Evaluation of Safety, Immunogenicity and Efficacy of an Attenuated Rotavirus Vaccine, RIX4414

ORIGINAL STUDIES

Evaluation of Safety, Immunogenicity and Efficacy of anAttenuated Rotavirus Vaccine, RIX4414

A Randomized, Placebo-Controlled Trial in Latin American InfantsBelen Salinas, MD,¶ Irene Perez Schael, MSc,* Alexandre C. Linhares, MD, PhD,†

Guillermo M. Ruiz Palacios, MD,‡ M. Lourdes Guerrero, MD, MS,‡ Juan Pablo Yarzabal, MD, MSc,§Yolanda Cervantes, MD,§ SueAnn Costa Clemens, MD, PhD,§ Silvia Damaso, MSc,�

Karin Hardt, PhD,� and Beatrice De Vos, MD�

Background: A live attenuated monovalent rotavirus vaccineRIX4414 was developed with a human strain of G1P1A P�8�specificity to reduce the rotavirus burden in children.Methods: A double blind, randomized, placebo-controlled studyevaluated the efficacy, immunogenicity, safety and reactogenicity of2 oral doses of RIX4414 (104.7, 105.2 or 105.8 focus-forming units)at 2 and 4 months coadministered with routine vaccinations and oralpoliovirus vaccine given for study purposes at least 14 days apart.The 2155 infants (1618 vaccine/537 placebo) enrolled in Brazil,Mexico and Venezuela were followed until 1 year of age.Results: Antirotavirus IgA seroconversion rates 2 months after dose2 ranged between 61% (10 4.7 ffu group) and 65% (105.8 ffu group),and most of the infants had seroprotective levels of antibodies tocoadministered routine vaccinations. The reactogenicity profile ofRIX4414 was similar to that of the placebo, and no vaccination-related serious adverse events were reported. Protective efficacyagainst severe and any rotavirus gastroenteritis from 15 days post-dose 2 was highest in the 105.8 ffu group �86%; 95% confidenceinterval (95% CI), 63–96% and 70% (95% CI 46–84%), P � 0.001,2-sided Fisher’s exact test�. The efficacy against hospitalization was79% (95% CI 48–92%) for pooled vaccine groups. Multiple rota-virus serotypes �G1 (50%), G9 (40%), G2, G3 and G4� were

identified from gastroenteritis stools (enzyme-linked immunosor-bent assay and reverse transcription-polymerase chain reaction)during the study period. For severe gastroenteritis caused by G9serotypes, the protection reached 77% (95% CI 18–96%) in the105.8 ffu group, providing proof of concept that the monovalentG1P1A P�8� human rotavirus vaccine elicits cross-protection againstthe G9 strain. A reduction in any and severe rotavirus gastroenteritiswas already observed at post-dose 1 (period: day of dose 1 to 14days post-dose 2) in vaccinees compared with placebo recipients.Conclusions: Two doses of RIX4414 are highly efficacious, pro-viding cross-protection (G1 and G9 strains, prevalent during thisstudy) and early protection against any and severe rotavirus gastro-enteritis and hospitalization to infants in Latin America.

Key Words: rotavirus, diarrhea, vaccine, efficacy, gastroenteritis

(Pediatr Infect Dis J 2005;24: 807–816)

Rotavirus is the leading cause of severe gastroenteritisamong children worldwide. Annually rotavirus is esti-

mated to cause 352,000–592,000 deaths, 2 million hospital-izations and 25 million clinic visits globally among childrenyounger than 5 years of age.1 Mortality and hospitalizationrates vary significantly depending on access to health care indeveloping settings. In Latin America, depending on thesetting and studies, rotavirus was found in 13–71% of diar-rhea cases requiring inpatient or outpatient medical care ininfants and young children.2–5 Consistent with this observa-tion, the general decline in childhood diarrheal deaths inMexico between 1989 and 1995 was much less evident forwinter seasonal diarrheal deaths in children younger than 2years of age when rotavirus infections have their seasonalepidemic.3 In Venezuela, 85% rotavirus hospitalizations oc-cur during the first year of life.4 It is clear that rotavirus is aserious public health problem in Latin American children.

The immunizing effect of natural rotavirus infec-tions6–10 was the basis for developing attenuated live oralvaccine candidates. Early monovalent rotavirus vaccine can-didates failed to protect in developing countries.11–13 The lowtiter tetravalent rhesus-human reassortant vaccine was effica-cious in the United States14 but showed insufficient protec-tion against rotavirus gastroenteritis in Peru15 and Brazil.16

Accepted for publication July 8, 2005.From *Instituto de Biomedicina, Caracas, Venezuela; †Instituto Evandro

Chagas, Secretaria de Vigila�ncia em Saude, MS, Belem, Brazil; ‡Insti-tuto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran,Mexico City, Mexico; §GlaxoSmithKline Latin America and Caribbean;�GlaxoSmithKline Biologicals, Belgium; and ¶Ciudad Hospitalaria DrEnrique Tejera-Insalud and Universidad de Carabobo, Valencia, Vene-zuela

Supported by GlaxoSmithKline Biologicals, Rixensart, Belgium.Presented in part at the Interscience Conference on Antimicrobial Agents and

Chemotherapy, September 27–30, 2002, San Diego, CA; the WorldSociety for Pediatric Infectious Diseases, November 18–21, 2002, San-tiago, Chile; the Eighth International Symposium on DS-RNA Viruses,Sep. 18–23 2003, Castelvecchio Pascoli-Lucca; Vaccines for EntericDiseases Congress, Montego Bay, Jamaica, April 28–30, 2004; and the24th International Congress of Pediatrics, Cancun, Mexico, August15–20, 2004.

