SUPPLEMENT ARTICLE. of Africa and Asia. The highest rates of rotavirus-associated mortality occur in sub-saharan Africa, where

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1 SUPPLEMENT ARTICLE Comparison of 2 Different Regimens for Reactogenicity, Safety, and Immunogenicity of the Live Attenuated Oral Rotavirus Vaccine RIX4414 Coadministered with Oral Polio Vaccine in South African Infants A. D. Steele, 1,a J. Reynders, 1 F. Scholtz, 1 P. Bos, 1 M. C. de Beer, 1 J. Tumbo, 2 C. F. Van der Merwe, 3 A. Delem, 4 and B. De Vos 4,a 1 MRC Diarrhoeal Pathogens Research Unit and Departments of 2 Family Medicine and 3 Gastroenterology, Medunsa Campus, University of Limpopo, Pretoria, Republic of South Africa; and 4 GlaxoSmithKline Biologicals, Rixensart, Belgium Background. A phase II, randomized, double-blind, placebo-controlled study was conducted in South Africa during to evaluate the safety, reactogenicity, and immunogenicity of 2 regimens of the live attenuated oral human rotavirus vaccine RIX4414 when coadministered with the Expanded Program on Immunization childhood vaccines, including oral polio vaccine. Methods. Healthy infants were randomized (2:2:1) to receive either 2 doses of RIX4414 ( n p 190; at 10 and 14 weeks, with placebo at 6 weeks), 3 doses of RIX4414 ( n p 189; at 6, 10, and 14 weeks), or 3 doses of placebo ( n p 96), all with concomitant routine vaccinations. The antirotavirus IgA seroconversion rate was assessed using enzyme-linked immunosorbent assay at 2 months after the last dose of RIX4414 or placebo. Antipolio types 1, 2, and 3 antibodies were measured using a virus neutralization assay. Solicited symptoms were recorded for 15 days after each dose. Results. The antirotavirus IgA seroconversion rates were similar in the RIX and 3-dose groups (44.3% and 44.4%, respectively; P p.544, by 1-sided Fisher exact test) and antirotavirus IgA geometric mean concentrations were also comparable. Seroprotection rates for antipolio types 1, 2, and 3 antibodies were high (93% 100%) and were not significantly different among groups. Solicited symptoms reported within 15 days after vaccination were similar in all groups. Conclusions. The immune seroconversion response to the RIX4414 vaccine with 3 doses was not superior to the 2-dose regimen. There was no interference by either regimen with antibody response to oral polio vaccine, and RIX4414 was well tolerated when given with routine vaccinations. Severe gastroenteritis due to rotavirus causes 1520,000 deaths among children!5 years of age annually [1, 2]. These deaths are disproportionately distributed globally, with 185% occurring in the developing countries of Africa and Asia. The highest rates of rotavirus-associated mortality occur in sub-saharan Africa, where Financial support: World Health Organization (V27/181/141), the Norwegian Programme for Development, Research and Higher Education (PRO 48/2002), and the South African Medical Research Council. Reprints or correspondence: Dr A. Duncan Steele, Vaccines and Immunization, PATH, 1455 Leary Way NW, Seattle, WA (dsteele@path.org). The Journal of Infectious Diseases 2010; 202(S1):S93 S by the Infectious Diseases Society of America. All rights reserved /2010/20205S1-0013$15.00 DOI: / of 7 countries with rates of 1300 deaths per 100,000 population are located [3]. The World Health Organization (WHO) has recommended the need for im- Potential conflicts of interest: A.D.S. has received honoraria and travel grants from GlaxoSmithKline. A.D. is an employee of GlaxoSmithKline Biologicals. B.D.V. was an employee of GlaxoSmithKline Biologicals at the time of design, conduct, completion, and reporting of the study. All other authors: none reported. Supplement sponsorship: This article is part of a supplement entitled Rotavirus Infection in Africa: Epidemiology, Burden of Disease, and Strain Diversity, which was prepared as a project of the Rotavirus Vaccine Program, a partnership among PATH, the World Health Organization, and the US Centers for Disease Control and Prevention, and was funded in full or in part by the GAVI Alliance. Presented in part: World Society for Paediatric Infectious Diseases, 1-4 September 2005, Warsaw, Poland; Asian Conference on Diarrhoeal Diseases and Nutrition, 8-10 March 2006, Bangkok, Thailand; Double Stranded RNA Virus Symposium, October 2006, Cape Town, South Africa. a Present affiliations: Vaccines and Immunization, PATH, Seattle, Washington (A.D.S.), and Sanofi Pasteur, Lyon, France (B.D.V.). Live Attenuated Oral Human Rotavirus Vaccine JID 2010:202 (Suppl 1) S93

