Clinical Studies of a Quadrivalent Rotavirus Vaccine

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1990,

p. 553-558

Vol. 28, No. 3

0095-1137/90/030553-06$02.00/0 Copyright © 1990, American Society for Microbiology

Clinical Studies of a Quadrivalent Rotavirus Vaccine in Venezuelan Infants IRENE PEREZ-SCHAEL,' MARIO BLANCO,' MARIA VILAR,' DORYS GARCIA,' LAURA WHITE,' ROSABEL GONZALEZ,' ALBERT Z. KAPIKIAN,2 AND JORGE FLORES2* Instituto de Biomedicina, Universidad Central de Venezuela and Ministerio de Sanidad Asistencia Social, Caracas, Venezuela,' and Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 208922 y

Received 16 August 1989/Accepted 9 November 1989

Phase I studies of an oral quadrivalent rotavirus vaccine were conducted in 130 Venezuelan infants 10 to 20 weeks of age. The vaccine consists of a mixture of equal amounts of rhesus rotavirus (RRV) vaccine (serotype 3 [VP7]) and each of three human rotavirus-RRV reassortant strains: D x RRV (serotype 1 [VP7]), DS1 RRV (serotype 2 [VP7]), and ST3 x RRV (serotype 4 [VP7]). Three different doses of the quadrivalent vaccine (0.25 x 104, 0.5 x 104, and 104 PFU of each component) were evaluated sequentially for safety and antigenicity in placebo-controlled, double-blind trials. Starting the day after vaccination, the infants were monitored by daily home visits for 7 days. Only minor reactions were observed during this period; these were limited to mild transient febrile episodes which began day 2 or 3 after vaccination and lasted 1 to 2 days in 15 to 30% of the infants. Serological studies demonstrated that 68 to 96% of the infants developed a rotavirus serum immunoglobulin A response following vaccination. However, when tested by plaque reduction neutralization assay against individual human rotavirus serotype 1, 2, 3, or 4, the response rates ranged from 4 to 23% with the low dose, 21 to 33% with the medium dose, and 32 to 58% with the high dose. Most (73 to 79%) infants developed neutralizing antibodies to RRV following administration of each dose schedule. Vaccine virus shedding was analyzed by utilizing tissue culture isolation of virus from stool. All of the infants who received the lower or medium dose and 89% of those fed the high dose shed one or more components of the vaccine. Analyses of rotavirus serotypes isolated from the stool of infants who received the 0.25 x 104-PFU dose revealed that DS1 RRV was the most commonly shed vaccine component, followed by RRV, D x RRV, and ST3 x RRV in that order. X

X

An effective rotavirus vaccine may have a great impact on the survival of hundreds of thousands of infants and young children in developing countries since rotaviruses are the most important etiologic agents of severe dehydrating diarrhea in this age group throughout the world (4). Because of the variable efficacy provided by monovalent rotavirus vaccines against heterotypic strains, as observed during different studies with the RIT 4237 and rhesus rotavirus (RRV) vaccines, it has become apparent that homotypic immunity against each of the four most commonly circulating rotavirus serotypes may be required to induce broad protection. For example, recent studies with the serotype 3 RRV vaccine revealed a significant degree of homotypic protection but lesser or no heterotypic protection (1, 6, 11, 13). The requirement for a polyvalent vaccine has led us to develop and evaluate, clinically, reassortant rotavirus vaccine candidates which have the neutralization specificity of the other three most commonly detected human rotavirus serotypes and the attenuation phenotype of RRV (16, 17). Previous studies in Venezuela have shown that the serotype 3 RRV vaccine as well as the serotype 1 (D x RRV) and 2 (DS1 x RRV) reassortants were safe and immunogenic when administered individually (5, 19) and that the serotype 3 RRV vaccine was effective in providing significant homotypic protection. The purpose of our study was to evaluate in phase I studies the safety and antigenicity of a quadrivalent vaccine composed of equal proportions of RRV vaccine and each of three single gene substitution reassortants in which the gene encoding the major neutralization protein of RRV *