Address for reprints: Irene Perez Schael, MSc, Instituto de Biomedicina-Fuvesin, A.P. 4043–Carmelitas, Caracas 1010 A, Venezuela. Fax 58 212864 1007; E-mail [email protected].

Copyright © 2005 by Lippincott Williams & WilkinsISSN: 0891-3668/05/2409-0807DOI: 10.1097/01.inf.0000178294.13954.a1

The Pediatric Infectious Disease Journal • Volume 24, Number 9, September 2005 807

Subsequently the 3-dose regimen of a higher titer of thetetravalent rhesus-human reassortant vaccine (RotaShield;Wyeth Laboratories)17–20 was licensed in 1998 for infantimmunization in the United States but was withdrawn withinthe first year after licensure because of the risk of intussus-ception.21 Efforts have since focused on new rotavirus vac-cines that would be safe and effective both in developing anddeveloped countries.

Researchers at Cincinnati Children’s Hospital andAVANT Immunotherapeutics developed the 89-12 humanrotavirus vaccine candidate (the isolate was recovered from astool specimen of a 15-month-old child) by multiple passagesin cell culture.22 Two doses of the 89-12 vaccine (G1P1AP[8] specificity) were well-tolerated, were immunogenic andprotected vaccinated infants against rotavirus disease duringa 2-year trial in the United States.23,24 The 89-12 vaccinestrain was further passaged in Vero cells and cloned atGlaxoSmithKline Biologicals, Rixensart, Belgium, to obtainthe new vaccine candidate strain designated as RIX4414.Two oral doses of RIX4414 live attenuated rotavirus vaccinewas tested in adults (Belgium), toddlers (Germany) andinfants (Finland) and were well-tolerated and immunogen-ic.25 In Finland, the vaccine was shown to be highly effica-cious during 2 epidemic seasons in industrialized setting.26

Along the recommendations of the World Health Or-ganization to evaluate new vaccines in developing countriesthat need them most,27,28 phase II evaluation of RIX4414vaccine was initiated in this setting. The results of a doubleblind, randomized, placebo-controlled study to determineefficacy, safety and immunogenicity of the RIX4414 humanrotavirus vaccine administered at 3 different virus concentra-tions along with routine infant vaccinations in 3 countries inLatin America are reported here.

MATERIALS AND METHODSThe study was approved by the ethics committee of

each study center and was conducted in accordance with theOctober 1996 version of the Helsinki Declaration.

Study ParticipantsThe clinical phase of the study was conducted at the

Ministry of Health’s national rotavirus reference center at theInstituto Evandro Chagas Secaao de Virologia in Belem,Brazil, at a pediatric clinic at the Instituto Nacional deCiencias Medicas y Nutricion, Salvador Zubiran in MexicoCity, Mexico and at the Hospital de Ninos Jorge Lizarraga dela Ciudad Hospitalaria Dr Enrique Tejera in Valencia, Ven-ezuela. These study sites were located in an urban area ofmiddle to low socioeconomic status in each country.

Potential study participants were healthy full term in-fants between 6 and 12 weeks of age who weighed �2000 gat birth. Infants were not recruited if they had a past historyof confirmed rotavirus gastroenteritis or other clinically sig-nificant history of gastrointestinal disease or malformation orhad received vaccines or treatment prohibited by the protocol,were immunocompromised or were in household contact withan immunosuppressed individual or pregnant woman.

Written informed consent was obtained from the par-ents or guardians of participating infants before enrollment.

Eligible infants were enrolled from May 25, 2001 to April 11,2002. The participating infants were randomly assigned toone of the 4 study groups (3 vaccine groups and a placebogroup) following a 1:1:1:1 allocation ratio according to acomputer-generated randomization list.

VaccinationThe live attenuated monovalent RIX4414 vaccine (Ro-

tarix; GlaxoSmithKline Biologicals) used in this study was alyophilized preparation to be given orally after reconstitutionwith a liquid calcium carbonate buffer. Infants in the vaccinegroups received 2 oral doses of the vaccine at the age of�2 and 4 months. Three concentrations, 104.7, 105.2 or 105.8

focus-forming units (ffu), of RIX4414 were tested. Vaccina-tion was postponed if the infant had an axillary temperatureof �37.5°C or a rectal temperature of �38°C or gastroenter-itis within 7 days before planned vaccination. There were norestrictions on feeding the infants before or after vaccination.An identical placebo that contained the same constituents as thevaccine except for the vaccine virus served as control. Doubleblinding was maintained during the entire study period.

Commercially available routine infant vaccination(Tritanrix-HB/Hib; GlaxoSmithKline Biologicals) againstdiphtheria, tetanus, pertussis, hepatitis B and Haemophilusinfluenzae type b (Hib) disease was given concomitantly witheach study vaccine dose. The third routine vaccination dosewas administered at the follow-up visit at 6 months of age.For study purposes, oral poliovirus vaccine (Polio Sabin;GlaxoSmithKline Biologicals) was given at least 14 daysbefore or after administration of study vaccine.

Postvaccination Reactogenicity and SafetyDiary cards were supplied to the parents to record

occurrence of specific solicited symptoms for 15 days aftereach vaccination. The use of medications such as antipyreticsand antibiotics was also recorded. Any other unsolicitedsymptoms were recorded during 43 days after each vaccina-tion. Serious adverse events were recorded throughout thestudy. The parents were asked to contact the study nurse orinvestigator if any symptoms appeared to be serious. Parents/guardians were informed about signs indicative of intussus-ception, and they were asked to contact the investigator ifintussusception was suspected. Follow-up and work-up pro-cedures for any intussusception cases were in place. Allserious adverse events were reviewed periodically by anindependent safety monitoring committee.