2 munogenicity and efficacy studies with rotavirus vaccines in the developing countries of Africa and Asia [4] and has prioritized the need for rotavirus vaccines to combat this deadly disease [2, 4]. Two rotavirus vaccines are now available and are being used in the routine immunization schedules in many countries, particularly in Latin America [5], and in several industrialized countries. The vaccines have also been licensed in 1100 countries worldwide and are used in the private sector in many of these. Clinical studies with these 2 vaccines are ongoing in Africa and Asia under the coordination and funding from the WHO and the GAVI Alliance funded rotavirus vaccine program PATH [6]. These studies are designed to investigate the clinical efficacy of the vaccines in infant populations in these 2 regions, as recommended by the WHO [7]. The results from these studies were published in 2010 and will inform global decision-making about the future introduction of these vaccines in developing countries, where the need for rotavirus immunization is highest. This study was conducted under the WHO Rotavirus Action Partnership for Immunization and Development (RAPID), which facilitated the conduct of rotavirus vaccine trials in developing countries, specifically in Africa and Asia, and investigated questions specific to populations in developing countries. RAPID consisted of public sector partners (including the WHO, US Agency for International Development, National Institutes of Health, PATH, and the US Centers for Disease Control and Prevention), academic institutions (International Centre for Diarrhoeal Disease Research, Bangladesh, and Medical University of Southern Africa), and GlaxoSmithKline Biologicals. This was the second immunogenicity study conducted in South Africa and complements the previous study and those in Bangladesh [8, 9] that examined the coadministration of oral polio vaccine (OPV) with the human monovalent rotavirus vaccine (Rotarix; GlaxoSmithKline Biologicals). The immune response to live oral rotavirus vaccines differs between infants in developed and developing countries. The immune seroconversion response observed in the previous rotavirus vaccine study in South Africa was lower than that seen in studies in Latin America [10 12]. In addition, early rotavirus vaccine trials in Africa, with alternative rotavirus vaccine candidates, demonstrated poor or no efficacy [13 15]. These factors resulted in the need to evaluate the immunogenicity of the new live oral rotavirus vaccines in the African setting. In South Africa, rotavirus infection has been shown to be associated with approximately one-third of all hospital admissions for diarrhea among children!2 years of age [16]. In addition, diarrheal disease is associated with relatively high mortality rates (16% among infants!1 year of age and 20% among children aged 1 4 years) [17]. Vaccination is the only control measure likely to have a significant impact on the rotavirus disease burden worldwide, when introduced as a universal childhood vaccine. A previous randomized, double-blind, placebo-controlled study in South Africa [8] demonstrated that 2 doses of RIX4414 can be given concomitantly with routine Expanded Program on Immunization (EPI) vaccines, including OPV. The aim of the present study was to evaluate the immunogenicity, reactogenicity, and safety of 2 regimens (2 vs 3 doses) of RIX4414 vaccine administered together with routine EPI vaccines (including OPV) in a randomized, double-blind, placebo-controlled trial. The rotavirus vaccination regimens used in this study were chosen on the basis of the results of the earlier South African study, which indicated that vaccination at 10 and 14 weeks of age tended to produce a better immune response than vaccination at 6 and 10 weeks of age [8] and that many children in developing countries have their EPI vaccinations later than the recommended schedule [18]. METHODS Study participants. Healthy full-term male or female infants aged 5 10 weeks at the time of their visit for