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Immunogenicity, reactogenicity and safety of the human rotavirus vaccine RIX4414 oral suspension (liquid formulation) in Finnish infants
Vaccine 29 (2011) 2079–2084
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Immunogenicity, reactogenicity and safety of the human rotavirus vaccine RIX4414 oral suspension (liquid formulation) in Finnish infants夽 T. Vesikari a,∗ , A. Karvonen a , A. Bouckenooghe b,1 , P.V. Suryakiran c , I. Smolenov b , H.H. Han d a
Vaccine Research Center, Medical School, University of Tampere, FIN-33014 Tampere, Finland GlaxoSmithKline Biologicals, Wavre, Belgium GlaxoSmithKline Pharmaceuticals Ltd., Bangalore, India d GlaxoSmithKline Biologicals, King of Prussia, USA b c
a r t i c l e
i n f o
Article history: Received 14 October 2010 Received in revised form 30 December 2010 Accepted 5 January 2011 Available online 14 January 2011 Keywords: RIX4414 Rotavirus vaccine Liquid formulation Finnish
a b s t r a c t The lyophilized formulation of a human rotavirus vaccine, RotarixTM (RIX4414) is highly immunogenic. In order to comply with the World Health Organization’s (WHO) recommendation, a liquid formulation of the vaccine that does not require reconstitution was developed. The immunogenicity, reactogenicity and safety of the liquid formulation were compared with lyophilized formulation in two Finnish studies. In Study A infants aged 6–12 weeks received two doses of the lyophilized or liquid formulation of the vaccine or placebo following a 0,1 month schedule. In Study B, infants aged 10–17 weeks received two doses of either liquid or lyophilized formulation of the vaccine. In both studies, anti-rotavirus IgA antibodies were assessed pre-vaccination and one month post-Dose 2. In Study A, the anti-rotavirus seroconversion rate was 90% (95% CI: 81.2–95.6%) and 83.7% (95% CI: 74.2–90.8%) in the groups that received the liquid and the lyophilized formulation of RIX4414, respectively; the respective anti-rotavirus IgA seroconversion rates in Study B were 88.6% (95% CI: 86.1–90.8%) and 90.5% (95% CI: 86.2–93.8%). Reactogenicity and safety profiles of the two vaccine formulations were similar. Liquid formulation of the rotavirus vaccine allows greater flexibility in supply and reduces logistical costs. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Worldwide, rotavirus is the most common cause of severe dehydrating gastroenteritis among children <5 years of age [1,2]. It has been estimated that annually rotavirus accounts for more than 25 million clinic visits, 2 million hospitalizations and 475,000–580,000 deaths among children aged <5 years [3]. Implementation of rotavirus vaccination is the only practical intervention for reducing the disease burden [4–7].
Abbreviations: WHO, World Health Organization; UNICEF, United Nations International Children’s Emergency Fund; GSK, GlaxoSmithKline; CCID, cell culture infective dose; DTPa-HBV-IPV-Hib, diphtheria toxoid, tetanus toxoid, acellular pertussis, hepatitis B, inactivated polio and Haemophilus influenzae type B; CI, confidence interval; ATP, according-to-protocol; GMC, geometric mean concentrations. 夽 ClinicalTrials.gov Identifier: 104480/NCT00137930 (Study A); 107876/NCT00382772 (Study B). ∗ Corresponding author. Tel.: +358 3 3551 8444; fax: +358 3 3551 8450. E-mail addresses: timo.vesikari@uta.fi (T. Vesikari), aino.karvonen@uta.fi (A. Karvonen), [email protected] (A. Bouckenooghe), [email protected] (P.V. Suryakiran), [email protected] (I. Smolenov), [email protected] (H.H. Han). 1 Current address: Sanofi Pasteur, Research and Development, Singapore. 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.01.004
RotarixTM (RIX4414, GlaxoSmithKline Biologicals), an oral human rotavirus vaccine containing G1[P8] strain is derived from the parent 89-12 strain [8–10]; the current vaccine was obtained by cloning at passage 43 and further passaging in cell culture which resulted in greater attenuation [11]. A lyophilized formulation of RotarixTM vaccine is currently licensed in over 110 countries. In pre-licensure studies, the lyophilized vaccine was found to be highly immunogenic, well tolerated and efficacious against severe rotavirus gastroenteritis and rotavirus gastroenteritis related hospitalizations [11–13]. The lyophilized formulation has to be reconstituted with a liquid calcium carbonate buffer prior to oral administration, which adds to complexity of vaccine administration. Furthermore, vaccine and buffer come in separate vials which make it bulky and consume cold storage space. Based on the recommendations of World Health Organization (WHO) and United Nations International Children’s Emergency Fund (UNICEF), a liquid formulation of RIX4414 vaccine that does not require reconstitution has been developed. The immunogenicity, reactogenicity and safety of the liquid formulation of RIX4414 were first evaluated in a feasibility study in Finland. Following positive experience, a larger non-inferiority trial was conducted. The results of the two Finnish trials are reported here.