ImmunogenicityBlood samples were collected from all infants imme-

diately before the first vaccination to rule out previous rota-virus infection. A subset of infants (N � 800) provided bloodsamples 2 months after the first dose (serology for antirota-virus IgA antibodies) and 2 months after the second dose(serology for antirotavirus IgA antibodies and antibodiesagainst antigens of routine infant vaccines). The first 200enrolled infants in each participating country constituted thissubset, and the remaining 200 infants were included accord-ing to the order of enrollment irrespective of country. At thefollow-up visit at 1 year of age, blood samples were collected

Salinas et al The Pediatric Infectious Disease Journal • Volume 24, Number 9, September 2005

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from all infants for antirotavirus IgA serology and from thesubset to measure antibodies against routine antigens.

Concentrations of antirotavirus IgA antibodies weremeasured at the laboratory of Dr R. Ward, Children’s Hos-pital Medical Centre, Cincinnati, OH, with the use of anenzyme-linked immunosorbent assay (ELISA).22 Concentra-tions of antibodies to administered routine infant vaccineantigens were measured at the laboratory of Dr MichaelPichichero, University of Rochester, Rochester, NY (diphthe-ria, tetanus, hepatitis B, Hib and the 3 poliovirus serotypes)and GlaxoSmithKline Biologicals (whole cell pertussis) withthe use of standard assays.29–31

One-half of the random serology subset of infants (N �400) sampled 2 months after each dose also provided stoolspecimens at day 0 and day 7 after each dose. The first 100enrolled infants in each participating country constituted thissubset, and the remaining 100 infants were included according tothe order of enrollment irrespective of country. The stool spec-imens were tested by ELISA22 at the laboratory of Dr R. Ward,Children’s Hospital Medical Centre, Cincinnati, OH, to detectrotavirus shedding. Positive specimens were tested by reversetranscription-polymerase chain reaction31,32 at GlaxoSmithKlineBiologicals to determine the G type. Sequence analysis was usedto differentiate vaccine and wild-type G1 strains.33

Vaccine take was defined as either seroconversionand/or viral shedding post-dose 1 and/or post-dose 2.

Efficacy AssessmentEfficacy surveillance started from day 0 (administration of

vaccine dose 1) and continued until the follow-up visit when thechild was 1 year old. Active surveillance for acute gastroenteritisepisodes was conducted via weekly visits by Health Workers.Gastroenteritis was defined as diarrhea characterized by 3 ormore looser than normal stools within a day. A minimum of 5days were required between episodes for them to be consideredas separate events. Diary cards were given to the parents to dailyrecord rectal temperature, number of vomiting episodes, numberof looser than normal stools, any treatment given and anyrehydration during each suspected episode of gastroenteritis.The information on diary cards was used to assess the severity ofeach gastroenteritis episode according to a widely accepted20-point scoring system (Ruuska and Vesikari34) that allowsclinically meaningful measurement of rotavirus gastroenteritisintensity. A total score of �7 was defined as mild, a score of7–10 as moderate and a score �11 as severe.34

Stool specimens were collected within 7 days of onsetof diarrhea and first tested for rotavirus with ELISA22 at thelaboratory of Dr R. Ward, Children’s Hospital Medical Cen-ter, Cincinnati, OH. All rotavirus-positive specimens weretested by reverse transcription-polymerase chain reaction atGlaxoSmithKline Biologicals to determine the G type.32,33

Any G1 rotavirus detected until 2 months after the seconddose were analyzed to differentiate between vaccine strainand wild G1 strains.

Statistical AnalysisData analysis was performed at GlaxoSmithKline Bio-

logicals with SAS software (version 8.2) and Proc StatXact-5on Windows NT 4.0.

All infants who had received at least 1 dose of the studyvaccine (total vaccinated cohort) were included in the pri-mary analysis of safety. The percentages of infants reportingsolicited symptoms after each dose were calculated by groupwith their exact 95% confidence intervals (95% CI).

Primary immunogenicity analysis was performed onthe according to protocol (ATP) immunogenicity cohort.Subjects from the immunogenicity subset were included inthe ATP cohort if they did not have any rotavirus other thanvaccine strain in stool specimens collected from first doseuntil 2 months after the second dose and if they compliedwith protocol eligibility criteria. Seroconversion rates werecalculated with exact 95% confidence intervals. Seroconver-sion was defined as the percentage of infants with post-antirotavirus IgA antibody concentration �20 units/mLamong those who were seronegative before the first dose. Forthe subset that provided stool specimens at specific timepoints, percentages of infants with vaccine virus shedding inpostvaccination stools and vaccine take (defined as antirota-virus IgA seroconversion and/or vaccine rotavirus sheddingin postvaccination stools) were calculated with exact 95%confidence intervals.