first vaccination and whose mothers had confirmed negative HIV status were enrolled at 7 clinics in South Africa from 5 September 2003 through 27 February Written informed consent was obtained from parents or guardians before study entry. Infants were excluded if they had previous rotavirus gastroenteritis or chronic gastrointestinal disease or malformation; had received immunoglobulins, blood products, vaccines (except bacille Calmette-Guérin, hepatitis B vaccine, and OPV vaccination at birth), immunosuppressants or immune-modifying drugs (except topical steroids), or antibiotics; had a history of/or intercurrent polio disease; of had allergies or any immunosuppressive or immunodeficient conditions. In addition, patients with acute disease at the time of enrollment or gastroenteritis (diarrhea) within 7 days before administration of the study vaccine were also excluded. Vaccinations were postponed if an infant had an axillary temperature 37.5 C or a rectal temperature 38 C or gastroenteritis within 7 days before the planned vaccination. There were no restrictions on feeding of infants before or after vaccination. Study design. This was a phase II, double-blind, randomized study (etrack /013/NCT ) with 3 parallel groups with a treatment allocation of 2:2:1 and 5 study visits: day 0 (dose 1), month 1 (dose 2), month 2 (dose 3), month 4 (2 months after the last dose of RIX4414 or placebo), and 6 months after the last dose of RIX4414 or placebo for safety. Group RIX4414_2D ( n p 190) received 1 dose of placebo at 6 weeks of age and 2 doses of vaccine at 10 and 14 weeks of age; group RIX4414_3D ( n p 189) received 3 doses of vaccine, S94 JID 2010:202 (Suppl 1) Steele et al

3 Table 1. Seroconversion Rates at 1 Month after the First Dose of Vaccine (RIX4414_3D) or Placebo (RIX4414_2D) and 2 Months after the Last Dose of Vaccine or Placebo and Geometric Mean Concentrations (GMCs) for Antirotavirus IgA Antibodies (Accordingto-Protocol Cohort for Immunogenicity) Group 1 Month after dose 1 2 Months after last dose No. of infants with seroconversion / total infants Seroconversion rate, a % (95% CI) No. of infants with seroconversion / total infants Seroconversion rate, a % (95% CI) GMC 2 months after last dose, b U/mL (95% CI) 2 Doses of RIX4414 c 3/ ( ) 58/ ( ) 29.3 ( ) 3 Doses of RIX / ( ) 59/ ( ) 30.7 ( ) Placebo 0/ ( ) 1/ ( )!20 NOTE. CI, confidence interval. a Percentage of infants initially negative for rotavirus with appearance of antirotavirus IgA antibody concentration 20 U/ml. b In all infants with available results. c Infants in this group received a placebo at 6 weeks as dose 1, RIX4414 as dose 2 at 10 weeks, and RIX4414 as last dose at 14 weeks. at 6, 10, and 14 weeks of age. The placebo group ( n p 96) received 3 doses of placebo, at 6, 10, and 14 weeks of age. Infants received routine vaccinations according to the local EPI schedule in South Africa. Bacille Calmette-Guérin and OPV vaccinations were given at birth; all other routine vaccinations (including diphtheria-tetanus toxoids-whole cell pertussis, hepatitis B, Haemophilus influenzae type b, and OPV) were administered concomitantly with the study vaccine. All of the infants received a dose of OPV concomitantly with each dose of study vaccine or placebo at all administration times. From the first 175 infants (a planned subset for stool analysis), stool samples were collected on the days of dose 1, 2, and 3 and on day 7 1 after each vaccine dose, to evaluate rotavirus antigen excretion. Study vaccines. The oral live attenuated human rotavirus vaccine RIX4414 was developed from the parent vaccine strain, which was cloned and passaged on Vero cells [19]. The viral concentration of 1 dose of RIX4414 vaccine contained at least median cell culture infective dose, and the lyophilised vaccine was reconstituted with calcium carbonate as buffer. The placebo composition was the same formulation without the viral antigen. This vaccine is commercially available today as Rotarix (GlaxoSmithKline). Laboratory assays. Blood samples (3 ml) were collected from all infants before dose 1 (at 6 weeks of age) for the determination of baseline antibody levels, at 1 month after dose 1 (at 10 weeks of age), and at 2 months after the last dose of vaccine (at 20 weeks of age). Antirotavirus IgA antibody