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2. Materials and methods
2.3. Assessment of immunogenicity
2.1. Subjects and study design
In both studies, serum samples were collected prior to Dose 1 and one month post-Dose 2 of vaccine/placebo to measure antirotavirus IgA antibody concentration using ELISA at GSK Biologicals, Belgium, adapting a previously described method [8,9]. The assay cut-off was 20 Units/milliliter (U/ml). Stool samples were collected from all infants in Study A at Day 0 (or Day 0 minus 1 day), Day 7 ± 1 day and 15 ± 1 day after each dose and one month post-Dose 2 (or one month post-Dose 2 minus 1 day) of vaccine/placebo. Stool samples were tested for rotavirus using ELISA at Dr. R. Ward’s laboratory at the Children’s Hospital Medical Center, Cincinnati, OH, United States. Presence of vaccine strain rotavirus in any of the stool samples collected at pre-determined time point indicated vaccine take. In study A, vaccine take was defined as appearance of serum IgA antibodies to rotavirus in post-vaccination sera at a concentration of ≥20 U/ml and/or presence of vaccine virus in any stool sample collected from Dose 1 of vaccine/placebo to one month post-Dose 2 in infants who were negative for rotavirus IgA antibody prior to Dose 1. In study B, vaccine take was determined by seroconversion only, as appearance of rotavirus IgA antibodies in infants who were rotavirus IgA negative prior to Dose 1.
The phase II feasibility study (Study A) and the phase III noninferiority study (Study B) were multi-center studies conducted in Finland. The phase II study was conducted in five centers between August 2005 and November 2005 and the phase III study was conducted in 12 centers from November 2006 to April 2007. The studies were performed following the Good Clinical Practice guidelines and the 1996 version of Declaration of Helsinki. Each study was approved by the Ethics Committee of the Pirkanmaa hospital district. Parents/guardians of infants provided written informed consent prior to enrolment. Healthy infants aged 6–12 weeks (Study A) and 10–17 weeks (Study B) with a birth weight >2 kg were enrolled. In Study A, infants were randomized into four groups (4:1:4:1) to receive two oral doses of either the liquid formulation of RIX4414/placebo or the lyophilized formulation of RIX4414 (RotarixTM )/placebo following a 0,1 month schedule. The study was double-blind with respect to each of the vaccine formulation and their respective placebo; however, blinding between the two vaccine formulations was not technically possible because of the difference in appearance of the vaccines. Infants in Study B were randomized into four groups (1:1:1:1) to receive two oral doses of either the liquid formulation (any of the three lots) or lyophilized formulation of RIX4414 vaccine concomitantly with routine childhood vaccines. The three lots of the liquid formulation of the vaccine were pooled to form a single pooled liquid group. The study was doubleblind with respect to the three lots of the liquid formulation and open label with respect to the liquid and the lyophilized formulation. The vaccine/placebo was administered following a 0,1 month schedule. A standard SAS® program was used for generating the randomization list and a block randomization was used in order to ensure that the balance between the treatment arms were maintained. A unique treatment number identified the vaccine/placebo doses that were to be administered to the infants. Infants were excluded from participating in the studies if they had received any other investigational drug or vaccine 30 days prior to the administration of the first dose of the study vaccine or had a history of allergy or rotavirus gastroenteritis. Infants with acute illness at the time of enrolment also could not receive the vaccine until the condition was resolved.