Only gastroenteritis episodes in which wild rotavirusother than the vaccine strain was identified in a stool speci-men were included in the efficacy analysis. Primary efficacyanalysis for the period from 15 days post-dose 2 until infantswere 1 year of age was performed on the ATP efficacy cohort(subjects who received 2 doses of study vaccine or placebo;who had no rotavirus other than vaccine strain in stoolsamples collected from the first dose until 2 months after thesecond dose; who entered the surveillance period and whohad follow-up beyond 15 days after the second dose). Effi-cacy analysis on the total cohort was also performed (inten-tion-to-treat analysis). To assess early efficacy after the firstvaccine dose, an exploratory efficacy analysis was also per-formed on gastroenteritis episodes reported between dose 1and dose 2 on the total vaccinated cohort. Vaccine efficacywith its 95% confidence interval was calculated for pooledvaccine groups and for each vaccine group. Vaccine efficacywas calculated with the formula 1 – (incidence of rotavirusgastroenteritis in the vaccine group/incidence of rotavirusgastroenteritis in the placebo group). The 95% confidenceinterval was derived from the exact confidence interval forcommon Poisson rate ratio. The 2-sided Fisher exact test (5%significance level) was used to compare rates betweengroups. To maintain the type I error at �0.05, hierarchicalstatistical testing was done starting with the pooled vaccinegroups and continuing with descending viral concentrationsonly if a significant difference was detected versus the pla-cebo.

Sample SizeThe sample size was calculated based on an anticipated

rotavirus attack rate of 12% for placebo recipients and avaccine efficacy rate of 70%. Based on these assumptions, theplanned sample size of 2076 infants was estimated to provide89% power to evaluate efficacy against any rotavirus gastro-enteritis (primary end point). With the observed rotavirusattack rate of 10.8% for placebo recipients during the study

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period, the actual sample size of 2155 infants provided �82%power to evaluate the primary end point.

RESULTS

Study PopulationThe study began in May 2001, and the efficacy and

safety follow-up at 1 year of age was completed in April2003. A total of 2155 infants were enrolled in the study (538in the 104.7 group, 540 in the 105.2 group, 540 in the 105.8

group and 537 in the placebo group). The 4 study groupswere comparable with respect to age, height and weight atfirst dose, female-to-male ratio and race (Table 1). Infantswere between 6 and 12 weeks of age at the time of the firstdose and between 13 and 28 weeks at the time of the seconddose. All enrolled infants received the first dose, and 2056

(95.4%) received both doses. Of the 2155 enrolled infants,there were 151 dropouts (infants unable to complete thefollow-up visit at 1 year of age): 80 moved outside the studyarea; parents of 52 infants withdrew consent; 6 were with-drawn after nonserious adverse events �1 infant had a vacci-nation related allergic reaction on the day of dose 1 (receivedRIX4414 and diphtheria-tetanus toxoids-whole cell pertussis-hepatitis B virus and Hib vaccines); and 5 infants had unre-lated adverse events as assessed by the investigator�; 5withdrew after advice from their family doctor; 3 were lost tofollow-up; 3 infants died of unrelated causes; and 2 did notreturn for follow-up visits. Thus the total study cohort was2004 infants who were followed up until 1 year of age.

All 2155 vaccinated infants were included in the safetyanalysis. Of these, 1526 were included in the primary ATP

TABLE 1. Characteristics of the Study Population by Group

Characteristics GroupRIX4414 104.7

GroupRIX4414 105.2

GroupRIX4414 105.8

GroupPlacebo

No. of infants enrolled and vaccinated 538 540 540 537Mean age (wk) at dose 1 8.3 (6–12)* 8.4 (6–12) 8.3 (6–12) 8.4 (6–12)Mean age (wk) at dose 2 17.7 (13–28) 18.0 (13–28) 17.9 (13–28) 18.1 (13–26)Gender (F/M) 240/298 265/275 254/286 267/270Mean height (cm) at dose 1 57.3 57.4 57.4 57.6Mean weight (kg) at dose 1 5.3 5.3 5.3 5.3Race

Black 21 17 19 13Caucasian 114 132 140 134Asian 0 0 0 1Other† 403 391 381 389

No. who completed efficacy surveillanceuntil 1 yr of age

500 499 499 506

*Numbers in parentheses, range.†Other indicates “mixed” or mestizo.

FIGURE 1. Trial profile.



immunogenicity analysis and 1846 subjects were included inthe primary ATP efficacy analysis (Fig. 1). Results of thetotal cohort efficacy and immunogenicity analyses (ITT) wereconsistent with the ATP analyses as evidenced by overlap-ping 95% confidence intervals (data not shown) indicatingthat deviations from the protocol were not treatment-relatedand did not lead to bias in the results.

Reactogenicity and SafetyIncidences of fever, diarrhea, vomiting, irritability, loss

of appetite and cough/runny nose reported during 15 daysafter any dose were similar among the 4 study groups.Overlapping 95% CI between each vaccine group and pla-cebo group suggested that there was no increase in reactoge-nicity either with subsequent doses or with higher viralconcentrations. Figure 2 presents the incidences of solicitedsymptoms reported during the 15 days after each dose foreach vaccine virus concentration and the placebo group. Asexpected, the concomitantly administered whole cell pertus-sis combination vaccine caused fever on the day of vaccina-tion at a similar rate in the vaccine and placebo groups.35,36

The percentage of infants who needed medication (antipyret-ics and antibiotics) after vaccination was similar betweenvaccine and placebo groups (data not shown).

The percentage of subjects reporting at least 1 unsolic-ited symptom was similar among the 4 study groups. Inparticular, the incidence of unsolicited symptoms related tothe gastrointestinal system was low and similar among allstudy groups (data not shown).