(VP7) is replaced by the corresponding gene of human rotavirus serotype 1, 2, or 4. MATERIALS AND METHODS Population studied. The study was conducted in Caricuao, a low-income area in the south of Caracas, Venezuela. Families were informed of the purpose of the study, the collaboration requested, and the risks and benefits of their participation. Written informed consent was given by the mothers. The study was approved by the Venezuelan Ministry of Health, the Institute of Biomedicine Ethical Committee, and the National Institute of Allergy and Infectious Diseases (National Institutes of Health) Clinical Research Subpanel. Children 10 to 20 weeks of age without a history of debilitating illnesses were recruited from the immunization clinics at the Hospital Materno Infantil de Caricuao, a facility of the Venezuelan Ministry of Health. Study design and vaccine administration. The study was conducted under a double-masked code; a random order was used to assign each child to a vaccine or placebo group. For safety reasons, a "low dose" of quadrivalent vaccine, 0.25 x 104 PFU of each serotype, was administered initially to 27 infants, whereas 23 received a placebo. This represented one-quarter of the dose evaluated previously for the individual components (RRV, D x RRV, DS1 x RRV, and ST3 x RRV). Later, the dose of each vaccine component was doubled (to 0.5 x 104 PFU of each component) and administered to 20 infants, while the same number were given a placebo. Finally, a dose of 104 PFU of each component was administered to 20 infants, and 20 additional infants received placebo. Prior to the oral administration of vaccine or

Corresponding author. 553

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TABLE 1. Clinical reactions to a low, medium, or high dose of quadrivalent rotavirus vaccine in 10- to 20-week-old Venezuelan infants during the week postvaccination No. of infants studied

No. of infants with fever '38.1°C

Low dose (0.25 x 104 PFU) Controls Vaccinees

23 27

0

8b

Medium dose (0.5 x 104 PFU) Controls Vaccinees

20 20

3 4

High dose (104 PFU) Controls Vaccinees

20 20

Study group

Day of fever onset (duration, if >1 day)

No. of infants with:

Liquid stools

Coughing

Rhinorrhea

2, 2, 2 (2); 3, 3, 3 (2); 3 (2); 4

4 3

12 10

4 1

1, 2 (2), 5 1, 1, 3, 5 (2)

1 2

5 5

2 1

0

1 3 6 1 5 4 a Quadrivalent vaccine contained D x RRV (serotype 1), DS1 x RRV (serotype 2), RRV (serotype 3), and ST3 x RRV (serotype 4). b Significantly different from corresponding control group (P < 0.01). The highest temperatures observed in this group of eight infants were 39.7°C in one infant and 39.1°C in another. 3

2, 3, 3 (2)