concentration in serum was measured using an enzyme-linked immunosorbent assay (ELISA; cutoff, 20 U/mL) based on the test designed by Ward [20, 21] and adapted by GSK Biologicals. Antipolio virus types 1, 2, and 3 antibody titers in serum samples collected 2 months after dose 3 were determined in Rixensart, Belgium, with use of a virus microneutralization test adapted from the WHO Guidelines for WHO/EPI Collaborative Studies on Poliomyelitis [22]. Stool samples were collected from a subset of patients on the day of or 1 day before each study vaccine dose and on day 7 1 after each dose and were analyzed at the laboratory of Dr. R. Ward, Children s Hospital Medical Centre (Cincinnati, OH), to detect the presence of rotavirus with use of ELISA [20] to assess rotavirus antigen excretion. In addition, stool samples collected during each episode of diarrhea that occurred from the first dose until 2 months after dose 3 were tested using ELISA to confirm the presence of rotavirus; if results were positive, the samples were tested by reverse-transcriptase poly- Table 2. Percentage of Infants with Rotavirus in Stool Samples Collected at Predetermined Times and Cumulative Results for All Times in the Stool Subset (According-to-Protocol Cohort for Immunogenicity) Group No. of infants with rotavirus detected in 1 stool sample/no. of infants tested Overall shedding Percentage of infants with rotavirus detected in 1 stool sample (95% CI) 2 Doses of RIX / ( ) 3 Doses of RIX / ( ) Placebo 0/ ( ) NOTE. CI, confidence interval. Live Attenuated Oral Human Rotavirus Vaccine JID 2010:202 (Suppl 1) S95

4 Figure 1. Trial profile and patient disposition merase chain reaction at GSK Biologicals (Rixensart, Belgium) to determine the VP7 and VP4 genotype. If any G1 type of rotavirus was detected, the vaccine virus was differentiated from the wild-type serotype with use of VP7 gene sequence analysis [23, 24]. Immunogenicity. The blood samples were analyzed using ELISA at GSK Biologicals with an assay cutoff of 20 U/mL. The primary outcome measure was the proportion of infantsin the vaccine groups who experienced seroconversion (antirotavirus serum IgA level, 20 U/mL) at 2 months after the last dose of RIX4414 or placebo. A secondary immunogenicity end point was the serum antirotavirus IgA antibody concentrations in all infants 1 month after dose 1 (day of dose 2) and 2 months after the last dose S96 JID 2010:202 (Suppl 1) Steele et al

5 Table 3. Seroprotection Rates and Geometric Mean Titers (GMTs) for Antipolio 1, 2, and 3 at 2 Months after Dose 3 (Accoring-to-Protocol Cohort for Immunogenicity) Antipolio antibody, vaccine group No. of infants with titer 8/no. of infants with available results Seroprotection rate, % (95% CI) GMT (95% CI) 1 2 Doses of RIX / ( ) ( ) 3 Doses of RIX / ( ) ( ) Placebo 52/ ( ) ( ) 2 2 Doses of RIX / ( ) ( ) 3 Doses of RIX / ( ) ( ) Placebo 55/ ( ) ( ) 3 2 Doses of RIX / ( ) ( ) 3 Doses of RIX / ( ) ( ) Placebo 54/ ( ) ( ) NOTE. CI, confidence interval. of RIX4414 or placebo. Secondary immunogenicity end points also included the proportion of all infants with antipolio type 1, 2, and 3 antibody titer 8 at 2 months after dose 3 and antibody titers for antipolio types 1,2 and 3 at 2 months after dose 3 in the RIX4414 and placebo groups. Other secondary outcomes included rotavirus antigen excretion and vaccine uptake after dose 1 and after the last dose in a subset of infants and the presence of rotavirus in diarrheal stool samples collected until 2 months after dose 3. Seroconversion was defined as an antirotavirus IgA antibody concentration 20 U/mL in infants who were initially seronegative at 6 weeks of age. Vaccine uptake was defined as seroconversion and/or rotavirus antigen excretion in any stool specimen collected between dose 1 and 2 months after the last dose of RIX4414 or placebo. Vaccine uptake was calculated only for infants in the stool analysis subset. Reactogenicity and safety. Infants were monitored for at least 30 min after each vaccination. Parents received a diary card to record information daily about solicited general symptoms (eg, fever, diarrhea, vomiting, fussiness or irritability, loss of appetite, and cough and/or runny nose) for 15 days after each dose of RIX4414 or placebo and any other adverse events occurring until the