Serious adverse events were reported for 220 children(52 in the 104.7 group, 55 in the 105.2 group, 49 in the 105.8

group and 64 in the placebo group) until 1 year of age,including 3 fatalities and 1 intussusception case. Of the 3deaths, 1 child from the placebo group died of generalizedvisceral congestion, the second death was in the 104.7 vaccinegroup because of sepsis and 1 infant from the 105.8 vaccinegroup died in an automobile accident. The intussusceptioncase was reported 6 months after the second dose of RIX4414104.7 ffu in a 10-month-old boy. The intussusception wasreduced surgically, and the child recovered completely. All

serious adverse events including the intussusception case, andthe fatalities were assessed as not related to study vaccinationby the respective investigators and by the independent mon-itoring committee.

Antirotavirus IgA Antibody ResponseAntirotavirus IgA antibody seroconversion rates were

similar in the 3 vaccine groups (Fig. 3). The antirotavirus IgAseroconversion rates ranged from 38 to 43% 2 months afterthe first dose and increased to between 61 and 65% 2 monthsafter the second dose. Antirotavirus IgA antibody geometricmean concentrations in seropositive subjects 2 months afterthe second dose were 162 (95% CI 124–211) units/mL in the104.7 group, 141 (95% CI 111–179) units/mL in the 105.2

group and 200 (95% CI 152–262) units/mL in the 105.8

group. For subjects seroconverting after the first dose, theantirotavirus IgA antibody geometric mean concentrationspost-first dose and post-second dose were similar (data notshown). Seven placebo recipients (5.3%) had detectable an-tirotavirus IgA antibodies 2 months after the second dosewith confirmed wild-type rotavirus strains in stools. At theend of the follow-up period, 73–77% of vaccine recipientsand 41% of the placebo recipients were seropositive forantirotavirus IgA antibodies.

Rotavirus Shedding and Vaccine TakeVaccine virus shedding was observed in 35–44% vac-

cinees on the 7th day after the first dose and in 11–21%vaccinees on the 7th day after the second dose (Fig. 4). Noneexcept 1 infant in the 105.8 vaccine group excreted vaccinestrain 60 days post-dose 1. Among placebo recipients, noneshed any rotavirus after the first dose whereas 1 shed G2wild-type rotavirus after the second dose.

The vaccine take data after each vaccination are shownin Figure 5. Vaccine take after 2 doses was observed in 65%(95% CI 55–73%) in the 104.7 group, 73% (95% CI 63–81%)

FIGURE 2. Solicited symptoms reported during the 15 daysafter each dose in vaccine groups versus placebo. Incidenceof the specific solicited symptom within 15 days after eachvaccine and placebo dose; pd1 and pd2 indicate post-dose1 and post-dose 2, respectively. N indicates total number ofvaccinated infants. Exact 95% CIs are shown.

FIGURE 3. Antirotavirus IgA seroconversion rates after eachvaccine dose in vaccine groups versus placebo. Blood sam-ples drawn 2 months after each dose; pd1 and pd2 indicatepost-dose 1 and post-dose 2, respectively. Seroconversionrate defined as percentages of infants with post-antirotavirusIgA antibody concentration �20 units/mL in infants whowere negative for rotavirus before the first dose of RIX4414or placebo. N indicates number of subjects with availableresults. Exact 95% confidence intervals are shown.



in the 105.2 group and 75% (95% CI 66–83%) in the 105.8

group.

Immunogenicity of Routine VaccinationsWhen given simultaneously, the rotavirus vaccine did

not have any impact on the immunogenicity of diphtheria,tetanus, pertussis, hepatitis B and Hib vaccinations as seenfrom similar seropositivity or seroprotection rates 2 monthsafter the second dose in the vaccine and placebo groups(Table 2). For study purposes, the oral poliovirus vaccine wasgiven at least 14 days apart from the rotavirus vaccine.Response to the 3 polio strains was also similar between thevaccine and placebo groups (Table 2). Persistence levels forantibodies to all routine infant vaccine antigens at the end ofthe efficacy period (4 months after completion of the 3-doseroutine vaccination course) were also similar between thevaccine and placebo groups.

EfficacyRotavirus Gastroenteritis Cases. During the follow-up periodfor the primary study end point (vaccine efficacy from 15days post-second dose until infants were 1 year of age; meanduration, 7 months), 1635 gastroenteritis episodes of anyetiology were reported in 787 infants. Of those, 1216 epi-sodes occurred in the 573 vaccine and 419 episodes in the 214placebo recipients. Stool ELISA results were available for1211 of 1635 (74%) reported gastroenteritis episodes. Noresults were available for 424 gastroenteritis episodes be-cause of insufficient quantity of stool samples or invalidresults. Rotavirus was detected in 109 of 1211 (9%) gastro-enteritis episodes reported in 107 infants. Two infants fromthe placebo group had 2 rotavirus gastroenteritis episodes (1infant had 2 G1 infections, whereas the other infant had firsta G9 infection and then a G1 infection). Of the 109 rotavirusgastroenteritis episodes reported, 19 (17%) were mild, 29(27%) were moderate and 61 (56%) were rated as severe.34 Inthe placebo cohort, gastroenteritis episodes of any etiology

FIGURE 4. Rotavirus shedding on day of vaccinations and7 days after each vaccine dose in vaccine groups versusplacebo. Rotavirus shedding on stool specimens collected onday of- and 7 days after each vaccination; pd1 and pd2indicate post-dose 1 and post-dose 2, respectively. N indi-cates number of subjects with available results.

FIGURE 5. Vaccine take after each vaccine dose in vaccinegroups versus placebo. Vaccine take defined as antirotavirusIgA seroconversion and/or vaccine rotavirus shedding in postvaccination stools; pd1 and pd2 indicate post-dose 1 andpost-dose 2, respectively. N indicates number of subjectswith available results. Exact 95% CIs are shown.