placebo, each child received 30 ml of formula (Similac; Ross Laboratories) which had been shown to lack rotavirusneutralizing activity; 400 mg of bicarbonate was added to the 30 ml of formula. A few minutes after drinking the buffered formula, two 0.5-ml aliquots containing vaccine or placebo were given to each child. The stock vaccine titers were 106 PFU for D x RRV, RRV, and ST3 x RRV and 104 PFU for DS1 x RRV. The 0.25 x 104-PFU regimen consisted of 0.5 ml of a mixture containing D x RRV, RRV, and ST3 x RRV vaccine stocks at a 1:200 dilution in the same formula and 0.5 ml of DS1 x RRV diluted 1:2 in a colored soft drink. For the 0.5 x 104-PFU dose, 0.5 ml of D x RRV, RRV, and ST3 x RRV vaccine stocks diluted 1:100 in formula and 0.5 ml of DS1 x RRV undiluted were administered. For the "high dose" of 104 PFU of each component, 1 ml of formula containing D x RRV, RRV, and ST3 x RRV vaccine stocks at 1:100 dilution was administered, followed by 1 ml of undiluted DS1 x RRV. Placebo for the low- and mediumdose groups consisted of 0.5 ml of formula followed by 0.5 ml of a colored soft drink; placebo infants for the high-dose group received 1 ml of formula and 1 ml of a colored soft drink. The mothers were asked not to feed their babies for 2 h before and 1 h after vaccination. Follow-up. Participating families were visited daily at home for 7 days by a physician beginning the day after vaccination. Rectal temperatures were obtained twice daily. Stool samples were collected daily for 7 days starting on the day of vaccination. A questionnaire on the health status of the children was completed during each visit. Codes were broken at the National Institutes of Health after completing analysis of the 1-week follow-up observations but were not disclosed to the field team. Laboratory studies. (i) Serology. Blood samples (2 to 3 ml) were obtained just before and 4 to 5 weeks after vaccination by antecubital venous puncture. Seroresponses to the vaccine were analyzed by an immunoglobulin A (IgA) enzymelinked immunosorbent assay (ELISA) (10) or by a plaque reduction neutralization (PRN) assay performed as described previously (10). RRV was used as the antigen in the IgA ELISA; PRN assays were performed with the following viruses: Wa (human serotype 1), DS1 (human serotype 2), P (human serotype 3), ST3 (human serotype 4), and RRV (simian serotype 3). Statistical analyses were done by using the two-tailed Fisher exact test. (il) Detection of rotavirus shedding. Stool specimens obtained on days 3 and 6 postvaccination were initially tested

by a confirmatory ELISA (14). For virus isolation in MA104 cell cultures, 100-p.l aliquots of 5 to 10% stool suspension were treated with trypsin (10 jxg/ml, 1 h at 37°C) and adsorbed for 1 h at 37°C in roller tubes of MA104 cells which were then washed and refed with minimal essential medium. Rotavirus was detected by testing combined cell lysate and supernatant in the confirmatory ELISA. In addition, stool

suspensions from days 3 and 6 after vaccination from the children who received the 0.25 x 104-PFU dose of each vaccine were directly plaqued in MA104 six-well cell culture plates by adsorbing stool suspensions onto the cells for 1 h and covering the monolayer with 0.7% agarose in minimal essential medium after washing the wells with minimal essential medium. Viral plaques were amplified in MA104 cells in roller tubes, and the yield was subjected to a VP7 serotyping ELISA, using monoclonal antibodies (20). RESULTS Reactions. The clinical reactions following administration of the quadrivalent rotavirus vaccine are summarized in Table 1. Overall, 15 of the 67 vaccinees and 3 of the 63 control infants in the three combined groups developed a short-lived febrile syndrome (P < 0.01). Of 27 infants who received the low dose of vaccine, 8 developed fever (.38.10C) 2 to 4 days after vaccination. Six were low grade (s38.5°C) and lasted for 1 day, except one which continued for 2 consecutive days. One vaccinee had a fever of 38.5 to 39.1°C (a 14-week-old infant) and another (12 weeks old) had a fever of 38.2 to 39.7°C; fevers lasted for 2 days in each individual. The latter child also had symptoms of an upper respiratory infection which lasted for 3 days. No other clinical findings were observed in the former infant. None of the controls developed fever. Twelve controls and 10 vaccinees were observed to cough, starting in most cases the day after vaccination (it should be noted that before vaccination several potential participants were rejected from the study for this symptom). Four controls and three vaccinees had liquid stools (one to two per day) lasting 1 day in each of the vaccinees and 1 to 3 days in the controls. In the medium-dose study, in which 20 infants received the 0.5 x 104-PFU dose of each vaccine component and 20 others received placebo, four vaccine and three placebo recipients developed a fever of >38.10C; only two of the controls and none of the vaccinees had fever of >38.50C. Again, no other clinical findings attributable to the vaccine were observed. In

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TABLE 2. Serological responses to a quadrivalent rotavirus vaccine consisting of 0.25 x 10' PFU of each components Response (no. of infants exhibiting a 4-fold or greater response/no. of infants tested) to:

Assay

PRN

Serotype 1

Serotype 2

Serotype 3

Serotype 4

6/26 (23%; Wa)b

3/26 (12%; DSl)

2/26 (8%; P) 19/26 (73%; RRV)

1/26 (4%; ST3)

IgA ELISA

25/26 (96%; RRV)

D x RRV, DS1 x RRV, RRV, and ST3 x RRV. b Percentages and virus used as antigen in each assay are given in

a

parentheses.

the high-dose group, three vaccinees developed mild fever which did not exceed 38.7°C and lasted for 1 or 2 days; three controls and five vaccinees had liquid or semiliquid stools (one or two per day). Serologic responses. Although 73% of the infants who received the low dose of quadrivalent vaccine developed a fourfold or greater rise in neutralizing antibody to RRV, only 4 to 23% of children developed such a response to human rotavirus prototype strain Wa, DS1, P, or ST3 (Table 2). Some 79% of infants fed the medium dose of quadrivalent vaccine developed a response by PRN assay to RRV, whereas 21, 32, 21, and 33% developed such a response to Wa, DS1, P, or ST3 virus, respectively (Table 3). In the high-dose group, 74% of the infants developed a neutralizing antibody response to RRV, while 58, 33, 42, and 32% responded to Wa, DS1, P, or ST3, respectively (Table 4). The responses to Wa, P, and ST3 in this group were significantly greater (P < 0.05, two-tailed Fisher exact test) than the corresponding responses in the infants who received the low dose of vaccine. The responses to Wa in the group of infants receiving the high dose were also significantly greater (P < 0.05) than the equivalent responses in the medium-dose group. A serum IgA response as determined by ELISA was observed in 96% of the infants who received the low dose and 68 and 74% of those who received the medium or the high dose, respectively. Of the 58 infants receiving placebo in the three combined studies, 6 developed an IgA response. Viral shedding. Rotavirus shedding, as determined by direct ELISA of stool, was detected on day 3 or 6 postvaccination in 12 of 23 infants who received the low dose of vaccine, in 6 of 14 who received the medium dose, and in 7 of 17 who received the high dose. When stool specimens were tested for virus in MA104 cell roller tubes, rotaviruses were recovered from stools collected on days 4 to 6 postvaccination from each of the 22 or 17 infants tested who received the low or medium vaccine dose, respectively, and from 16 of 18 (89%) infants tested who received the high dose. In an attempt to determine the VP7 serotype specificity of virus shed, 5 to 10% stool suspensions from the

infants who received the low dose were inoculated directly onto six-well plates of MA104 cells. Plaques were picked and amplified by a single passage in MA104 cells and serotyped with monoclonal antibodies (20). Table 5 shows that DS1 x RRV was isolated from each of 20 infants tested, while RRV, D x RRV, and ST3 x RRV were recovered from 50 to 90% of the vaccinees. The medium- and high-dose recipients were not tested. DISCUSSION The "Jennerian" strategy of using an animal virus to induce resistance to a related human virus has been used in the development and evaluation of three candidate rotavirus vaccines (12): the Smith-Kline-RIT 4237 and Wistar WC3 bovine vaccines and the National Institutes of Health RRV vaccine. The results of clinical trials have been variable. Whereas the RIT 4237 vaccine was highly effective in preventing clinically significant rotavirus diarrhea in infants in Finland (21, 22), it failed to induce protection in studies carried out in two developing countries, Rwanda (3) and The Gambia (8). Although preliminary studies of the bovine WC3 vaccine in infants in Philadelphia, Pa., show promise (2), this vaccine needs to be evaluated in younger infants in developing countries. The RRV vaccine induced a significant level of protection in Venezuelan infants against diarrhea during a period when the homotypic serotype 3 human rotavirus was circulating (6). However, in parallel studies, the vaccine was not effective in Rochester, N.Y., and in Arizona when the predominant circulating rotavirus serotype was not that of the vaccine strain (1, 13; M. Santosham et al., manuscript in preparation). Thus, although the RRV vaccine is able to infect completely susceptible infants efficiently, even in the presence of maternal antibodies, its ability to induce heterotypic human rotavirus antibodies is insufficient to provide adequate protection. Therefore, a more cross-reactive vaccine candidate must be sought or a polyvalent vaccine must be used. The ability of rotaviruses to reassort during coinfection has made it possible to construct human-RRV reassortants