next study visit. Weekly supervision was conducted by health care workers, and the study physician or his staff questioned the parents on their child s health and verified the completed diary card at each visit. Infants who experienced a serious adverse event and required hospitalization were admitted at the local district hospital in the study sites or at Ga-Rankuwa Hospital (Pretoria), the referral hospital for the study site and surrounding areas. Any suspected cases of intussusception were immediately referred to Ga-Rankuwa Hospital. All serious adverse events were reported to the sponsor and the ethics committees and were followed up until resolution. Hospital records and autopsy reports were obtained as applicable, and a standard verbal autopsy questionnaire was to be completed whenever possible for all deaths. Parents were followed up for safety on a weekly basis for 6 months after the last dose of RIX4414 or placebo to obtain information on any serious adverse events since the final study visit. All serious adverse events were reviewed periodically by an independent safety monitoring committee. Statistical analyses. Data analysis was performed at GSK Biologicals with use of SAS software (SAS) and Proc-StatXact 5 on Windows NT. The primary immunogenicity analysis was performed for the according-to-protocol immunogenicity cohort. The according-to-protocol cohort included all infants enrolled in the study who met the criteria defined in the protocol for the considered analysis (safety or immunogenicity). All infants who received at least 1 dose of RIX4414 or placebo (total vaccinated cohort) were included in the primary analysis of reactogenicity. The target enrollment of 475 infants was estimated to provide 80% power to detect a 15% increase in antirotavirus IgA antibody seroconversion at 2 months after the last dose of study vaccine in the RIX4414_3D group, compared with the RIX4414_2D group, with use of a 1-sided Fisher exact test (significant level of a, 0.05) and assuming a 45% seroconversion rate in the RIX4414_2D group [8]. Seroconversion rates and antibody geometric mean concentrations for antirotavirus IgA antibodies were calculated with exact 95% confidence intervals (CIs). Antigen excretion at combined times in the stool analysis subset, seroprotection rates, and geometric mean titers for antipoliovirus types 1, 2, and 3 antibodies at 2 months after dose 3 were calculated with 95% CIs. The percentage of doses and of infants with specific solicited symptoms reported during the Live Attenuated Oral Human Rotavirus Vaccine JID 2010:202 (Suppl 1) S97

6 Figure 2. Percentage of infants for whom solicited general symptoms were reported during the 15-day follow-up period after any dose (total vaccinated cohort for safety). 15-day follow-up period and the unsolicited symptoms reported during the 43-day follow-up period after any dose were tabulated with their 95% CIs. The number of serious adverse events occurring during the study up to 6 months after the last dose of vaccine or placebo was reported. RESULTS Study population. Four hundred seventy-five infants were randomized to receive either 1 dose of placebo and 2 doses of vaccine (RIX4414_2D; n p 190), 3 doses of vaccine (RIX4414_3D; n p 189), or 3 doses of placebo ( n p 96). The clinical phase of the study commenced on 5 September 2003 and was completed on 25 October The study groups were similar with respect to age, height, weight, sex, and ethnicity at first dose. The mean age in the according-to-protocol cohort was 6.3 weeks at the time of dose 1, 10.2 weeks at the time of dose 2, and 14.3 weeks at the time of dose 3. The majority of infants (80.3%) in the study population were of African ethnicity. All infants received their EPI scheduled OPV doses concomitantly with the study vaccine. Of the 475 enrolled infants, 420 (88.4%) completed the study and 55 dropped out (Figure 1). Response to rotavirus vaccination. Two months after the last dose of RIX4414 or placebo, the antirotavirus IgA seroconversion rates were similar in the RIX4414_2D and RIX4414_3D groups (44.3% and 44.4%, respectively) (Table 1). In addition, the antirotavirus IgA geometric mean concentrations after the last dose of RIX4414 or placebo calculated in all infants were similar in the 2 vaccine groups (Table 1). One