TABLE 2. Seropositivity Rates to Routine Infant Vaccine Antigens

Antibodies

2 mo Post Dose 2 of Routine Infant Vaccines 4 mo Post Dose 3 of Routine Infant Vaccines

Pooled Vaccine Groups Placebo Group Pooled Vaccine Groups Placebo Group

N* % † N % N % N %

Anti-diphtheria*§ 412 66 (61–71)‡ 133 71 (62–78) 455 70 (65–74) 149 74 (67–81)Anti-tetanus§ 414 99 (97–100) 134 99 (96–100) 459 100 (98–100) 149 100 (98–100)Anti-BPT� 409 63 (58–68) 130 62 (53–70) 460 75 (71–79) 148 72 (64–79)Anti-HBs¶ 417 98 (96–99) 133 98 (95–100) 458 97 (94–98) 149 97 (92–99)Anti-PRP# 412 100 (98–100) 131 99 (96–100) 457 100 (98–100) 149 100 (98–100)Anti-poliovirus type 1** 415 98 (97–99) 130 99 (96–100) 429 100 (99–100) 138 100 (97–100)Anti-poliovirus type 2** 415 99 (98–100) 130 99 (96–100) 451 100 (98–100) 148 99 (96–100)Anti-poliovirus type 3** 415 87 (83–90) 130 85 (77–90) 451 93 (90–95) 148 93 (88–97)

*N indicates number of infants.†Percentage of infants with antibody titer above the assay cutoff.‡Numbers in parentheses, exact 95% CI.§ELISA, cutoff at 0.1 IU/mL.�ELISA, cutoff at 15 EL.U/mL.¶AUSAB, Abbott Laboratories cutoff at 10 mIU/mL.#ELISA, cutoff at 0.15 �g/mL.**Virus microneutralization cutoff titer �8.Cutoff levels for anti-diphtheria, anti-tetanus, anti-HBs, anti-PRP and anti-poliovirus types 1, 2 and 3 are seroprotective.Anti-BPT indicates anti-Bordetella pertussis; anti-HBs, anti-hepatitis B surface antigen; anti-PRP, anti-phosphoribosyl phosphate.



were mild to moderate in 74% and severe in 26% of cases. Incomparison, gastroenteritis episodes caused by rotavirus inthis cohort were classified as mild to moderate in 33% and assevere in 67% of cases. This observation highlights andconfirms the importance of rotavirus causing severe illness inyoung infants.

The predominant rotavirus serotypes identified duringthe study were wild-type G1 identified in stools from 55episodes (50%) and G9 identified in stools from 44 episodes(40%). In addition, G2 (4 isolates), G3 (3 isolates) and G4 (1isolate) strains were identified. One of the episodes was amixture of G4 and G9. Two isolates could not be typed, and1 case was attributed to canine rotavirus. Table 3 presents thecharacteristics of the rotavirus gastroenteritis episodes re-ported in each.Vaccine Efficacy Against Rotavirus Gastroenteritis. The vac-cine efficacy results of the total cohort (data not shown) wereconsistent with those observed in the ATP cohort. Table 4presents vaccine efficacy (ATP cohort) against rotavirus gas-troenteritis for each vaccine group versus placebo (hierarchi-cal statistical testing starting with the pooled vaccine groupsand continuing with descending viral concentrations showeda power of P � 0.001 for each comparison versus theplacebo). Compared with the placebo group, infants in each

vaccine group had significantly fewer rotavirus gastroenteritisepisodes of any severity (P � 0.001). Although the vaccinewas effective at all concentrations tested, vaccine efficacyagainst severe rotavirus gastroenteritis (66–86%) was greaterwith vaccine virus titers of 105.2 ffu or higher. Vaccineefficacy rates against any rotavirus gastroenteritis rangedfrom 56 to 70% with the higher concentration.

Vaccine efficacy against severe gastroenteritis causedby G1 type as well as non-G1 types could be evaluatedbecause of the multiple serotypes detected during this trial.This G1 rotavirus vaccine effectively protected against theglobally dominant G1 type and also induced significant pro-tection against gastroenteritis associated with the emergingG9 type (Table 5). Vaccine efficacy was 88% (95% CI48–99%) for the 105.8 ffu group against severe gastroenteritiscaused by G1 types and 77% (95% CI 18–96%) for the 105.8

ffu group against severe gastroenteritis caused by the G9type. Because of the small numbers of other serotypes,type-specific efficacy could not be calculated. However, ofthe 4 infections with G2, 3 occurred in the 537 placebo andonly 1 in the 1618 vaccine recipients.

Between the first dose of vaccine at 8 weeks of age andthe second vaccine dose, 16 of 537 (3%) placebo recipientsreported rotavirus gastroenteritis of which one-half were

TABLE 3. Features of Rotavirus Gastroenteritis Episodes Reported During the Study

RIX4414 104.7 ffu(N � 468)*

RIX4414 105.2 ffu(N � 460)

RIX4414 105.8 ffu(N � 464)

Placebo(N � 454)

Any rotavirus gastroenteritis 21 22 15† 51

No. of episodes with specific feature among all rotavirusgastroenteritis episodes reported

Severity scores34

�7 4 (19)‡ 8 (36) 2 (13) 5 (10)7–10 5 (24) 4 (18) 8 (53) 12 (24)�11 12 (57) 10 (45) 5 (33) 34 (67)

Identified rotavirus serotypesWild G1 12 (57) 6 (27) 7 (47) 30 (59)G2 0 0 1 (7) 3 (6)G3 1 (5) 0 0 2 (4)G4 0 0 1 (7) 0G9 8 (38) 14 (64) 7 (47) 15 (29)Canine 0 0 0 1 (2)Unknown 0 2 (9) 0 0

*N indicates number of infants.†G4 and G9 rotavirus were isolated from 1 episode.‡Numbers in parentheses, percent.