TABLE 3. Serological responses to a quadrivalent rotavirus vaccine consisting of 0.5 x 104 PFU of each component' Assay

PRN

Response (no. of infants exhibiting a 4-fold or greater response/no. of infants tested) to:

Serotype 2 6/19 (32%; DS1)

Serotype 1

4/19 (21%; Wa)b

Serotype 4

4/19 (21%; P)

4/12 (33%)

15/19 (79%; RRV) 13/19 (68%; RRV)

IgA ELISA D x RRV, DS1 x RRV, RRV, and ST3 x RRV. b Percentages and virus used as antigen in each assay

Serotype 3

a

are

shown in

parentheses.

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TABLE 4. Serological responses to a quadrivalent rotavirus vaccine consisting of 104 PFU of each components Response (no. of infants exhibiting a 4-fold or greater response/no. of infants tested) to:

Assay

PRN

Serotype 1

Serotype 2

Serotype 3

Serotype 4

11/19 (58%; Wa)b

6/18 (33%; DS1)

8/19 (42%; P) 14/19 (74%; RRV)

6/19 (32%; ST3)

14/19 (74%; RRV)

IgA ELISA D x RRV, DS1 x RRV, RRV, and ST3 x RRV. b Percentages and virus used as antigen in each assay are shown in parentheses.

a

with the neutralization specificity (VP7) of human serotype 1, 2, or 4. Administered together with the serotype 3 RRV vaccine in the form of a quadrivalent vaccine, such a combination could cover the serotypic spectrum of epidemiologically important rotaviruses. In each of these reassortant candidate vaccines, 10 genes are derived from RRV, while the gene encoding the major neutralizing protein VP7 is derived from the human strain D (serotype 1), DS1 (serotype 2), or ST3 (serotype 4). This gene combination appears to be adequate to maintain the attenuation phenotype of RRV in the resulting reassortants. In the present study, three different doses of quadrivalent vaccine were tested in 10- to 20-week-old infants. Seroresponses were detected in most of them by a rotavirus IgA ELISA, which is not obscured by preexisting maternal antibody since IgA does not cross the placenta. Shedding of one or more components of the candidate vaccine was detected in stool samples obtained 3 to 6 days postvaccination from all but two of the infants tested, suggesting that virus replicated in the intestines. The reactions to the vaccine were minimal. Overall, a self-limited fever (38.1°C TABLE 5. Serotype of rotaviruses isolated from the stools of infants receiving a quadrivalent vaccine containing 0.25 x 104 PFU of each component: D x RRV, DS1 x RRV, RRV, and ST3 x RRV Child no.

5 6 7 9

il 16 17 19 21 25 26 29 31 32 34 35 40 42 43 46 No. of children shedding virus/no. of children tested