2.2. Vaccine The study vaccines were provided by GlaxoSmithKline (GSK) Biologicals, Belgium. Each dose of RIX4414 oral suspension (liquid formulation) (RIX4414 liq; 1.5 ml) and RIX4414 (RotarixTM ) lyophilized formulation (RIX4414 lyo; 1 ml after reconstitution with calcium carbonate buffer) contained at least 106 median cell culture infective dose (CCID50 ) of live attenuated RIX4414 human rotavirus strain. The liquid formulation of RIX4414 contained sucrose as excipient and the content of sucrose in the liquid formulation is higher than one in the lyophilized formulation. The placebo preparation had the same constituents as the active vaccines but it did not contain the viral strain. In study A, the minimum time interval between study vaccine/placebo and routine infant vaccine administration was 14 days. Infants in Study B concomitantly received a combined diphtheria toxoid, tetanus toxoid, acellular pertussis, hepatitis B, inactivated polio and Haemophilus influenzae type B vaccine (DTPaHBV-IPV-Hib, Infanrix hexaTM ).
2.4. Assessment of reactogenicity and safety Parents/guardians of infants were provided diary cards to record solicited general symptoms (loss of appetite, fussiness/irritability, fever, diarrhea, vomiting, and cough/runny nose) during a 15-day (Study A) and 8-day (Study B) post-vaccination follow-up period. The intensity of each adverse event was assessed using a 4-point scale where “0” refers to ‘absent’ and “3” refers to ‘severe’. Grade 3 solicited general symptoms were defined as not eating at all (loss of appetite), preventing normal activities (fussiness/irritability, cough/runny nose), axillary temperature >39.0 ◦ C (fever), ≥6 looser than normal stools per day (diarrhea) and ≥3 episodes of vomiting per day (vomiting). Unsolicited symptoms were recorded for the 31-day follow-up period after each dose and serious adverse events (SAEs) were recorded throughout the study period. 2.5. Assessment of gastroenteritis Gastroenteritis was defined as diarrhea (≥3 looser than normal stools) with or without vomiting. Parents/guardians of infants were advised by the investigator to collect stool samples as soon as possible during each gastroenteritis episode between Dose 1 and one-month post-Dose 2. Gastroenteritis stool samples were tested for rotavirus by ELISA (Ward laboratory for Study A and GSK laboratory for Study B). Rotavirus positive stool samples were further tested to determine the G and P type using reverse transcriptase polymerase chain reaction (RT-PCR) at Delft Diagnostic Laboratory, Delft, the Netherlands [14]. Wild-type G1 rotavirus was differentiated from the vaccine G1 strain by sequencing. 2.6. Statistical analyses All statistical analyses were performed using SAS 8.2 and 95% confidence interval (CI) calculated using Proc StatXact-5. A sample size of 250 infants (100 infants each in RIX4414 liq and RIX4414 lyo groups and 25 infants each in the respective placebo groups) in Study A was planned to have at least 190 evaluable infants and a power of 80% to detect a difference in vaccine take between each of the vaccine formulations and the placebo groups (˛ = 5%, one-sided standardized asymptotic score test). In Study B, a sample size of 1200 infants (900 infants in the RIX4414 liq group and 300 infants in the RIX4414 lyo group) was
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Fig. 1. Number of infants enrolled and included in the ATP immunogenicity cohort (Study A).
planned to have at least 960 evaluable infants and 94% power to rule out that the seroconversion rate in the RIX4414 liq group was decreased by more than 10% as compared to the RIX4414 lyo group (one-sided test, ˛ = 2.5%, Bonferroni adjustment of beta). Immunogenicity analysis was performed on the according-toprotocol (ATP) cohort which included infants who complied with the protocol and for whom immunogenicity data was available at pre-vaccination and one month after the completion of vaccination. Safety analysis was performed on the total vaccinated cohort that included infants who received at least one vaccine/placebo dose.