month after the first dose of RIX4414 or placebo received at 6 weeks of age, the seroconversion rate was 19% in the RIX4414_3D group (first dose was RIX4414) and 2.1% in the RIX4414_2D group (first dose was a placebo); no seroconversion was observed in the placebo group. Rotavirus antigen excretion. Rotavirus antigen excretion in the stool analysis subset ( n p 175) was predominantly ob- served on day 7 after the first dose of RIX4414 vaccine (35% in the RIX4414_2D group [ n p 48] and 13% in the RIX4414_3D group [ n p 61]). The cumulative rate of rotavirus antigen excretion at all times was higher in the RIX4414_2D vaccine group (46%; n p 48) than in the RIX4414_3D vaccine group (31%; n p 61) (Table 2). Rotavirus antigen was not de- tected in stool samples from the placebo group, which suggests that wild-type rotavirus infection was infrequent in the population (Table 2). One case of G1 wild-type rotavirus was detected in a rotavirus-positive stool sample from an infant in the RIX4414_2D vaccine group. In addition, 1 infant in the RIX4414_3D vaccine group whose stool samples had previously been negative for rotavirus had a rotavirus-positive stool sample at the day of dose 3 administration of RIX4414; the strain was a G1 vaccine strain. Vaccine uptake. Vaccine uptake of combined doses 1, 2, and 3 (stool analysis subset; according-to-protocol cohort for immunogenicity) was similar in the vaccine groups (59.1% [95% CI, 43.3% 73.7%] in the RIX4414_2D group [ n p 44] and 55.6% [95% CI, 41.4% 69.1%] in the RIX4414_3D group [ n p 54]). Response to polio vaccination. No statistically significant differences in the response to OPV in terms of antipoliovirus types 1, 2, and 3 seroprotection rates or geometric mean titers of polio antigens 2 months after the last dose were detected between the placebo group and each RIX4414 vaccine group (Table 3). Postvaccination reactogenicity and safety up to 6 months after the last dose. Data on solicited symptoms were available for all 475 patients (100%) in the total vaccinated cohort. Coadministration of RIX4414 with OPV was not associated with an increase in adverse effects, compared with placebo, and the incidence of symptoms did not increase with successive doses. With respect to the incidence rates of solicited symptoms reported within 15 days after vaccination, including those rated as grade 3, a statistically significant difference was detected between the RIX4414_2D vaccine group and the placebo group for the percentage of infants reporting any cough and/or runny nose, any diarrhea, and any fever. For the other solicited symptoms, no other statistically significant differences were detected among all groups (Figure 2). Moreover, the incidences of any unsolicited symptoms within 43 days after vaccination, including those rated as grade 3, were 70% (95% CI, 62.9% 76.4%) in the 2-dose group, 54% (95% CI, 46.6% 61.2%) in the 3- S98 JID 2010:202 (Suppl 1) Steele et al

7 dose group, and 59% (95% CI, 48.9% 69.3%) in the placebo group. In total, 24 (5.1%) of the 475 infants (10 [5.3%] in the RIX4414_2D group, 9 [4.8%] in the RIX4414_3D group, and 5 [5.2%] in the placebo group) reported 40 serious adverse events. Only one case, gastroenteritis in an infant in the placebo group who recovered without sequelae and whose stool sample collected during the severe adverse event was negative for rotavirus, was determined by the investigator as causally related to study vaccination. A total of 3 deaths occurred during the study; all occurred 142 days after the last dose of study vaccine and were judged to be unrelated to vaccination. One fatal case of gastroenteritis combined with bronchopneumonia was recorded in the RIX4414_2D group 46 days after the last dose of RIX4414; no stool sample was collected, and therefore, the etiology of the diarrhea was unknown. Another infant in the 2-dose group died of bronchopneumonia 124 days after the last dose of RIX4414. One fatal case of gastroenteritis and bronchopneumonia was recorded in the RIX4414_3D group 77 days after the last dose of RIX4414; the stool sample tested locally was found to be negative for rotavirus. No cases of intussusception were reported during the study. DISCUSSION Overall, this study showed that 2 doses of RIX4414 administered at 10 and 14 weeks of age