No. of rotavirus plaques typed

1

2

3

4

12 24 7 45 35 29 30 29 35 20 16 27 29 15 3 14 12 28 12 19

0 il 2 5 2 0 1 3 2 2 0 4 0 2 1 6 0 6 0 2

5 5 3 25 14 29 10 14 12 il 13 22 8 4 1 5 1 7 1 14

7 8 2 12 14 0 19 12 20 6 3 0 18 7 1 1 9 15 9 3

0 0 0 2 1 0 0 0 1 1 0 1 3 2 0 2 2 0 2 0

14/20

20/20

18/20

10/20

No. of plaques with given

serotype specificity

or greater) developed in 15 of 67 infants receiving any dose of vaccine and in 3 of 63 controls. Two vaccinees and two control infants had temperature rises >38.5°C. No differences in the occurrence of gastrointestinal or respiratory symptoms were observed between the vaccine and placebo groups. The neutralizing antibody response to each component of the low-dose quadrivalent vaccine as measured by PRN assay was disappointing (only 4 to 23% of the vaccinees developed a seroresponse to human rotavirus serotype 1, 2, 3, or 4). However, most children responded to RRV (an animal serotype 3 virus strain), strongly suggesting that (i) such neutralizing responses were mediated through antibodies directed to the VP4 rotavirus protein, which in each of the four viruses was derived from RRV; and (ii) the VP4 protein is more immunogenic than the VP7 protein. Previous studies with the individual RRV vaccine reassortants at a dose of 104 PFU or with a combination of RRV and one reassortant (D x RRV) at a dose of 0.5 x 104 PFU of each component (5) indicated that the reassortants were able to induce a neutralizing antibody response with reasonable frequency. To be on the safe side, a dose of 0.25 x 104 PFU of each vaccine component was used initially in order not to exceed the 104-PFU dose used previously for the monovalent and bivalent vaccines. In an attempt to increase the frequency of antibody responses to VP7, and in view of the low reactions to the 0.25 x 104-PFU dose, a second group of children was given a 0.5 x 104-PFU dose of each component. Again, neutralizing antibody responses to the RRV (most likely through VP4) were observed in the majority of these infants, but this time it was also possible to detect VP7 responses by PRN assay against human serotypes 1, 2, and 3 in 21 to 33% of the infants. Since this level was not considered sufficient to justify a field trial, a third group of infants was tested with a full dose of 104 PFU of each vaccine component. The responses to Wa with this dose were significantly greater than those observed with the medium and low doses. In addition, the responses to human serotypes 3 (P) and 4 (ST3) were also significantly greater in the high-dose group than in the group of infants who received the low dose of vaccine; the responses to serotype 2 (DS1) were also greater but not statistically significant. Even after this increase in dose the response to serotype 2 was lower than that observed when this virus was administered individually in previous studies (5). This suggested that interference by one or more of the other components took place, since response to the other serotypes was comparable to that of infants who received a monovalent formulation of D x RRV or RRV. The basis for this possible interference is not understood at this time. Additional adjustments in the total or the relative amounts of the different components of the quadrivalent vaccine may be required to induce satisfactory neutralizing seroresponses to

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all VP7 components. Our minimal goal is to achieve a seroresponse rate of about 50% against each serotype component. However, it is possible that protection might be afforded without necessarily inducing detectable antibody responses if rotavirus vaccines behave similarly to poliovirus vaccine, in that protection may be induced in the absence of a detectable neutralizing response, and if a better correlation exists between vaccine virus shedding and induction of resistance (7). Analysis of shedding of the different viruses of the quadrivalent vaccine by infants who received the low dose suggested that each of the four strains infected most children (Table 5) at least at the low dose tested and likely (although not evaluated) at the higher doses. Of interest, the serotype 2 reassortant was shed more often than the others, and yet response to VP7 of that virus was quite low (12%) despite the finding that preexisting antibody levels against it were low or undetectable in most infants. Serotype 3 RRV was shed by a majority of the infants, and yet a response to its VP7 was only seen in 2 of 25 infants tested. Caution should be exercised when interpreting these shedding differences since in vitro studies of competition among the four components of the quadrivalent vaccine have not been carried out and it is possible that the efficiency of detection may reflect differences among the four viruses in their ability to grow in vitro rather than in vivo. The significance of neutralizing antibody responses to VP4 is of interest in view of the observation that response to this viral component occurred in most infants. Studies of monoclonal antibodies and analysis of cross-reactivity among rotavirus reassortants and their parental viruses have led to the detection of neutralization antigenic sites on VP4 (9, 18). It can be deduced from RRV vaccine trials in which protection was not observed that the VP4 as well as the VP7 of circulating rotaviruses were antigenically different from those of RRV. Antibodies directed to VP4 appear to play only a minor role in the neutralizing antibody responses detected in serum after parenteral immunization of animals. Such sera have been used for the classification of rotaviruses into different serotypes (10), and therefore the current classification is based primarily on VP7 specificities. The serotype spectrum of the VP4 component of human rotaviruses has not been thoroughly studied in view of its low antigenicity when the virus is administered parenterally in experimental animals. It is possible that neutralization epitopes on VP4 may induce cross-reactive immunity across different rotavirus serotypes (VP7 serotypes) after natural or experimental intestinal infection. Analysis of serological responses to natural rotavirus infection in young infants may be helpful in predicting which VP4 component of a potential vaccine candidate will induce significant cross-reactivity with human strains. Meanwhile, the concept of a quadrivalent rotavirus vaccine appears to be a promising strategy for immunoprophylaxis of severe rotavirus diarrhea caused by the four epidemiologically important serotypes. Perhaps a two-dose schedule of the current high-dose quadrivalent vaccine formulation or a further increase in the dose of certain components (for single-dose administration) is necessary to achieve maximum protection. Multidose schedules of the high-dose quadrivalent vaccine as well as adjustments to the dose of individual components (as has been necessary for oral poliovirus vaccine) are being considered. However, because of the practical difficulties in administering multidose vaccines to infants in the developing areas of the world, our preference is the administration of a safe, antigenic, effective