In Study A, the percentage of infants with vaccine take after combined doses was calculated with their exact 95% CI. In Study B, non-inferiority of RIX4414 liq to RIX4414 lyo in terms of antirotavirus IgA seroconversion rate one month post-Dose 2 was established if the upper limit of the two-sided asymptotic standardized 95% CI for the difference in seroconversion rates between the RIX4414 lyo and (minus) the RIX4414 liq group was ≤10%. In both studies, seroconversion rates and geometric mean concentrations (GMCs) one month post-Dose 2 were tabulated with 95% CI. Percentage of doses followed by each of the solicited general symptom including those graded 3 in intensity during the 15-
Fig. 2. Number of infants enrolled and included in the ATP immunogenicity cohort (Study B).
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day/8-day post-vaccination follow-up period was tabulated with 95% CI. The percentage of infants with gastroenteritis episodes was also tabulated. During the 31-day follow-up period, the percentage of doses followed by unsolicited symptoms was tabulated with 95% CI. All SAEs reported during the entire study period were recorded. 3. Results 3.1. Study population and demography In Study A, 250 infants were enrolled (RIX4414 liq = 100, RIX4414 lyo = 100, Placebo liq = 25 and Placebo lyo = 25; pooled placebo = 50), and in Study B 1200 infants (RIX4414 liq = 900 and RIX4414 lyo = 300) were enrolled. The number of infants included in each of the study cohorts is presented in Figs. 1 and 2. Mean age of infants at Dose 1 in Study A was 9.1 weeks with a standard deviation of 1.94 weeks; in Study B, mean age was 11.6 with a standard deviation of 1.25 weeks (total vaccinated cohort). The distribution of males and females in the study groups was similar and majority (>98%) of the infants were white in both studies. 3.2. Immunogenicity Vaccine take on combined doses in Study A was 93.4% (95% CI: 86.2–97.5%) in RIX4414 liq group (N = 91) and 89.4% (95% CI: 81.3–94.8%) in RIX4414 lyo group (N = 94). By rotavirus IgA seroconversion only, vaccine uptake in RIX4414 liq group was 90% (95% CI: 81.2–95.6%) and in RIX4414 lyo group 83.7% (95% CI: 74.2–90.8%) (Table 1). None of the infants seroconverted in the pooled placebo group (Table 1). Stool samples collected at pre-determined time points showed that rotavirus vaccine shedding peaked on Day 7 after Dose 1 of vaccination (57.5% in the RIX4414 liq group 58.5% and in the RIX4414 lyo group). On day 15 post-Dose 1, 32.2% of the infants in the RIX4414 liq group and 25.6% of infants in the RIX4414 lyo group still shed the vaccine virus. In study B, seroconversion by rotavirus IgA antibody was seen in 90.9% (95% CI: 86.6–94.2%), 90.4% (95% CI: 86.1–93.7%) and 84.4% (95% CI: 79.3–88.7%), respectively, in infants receiving the three lots of liquid vaccine one month after Dose 2. The three groups were pooled for comparison with lyophilized formulation. The seroconversion rate in the group receiving RIX4414 lyo was 90.5% (95% CI: 86.2–93.8%). The GMCs in the recipients of the both vaccine formulation in each study are shown in Table 1. Non-inferiority of the RIX4414 liquid formulation to that of RIX4414 lyophilized formulation in terms of seroconversion rate was demonstrated as the upper limit of the 2-sided standardized asymptotic 95% CI for the group difference was <10% (1.87% [95% CI: −2.85% to 5.83%]).
Fig. 3. Solicited general symptoms-overall/dose during the 15-day post-vaccination follow-up period (Study A) (total vaccinated cohort).