TABLE 4. Protective Efficacy of 2 Doses of RIX4414 Human Rotavirus Vaccine Against Rotavirus Gastroenteritis

N*

Any RotavirusGastroenteritis

Severe34 RotavirusGastroenteritis

Hospitalization forRotavirus Gastroenteritis

n Efficacy n Efficacy n Efficacy

Pooled vaccine groups 1392 58 (4)†‡ 61 (42–74)§ 27 (2)†‡ 74 (56–85)§ 9 (0.6)†‡ 79 (48–92)§

RIX4414 105.8 ffu 464 15 (3)† 70 (46–84) 5 (1)† 86 (63–96)RIX4414 105.2 ffu 460 22 (5)† 56 (25–75) 10 (2)† 71 (40–87)RIX4414 104.7 ffu 468 21 (4)† 58 (29–76) 12 (3)† 66 (32–84)Placebo 454 49 (11) — 34 (7) — 14 (3) —

*N indicates number of subjects. n indicates number of subjects reporting at least 1 specified rotavirus gastroenteritis episode.†P � 0.001 for each comparison between the vaccine and placebo groups by 2-sided Fisher’s exact test (significant level of � � 0.05).‡Numbers in parentheses, percent.§Numbers in parentheses, exact 95% CIs.



severe cases34; compared with 18 of 1618 (1%) vaccinees ofwhich only 5 cases (28%) were severe.34 All cases in thevaccine and placebo group were serotype G1. A reduction inany and severe rotavirus gastroenteritis episodes translatesinto a protective efficacy of 86% (95% CI �11–100%)against severe and 69% (95% CI 11–91%) against any rota-virus gastroenteritis for the 105.2 ffu group.

DISCUSSIONSeveral studies have indicated that the majority of

rotavirus gastroenteritis occurs during infancy and earlychildhood37–41; therefore protection at an early age beforemost illness occurs is crucial. This study has been able todemonstrate a statistically significant decrease in the percent-age of infants developing rotavirus gastroenteritis when vac-cinated with 2 oral doses of RIX4414 for all 3 studied viralconcentrations. Higher titers of RIX4414 (105.2 and 105.8 ffu)displayed an increased efficacy against rotavirus gastroenter-itis. The protective efficacy against more severe illness wasmore pertinent. In fact, the enhanced protection against se-vere rotavirus gastroenteritis observed with this vaccine isconsistent with observations from a study in Mexican chil-dren in which complete protection against moderate to severedisease was observed after 2 symptomatic or asymptomaticnatural rotavirus infections.8 Other longitudinal studies havealso shown that repeated and early rotavirus infections impartnatural immunity among children and protect them againstsubsequent severe disease.6,7,9

Most of the rotavirus strains identified during the othertrials25,26 were of G1 type, whereas in this study G9 ac-counted for 40% of all infections. The protection by RIX4414vaccine against severe disease caused by the homologous G1type observed previously in Finland26 and now confirmedwith this trial in Latin America is an important piece ofinformation given the global predominance of the G1 type.Furthermore and of critical importance for a monovalent

G1P1A P�8� vaccine was clinical proof of cross-protectionagainst the globally emerging G9 strain. The complex diver-sity of circulating strains and increased prevalence of otherstrains including the emerging G9 type42,43 has emphasizedthe need for vaccines to protect against multiple serotypes.This protection elicited by 2 doses of monovalent RIX4414vaccine against both G1 and G9 strains could be correlatedwith observations reported with natural infection where sub-sequent infections broaden the initial homotypic response todevelop heterotypic responses.44

Given the need for early protection in developing coun-tries,37 an efficacy analysis was also performed for the periodfrom dose 1 to dose 2. During this period, a reduction inrotavirus gastroenteritis and specifically severe cases wasobserved in the vaccine groups as compared with placebo.

The overall efficacy results in this study performed withthe 2-dose regimen of RIX4414 in 3 developing countries inLatin America are comparable with results of a similar studywith RIX4414 in Finland,26 the parent 89-12 vaccine in theUnited States23,24 and 3-dose regimen of RotaShield datafrom a trial in Venezuela.20 Although previously evaluatedrotavirus vaccines induced good protection in developedsettings, they have shown variable efficacy in the developingcountries. Both bovine rotavirus vaccines, RIT423745,12 andWC3,13 lacked satisfactory efficacy in developing countries.Three doses of RotaShield vaccine at a concentration of4 � 104 plaque-forming units/dose showed poor efficacy inPeru15 and Brazil,16 whereas a trial in Venezuela20 with a10-fold higher dose of RotaShield was effective.

In this study, all evaluated immunologic markers suchas serum antirotavirus IgA, rotavirus antigen shedding instools and vaccine take showed a distinct immune responsefor all vaccine virus concentrations, but again better resultswere observed with higher virus concentrations. The influ-ence of the second dose to the overall immune response canbe described as a catch-up phenomenon as indicated by theincreased rate of seroconverters rather than an increase oftiters. Although specific immune correlates of protection stillremain elusive, high antirotavirus IgA antibody concentra-tions are considered a good marker of protection.46,47 Like-wise seroconversion after 2 doses of RIX4414 correlated wellwith decreased likelihood of developing rotavirus illness inthe vaccine groups. In the pooled vaccine groups, only 1.6%of seroconverted infants developed rotavirus gastroenteritisversus 11.2% of infants who had failed to seroconvert afterthe 2 doses (data not shown).