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quadrivalent vaccine in a single dose in the first few months of life. ACKNOWLEDGMENTS We acknowledge the excellent technical support provided by Anne Pittman, Harvey James, Johnna Sears, Cidalia Urbina, and Jordi Boher. This study was partially supported by the Agency for International Development Vaccine Program through a PASA agreement with the National Institute of Allergy and infectious Diseases. LITERATURE CITED 1. Christy, C., H. P. Madore, M. E. Pichichero, C. Gala, P. Pancus, D. Vosefski, Y. Hoshino, A. Z. Kapikian, R. Dolin and Elmwood and Panorama Pediatric Groups. 1988. Field trial of Rhesus Rotavirus Vaccine in infants. Pediatr. Infect. Dis. J. 7:645-650. 2. Clark, H. F., F. E. Borian, L. M. Bell, K. Modesto, V. Gouvea, and S. Plotkin. 1988. Protective effect of WC3 vaccine against rotavirus diarrhea in infants during a predominantly serotype 1 rotavirus season. J. Infect. Dis. 158:570-587. 3. De Mol, P., G. Zissis, J. P. Butzler, A. Mutwewingabo, and F. E. Andre. 1986. Failure of live, attenuated oral rotavirus vaccine. Lancet ii:108. 4. De Zoysa, I., and R. G. Feachem. 1986. Interventions for the control of diarrhoeal diseases among young children: rotavirus and cholera immunization. Bull. W.H.O. 63:569-583. 5. Flores, J., I. Perez-Schael, M. Blanco, M. Vilar, D. Garcia, M. Perez, N. Daoud, K. Midthun, and A. Z. Kapikian. 1989. Reactions to and antigenicity of two human-rhesus rotavirus reassortant vaccine candidates of serotypes 1 and 2 in Venezuelan infants. J. Clin. Microbiol. 27:512-518. 6. Flores, J., I. Perez-Schael, M. Gonzalez, D. Garcia, M. Perez, N. Daoud, W. Cunto, and A. Z. Kapikian. 1987. Protection against rotavirus diarrhoea by rhesus rotavirus vaccine in Venezuelan children. Lancet i:882-884. 7. Halsey, N., and A. Galazka. 1985. The efficacy of DPT and oral poliomyelitis immunization schedules initiated from birth to 12 weeks ofage. Bull. W.H.O. 63:1151-1169. 8. Hanlon, P., L. Hanlon, W. Marsh, P. Byass, F. Shenton, M. Hassan-King, O. Hobe, H. Sillah, R. Hayes, B. H. M. Boge, H. C. Whittle, and B. M. Greenwood. 1987. Trial of an attenuated bovine rotavirus vaccine (RIT 4237) in Gambian infants. Lancet

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