In Study B, vomiting was reported following 13.9% (95% CI: 12.3–15.7%) of combined doses in the RIX4414 liq and 15.4% (95% CI: 12.5–18.6%) of combined doses in the RIX4414 lyo group; diarrhea was reported after 2.6% (95% CI: 1.9–3.5%) of combined doses and 1.8% (95% CI: 0.9–3.2%) of combined doses in the respective groups (Fig. 4). Gastroenteritis episodes were reported in 9.7% (95% CI: 7.8–11.8%) of infants in RIX4414 liq group and 7.3% (95% CI: 4.7–10.9%) of infants in RIX4414 lyo group throughout the study period. Two stool samples collected in infants from RIX4414 liq were positive for rotavirus and the G1P [8] vaccine strain was isolated from both the samples. One infant experienced diarrhea and mild fever during the first seven days after Dose 1. The child simultaneously received a 10-day course of antibiotic for prophylaxis of Streptococcus tonsillitis which started 2 days before onset of diarrhea. Gastroenteritis and fever were assessed to be related to vaccination. The other infant reported gastroenteritis 23 days postDose 2 of vaccination. This episode was assessed to be unrelated to vaccination by the investigator.
3.3. Reactogenicity and safety In study A, during the first 15 days period (overall/dose), vomiting was reported after 14.2% (95% CI: 9.7–19.9%) of doses in the RIX4414 liq group, 21.1% (95% CI: 15.7–27.4%) in the RIX4414 lyo group, and 13.1% (95% CI: 7.2–21.4%) of doses in pooled placebo group. Diarrhea was reported following 2.0% (95% CI: 0.6–5.1%), 5.5% (95% CI: 2.8–9.7%) and 4.0% (95% CI: 1.1–10.0%) of doses in the three groups, respectively (Fig. 3). The percentage of infants with gastroenteritis episodes reported between Dose 1 until one month post-Dose 2 was 6% (95% CI: 2.2–12.6%) in RIX4414 liq, 11% (95% CI: 5.6–18.8%) in RIX4414 lyo and 12% (95% CI: 4.5–24.3%) in the pooled placebo groups in Study A. Of these gastroenteritis episodes, rotavirus was isolated from four cases in RIX4414 lyo group; G1P [8] vaccine strain was isolated from one stool sample and the rotavirus serotype was unknown in the remaining three cases.
Fig. 4. Solicited general symptoms-overall/dose during the 8-day post-vaccination follow-up period (Study B) (total vaccinated cohort).
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Table 1 Anti-rotavirus IgA seroconversion rate and GMCs one month post-Dose 2 (ATP cohort for immunogenicity). N (n) Study A
RIX4414 liq RIX4414 lyo Pooled placebo
Study B
RIX4414 RIX4414 RIX4414 RIX4414 RIX4414
liqA liqB liqC liq* lyo
% (95% CI)
GMC value (95% CI)
80 (72) 86 (72) 44 (0)
90 (81.2–95.6) 83.7 (74.2–90.8) 0 (0–8.8)
301.3 (205.4–442.0) 360.6 (236.4–549.8) <20
242 (220) 260 (235) 244 (206) 746 (661) 252 (228)
90.9 (86.6–94.2) 90.4 (86.1–93.7) 84.4 (79.3–88.7) 88.6 (86.1–90.8) 90.5 (86.2–93.8)
384.4 (309.1–478.2) 418.8 (337.8–519.1) 324.4 (253.4–415.3) 374.7 (328.8–426.9) 331.8 (265.0–415.4)
N, number of infants with available results; n, number of infants with anti-rotavirus antibody concentration above the cut-off; %, percentage of infants with anti-rotavirus antibody concentration above the cut-off; 95% CI, 95% confidence interval; GMC, geometric mean concentration in U/ml; RIX4414 liqA, RIX4414 liqB, RIX4414 liqC: three lots of the RIX4414 liquid vaccine formulation; RIX4414 liq*: the three lots of the liquid formulation of RIX4414 are pooled to form the pooled liquid group.