This study showed there was no interference ofRIX4414 vaccine on coadministered routine diphtheria, teta-nus, pertussis, hepatitis B and Hib vaccines. For oral polio-virus vaccine (OPV), which was administered 2 weeks apartfrom the RIX4414 in the present study, there was equally noevidence of interference. Because this form of administrationis not in line with common Expanded Program on Immuni-zation schedules, another study in South Africa specificallyaddressed the question of interference when RIX4414 andOPV vaccine were administered concomitantly.48 Controlgroups in that study received either RIX4414 and inactivatedpoliovirus vaccine concomitantly or OPV and RIX4414 pla-cebo. The study showed that 2 doses of RIX4414 coadmin-

TABLE 5. Protective Efficacy of 2 Doses of RIX4414Human Rotavirus Vaccine Against Serotype-SpecificSevere Rotavirus Gastroenteritis

N*Severe Rotavirus Gastroenteritis

n Efficacy P†

G1 wild-type rotavirusPooled vaccine groups 1392 13 (0.9)‡ 74 (41–88)§ �0.001RIX4414 105.8 ffu 464 2 (0.4) 88 (48–99) �0.001RIX4414 105.2 ffu 460 4 (0.9) 75 (24–94) 0.006RIX4414 104.7 ffu 468 7 (1.5) 58 (�9–85) 0.057Placebo 454 16 (4) — —

G9 rotavirusPooled vaccine groups 1392 13 (0.9) 67 (24–86) 0.005RIX4414 105.8 ffu 464 3 (0.6) 77 (18–96) 0.011RIX4414 105.2 ffu 460 6 (1.3) 54 (�28.5–86) 0.109RIX4414 104.7 ffu 468 4 (0.9) 70 (3–93) 0.027Placebo 454 13 (2.9) — —

*N indicates number of infants. n indicates number of subjects reporting at least 1specified rotavirus gastroenteritis episode.

†Two-sided Fisher’s exact test (significant level of � � 0.05) used for each comparisonbetween the vaccine and placebo groups.

‡N numbers in parentheses, percent.§Numbers in parentheses, exact 95% CI.



istered with OPV did not impact on the seroprotection ratesinduced by any of the 3 poliovirus serotypes when comparedwith the 2 control groups, nor was there any interference ofOPV or inactivated poliovirus vaccine on the immunogenic-ity of RIX4414. Moreover no increase in side effects wasseen because of coadministration of routine vaccines. Theseobservations are important for the integration of the RIX4414vaccine into the routine immunization schedule.

The present trial confirmed the low reactogenicity ofRIX4414 observed previously in Finland,25,26 with no in-crease in any adverse events including fever, diarrhea andvomiting as compared with the placebo. The overall reacto-genicity profile of the RIX4414 vaccine was very mild, andthere were no serious medical concerns after vaccination.RotaShield, on the other hand, was associated with fever49

during prelicensure studies and analysis of postmarketingadverse events has indicated that the vaccine was associatedwith gastrointestinal-related illness.50 Bloody diarrhea and/orbloody stools reported following administration of the rhesus-human reassortant vaccine are not typical symptoms of wildrotavirus disease50 and were not observed in this trial. Theapparent lack of any link between wild rotavirus and intus-susception51 offers the potential for attenuated human rota-virus to be a safe vaccine candidate. Whether the low reac-togenicity of RIX4414 means lower risk of intussusception isbeing studied in larger trials presently ongoing worldwide.

In conclusion, this study conducted in 3 countries inLatin America where multiple rotavirus serotypes were cir-culating showed that 2 doses of RIX4414 vaccine is highlyeffective in preventing severe rotavirus gastroenteritis andrelated hospitalizations and has demonstrated that this mono-valent vaccine could afford clinical cross-protection early ininfancy. If integrated in routine infant immunization sched-ules, RIX4414 may impact on the reduction of the globalrotavirus disease burden. In the meantime, further evaluationof the RIX4414 vaccine is continuing towards this goal.

ACKNOWLEDGMENTSWe thank Dr R. L. Ward, Cincinnati, OH, for perform-

ing the ELISAs and Dr Michael Pichichero, University ofRochester, Rochester, NY, for performing assays on antibod-ies to administered routine infant vaccine antigens.

We thank the pediatricians and microbiologists whocontributed to this study. For Venezuela: Marıa AngelaTomat, Marıa Peiro, Merly Villarroel, Miguel Gonzalez andBelkis Benites de Rada (pediatricians); German Gonzalez,Angela Fierro, Giconda Clemente and Rosabel Gonzalez(microbiologists). For Brazil: Consuelo S. Oliveira, ElieteAraujo, Rosa Helena P. Gusmao, Maria Cleonice A. Justino(pediatricians); Edvaldo C. Brito Loureiro, Va�nia N. Caval-cante, Yvone Gabbay Mendes, Joana D’Arc P. Mascarenhas(microbiologists). For Mexico: Aurora Bautista-Marquez,Maria Edilia Luna-Cruz, Juan Pablo Jorba-Aliacar (pedia-tricians); Fernando Tuz-Dzib, Leticia Reyes Gonzalez (mi-crobiologists).

We thank Fernando Aguirre, Pascale Dieryck, SilvinaSantucho and the local clinical research assistants, Glaxo-SmithKline Biologicals, for study coordination.

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Evaluation of Safety, Immunogenicity and Efficacy of an Attenuated Rotavirus Vaccine, RIX4414

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