 9

0

5

5

6

4

10

49

0 0 0 0

64

64

21 10 69

21 10 69

44 12 17 48

51 16 9 58

95 28 26 106

31* 23 61* 33*

1 or type 3 virus. This protection (100% vs. total rotavirus disease) is slightly superior (76% vs. all rotavirus disease, 100 % vs. moderate to severe disease) to that previously reported for infants immunized with WC3 rotavirus [11]. However, the results are not directly comparable because the infants described in the current study received two doses of WI79-9 and the previously reported trial was done with infants given a single WC3 dose. The protection observed in WI79-9-vaccinated infants against symptomatic rotavirus disease was not associated with a reduction in the overall rate of rotavirus infections in vaccinees as determined by post-rotavirus season serum antibody analysis. This observation is in agreement with previous observations that WC3 rotavirus vaccine protected against clinical expression of rotavirus infection but not infection itself in a trial done in suburban Philadelphia [11]. In this trial, we observed an unusually high (97 %) immune response rate to the serotype 6 (vp4, WC3) immune component of WI79-9 but only a disappointingly low (22 %) immune response rate to the serotype 1 (vp7, WI79) component of this reassortant. This immune response rate to serotype 1 was higher than that previously obtained with WC3. Given the protective efficiency of the vaccine, it is possible that a serotype l-specific immune response may be induced that is not detected by the serum PRN antibody test (such as type-specific cellular responses). The potential for inducing heterotypic immune protection with rotavirus vaccine has been clearly demonstrated in vaccine trials wherein infants were protected against serotype 1 rotavirus disease by serotype 6 rotavirus vaccines of either strain RIT4237 [16] or strain WC3 origin, as well as in certain experimental animal studies (reviewed in [17]). However, immune protection of infants with serotype 3 primate-origin rotavirus vaccine MMU 18006 has been presumed to be serotype-limited in most but not all clinical trials. [7-9]. In addition, in this study we confirmed in a placebocontrolled vaccine trial previous observations from phase I

trials that a reassortant rotavirus WI79-9, containing gene 9 of human type 1 virus and 10 genes of type 6 strain WC3 virus, causes no symptoms in infants 2-11 months old. Such a result was anticipated because the reassortant WI79-9 virus contains the major gene complement from rotavirus strain WC3 (including gene 4, which is strongly associated with control of virulence) [18]. The WC3 strain has now been shown to be nonreactogenic in studies of > 300 infants. Whether the incorporation of human serotype-specific antigens into the WC3 rotavirus virion is useful and necessary for optimal immune protection must be determined by further comparative trials of WC3 rotavirus vaccine and its reassortant derivatives. The present report indicates that the production of a safe rotavirus vaccine that efficiently protects against infant disease is possible. In the suburban American population tested in this study, prevention of rotavirus disease led to significant reduction in the days of gastroenteritis (regardless of cause) in vaccinated infants, suggesting that an effective vaccine will find a high degree of acceptance and will be cost-effective in developed nations.

Acknowledgment We thank the staff and physicians at Drexel Hill Pediatric Associates, Andorra Pediatrics, and Cirotti-Sohn Associates of Willow Grove (all Pennsylvania).

References 1. BartlettAV m,Bednarz-Prashad AJ, DuR>ntHL, PickeringLK. Rotavirus gastroenteritis. Annu Rev Med 1987;38:399-415 2. DeZoysa I, Feachem RG. Interventions for the control of diarrhoeal diseases among young children: rotavirus and cholera immunization. Bull WHO 1985;63:569-583 3. De Mol P, Zissis G, Butzler lP, Mutwewingabo A, Andre FE. Failure of live. attenuated oral rotavirus vaccine [letter]. Lancet 1986;2:108 4. Hanlon P, Marsh V, Shenton F, lobe 0, Hayes R, Whittle HC, Hanlon L, Byass P, Hassan-King M, Sillah H, M'Boge BH, Greenwood BM.

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• P < .05.

1104

5.

6.

7.

8.

10.

11.

Trial of an attenuated bovine rotavirus vaccine (RIT 4237) in Gambian infants. Lancet 1987;1:1342-1345 Losonsky GA, Rennels MB, Kapikian AZ, Midthun K, Ferra PJ, Fortier DN, Hoffman KM, Baig A, Levine MM. Safety, infectivity, transmissibility and immunogenicity of rhesus rotavirus vaccine (MMU 18006) in infants. Pediatr Infect Dis J 1986;5:25-29 Vesikari T, Kapikian AZ, Delem A, Zissis G. A comparative trial of rhesus monkey (RRV-l) and bovine (RIT 4237) oral rotavirus vaccines in young children. J Infect Dis 1986;153:832-839 Christy C, Madore HP, Pichichero ME, Gola C, Pincus P, Vosefski D, Hoshino Y, Kapikian A, Dolin R, Elmwood and Panorama Pediatric Groups. Field trial of rhesus rotavirus vaccine in infants. Pediatr Infect Dis J 1988;7:645-650 Flores J, Gonzalez M, Perez M, Cunto W, Perez-Schael I, Gracia D, Daoud N, Chanock RM, Kapikian AZ. Protection against severe rotavirus diarrhoea by rhesus rotavirus vaccine in Venezuelan infants. Lancet 1987;1:882-884 Gothefors L, Wadell G, Juto P, Taniguchi K, Kapikian AZ, Glass RI. Prolonged efficacy of rhesus rotavirus vaccine in Swedish children. J Infect Dis 1989;159:753-757 Clark HF, Furukawa T, Bell LM, Offit PA, Perrella PA, Plotkin SA. Immune response of infants andchildren to low-passagebovine rotavirus (strain WC3). Am J Dis Child 1986;140:350-356 Clark HF, Borian FE, Bell LM, Modesto K, Gouvea V, Plotkin SA. Protective effect ofWC3 vaccine against rotavirus diarrhea in infants during a predominantly serotype 1 rotavirus season. J Infect Dis 1988;158:570-587

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12. Clark HF, Borian FE, Modesto K, Plotkin SA. Serotype 1 reassortant of bovine rotavirus WC3, Strain WI79-9, induces a polytypic antibody response in infants. Vaccine 1990 (in press) 13. Sanbourg M, Goudeau A, Courant C, Pinon G, Denis E Direct appraisal of latex agglutination testing, a convenient alternative to enzyme immunoassay for the detection of rotavirus in childhood gastroenteritis, by comparison of two immunoassays and two latex kits. J Clin Microbiol 1985;21:622-625 14. Dolan KT, Twist EM, Horton-Slight P, Forrer C, Bell LM, Plotkin SA, Clark HE Epidemiology of rotavirus electropherotypes determined by a simplified diagnostic technique with RNA analysis. J Clin MicrobioI1985;21:753-758 15. Offit PA, Clark HF, Plotkin SA. Response of mice to rotaviruses of bovine or primate origin assessed by radioimmunoassay, radioimmunoprecipitation, and plaque reduction neutralization. Infect Immun 1983;42:293-300 16. Vesikari T, Isolauri E, Delem A, d'Hondt E, Andre FE, Beards GM, Flewett TH. Clinical efficacy of the RIT 4237 live attenuated bovine rotavirus vaccine in infants vaccinated before a rotavirus epidemic. J Pediatr 1985;107:189-194 17. Clark HE Rotavirus vaccines. In: Plotkin SA, Mortimer EA Jr, eds. Vaccines. Philadelphia: W. B. Saunders, 1988:517-525 18. Offit PA, Blavat G, Greenberg HB, Clark HE Molecular basis of rotavirus virulence: role of gene segment 4. J ViroI1986;57:46-49

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9.

Clark et al.