provided antirotavirus IgA seroconversion rates and rotavirus vaccine uptake similar to those of 3 doses of RIX4414 given at 6, 10, and 14 weeks of age. Seroconversion rates were similar to results obtained previously with 2 doses in the same population in South Africa [8] but slightly lower than results observed in Latin American infants. In Latin America, the schedule for childhood immunizations is 2, 4, and 6 months of age (ie, 8 and 16 weeks for the 2-dose rotavirus vaccine); 55% 74% of infants experienced seroconversion after 2 doses of RIX4414, depending on the viral concentration [10, 11]. The point estimate of efficacy against severe rotavirus gastroenteritis was demonstrated to be higher than the percentage of seroconversion in these studies. In the Latin America study, OPV was administered 2 weeks apart from human rotavirus vaccine. Several factors may explain the lower seroconversion rates observed in this study, such as immature gut or immune system [25], concurrent infection with other enteric pathogens [26], or the presence of rotavirus-specific maternal antibodies. A possibility is the concomitant administration with OPV. The seroconversion rates in the 2 groups 1 month after the first dose of placebo or RIX4414 administered at 6 weeks concomitantly with OPV was low (only 2.1% in RIX4414_2D group in which the first dose was a placebo and, thus, anticipated to be low, and 19.1% in the RIX4414_3D group). This latter seroconversion rate after a single dose with OPV at 6 weeks of age was similar to the response rate (13.1%) observed in the previous study conducted in South Africa in which OPV was also coadministered [8]. In this earlier study, in which RIX4414 was administered either concomitantly with OPV or with inactivated polio vaccine, it was shown that OPV interfered with the first-dose RIX4414 response, although this low immune response to RIX4414 was overcome by the second vaccination with RIX4414 and OPV. This phenomenon of OPV interference with the rotavirus vaccine response at the first dose has been observed with other rotavirus vaccine candidates [27 29]. Seroprotection rates and geometric mean titers for the antipolio types 1, 2, and 3 antibodies were high and similar in all groups, which indicated that the immune response to OPV was not impaired by coadministration with RIX4414 vaccine. These findings were consistent with previous studies of RIX4414 [8, 9] and animal-based rotavirus vaccines [27 29]. In the placebo group, wild-type rotavirus was not detected at any of the predetermined times, confirming low circulation of rotavirus during the study period. The cumulative rate of rotavirus vaccine shedding after all doses of the vaccine at all times ranged from 31% in the RIX4414_3D group to 46% in the RIX4414_2D group. Rotavirus excretion was observed to be higher on day 7 after dose 2 in the RIX4414_2D group (ie, the first dose of RIX4414) than on day 7 after dose 1 in the RIX4414_3D group. This may have been the result of age difference at the time of first RIX4414 administration. Finally, vaccination with RIX4414 was well tolerated and showed a good safety profile for up to 6 months after the last dose of RIX4414 or placebo when given with other routine vaccinations under the EPI schedule. In conclusion, the immunological response to the RIX4414 vaccine administered as a 3-dose regimen at 6, 10, and 14 weeks was not superior to 2 doses when given at 10 and 14 weeks of age. Efficacy studies with this vaccine are being conducted in developing countries that still use OPV and will investigate whether the similar immune response in both groups also translates into comparable clinical protection. Acknowledgments We thank the infants and their families, for participating in this trial; all investigators; the study nurses and other staff members, for contributing to this study; Dr M. Pichichero, for performing the mirconeutralization assays; Dr R. L. Ward, for performing the enzyme-linked immunosorbent assay; B. Cheuvart, for contribution to study design; S. Damaso, for statistical analysis; A. Dujardin and the local clinical research associate; S. Lancaster, for contributing to technical writing aspects; and S. Baronikova and N. Van Driessche, for editorial assistance. 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