The percentage of doses followed by at least one unsolicited symptom was similar in the two studies (data not shown). In Study A, two SAEs (bronchiolitis and otitis media) in RIX4414 liq and one SAE (otitis media) in RIX4414 lyo groups were reported. A total of 20 SAEs (mainly pneumonia, otitis media, gastroenteritis, bronchitis and laryngitis) in 18 infants were reported until one month after Dose 2 of vaccination in Study B. One SAE (infantile spasms) required long-time therapy in an infant belonging to RIX4414 lyo group and the infant was withdrawn from the study as a result of this SAE. No cases of intussusception were reported during the entire study period.
4. Discussion Higher manufacturing output, easy handling and low logistical costs of the liquid formulation of RIX4414 formed the basis for the manufacturing transition of the RIX4414 rotavirus vaccine from the lyophilized formulation to a liquid formulation. Although the liquid formulation has the same live vaccine virus at the same viral concentration as the licensed lyophilized formulation of RIX4414 (RotarixTM ), there was a need to assess the immunogenicity and safety of the new vaccine formulation in the target population. Immunogenicity was assessed in terms of rotavirus IgA antibody response. Rotavirus IgA antibodies, as measured by ELISA, have been used as an immunogenicity end point in all recent rotavirus vaccine studies and also as a proxy correlate of protection. While many factors may contribute to protection against rotavirus, a high titer of rotavirus serum IgA antibody is generally accepted as a surrogate marker for protective immunity [15–17]. The results of the phase II feasibility (Study A) study suggested that the RIX4414 liquid formulation was highly immunogenic and non-inferior to the RIX4414 lyophilized formulation of the vaccine. In Study A, the anti-rotavirus seroconversion rate was slightly higher in the RIX4414 liq group (90%) when compared to the RIX4414 lyo group (83.7%); the GMCs were higher in the RIX4414 lyo group. However, the difference was smaller when vaccine virus shedding was also considered as an end point. In Study B, the seroconversion was higher in the RIX4414 lyo group (90.5%) when compared to the RIX4414 liq group (88.6%). In earlier immunogenicity studies in Finland with the lyophilized rotavirus vaccine formulation, the seroconversion rate following two doses ranged between 80% and 96%, which is in line with what was observed in both the studies presented in this paper using liquid formulation [11,12]. Including the vaccine virus shedding in the study with lyophilized vaccine, the uptake was 96% [11]. Co-administration of routine vaccines with rotavirus vaccines in Study B did not have an impact on the immune response generated by both the rotavirus vaccine formulations. Even so, the uptake rate might have been higher if the vaccine virus shedding had been included as an end point.
Since the vaccine virus titer of the liquid formulation is similar to that in the lyophilized formulation, a similar safety profile is expected. At most, the different constituents of the two formulations might be associated with different side effects. Nevertheless, no difference in side effects was observed between the infants who received liquid formulation and those that received the lyophilized formulation in the two studies presented here. The percentage of infants reporting solicited and unsolicited symptoms and SAEs were similar between the two vaccine formulations. Keeping the advantages of using a liquid formulation of the vaccine in perspective and considering the high global demand and a higher manufacturing capacity, RIX4414 liquid formulation holds the promise to become a formulation of choice for global use.
Acknowledgements We thank the infants and their families for participating in this trial; all investigators, the study nurses and other staff members for contributing in many ways to this study. We acknowledge DDL Diagnostic Laboratory (the Netherlands) for performing reverse transcriptase polymerase chain reaction (RT-PCR) followed by reverse hybridization to determine rotavirus G and P types. The authors thank Silvia Damaso for her contribution to study design; to Rosalia Calamera and their team for acquisition of data; to Yolanda Guerra and safety team for management of safety information, to Bruno Anspach for global study management, to Satu Sumanen and Markku Pulkkinen for country study management, Geetha Subramanyam, Nancy Van-Driessche and Manjula K for providing technical assistance in the preparation of this manuscript. Rotarix and Infanrix hexa are trademarks of GlaxoSmithKline group of companies. Conflicts of interest statement: Prof. Vesikari T is on advisory boards of GSK, SP-MSD, Merck, Novartis and MedImmune and has received consultation and lecture fees from the same; Dr. Karvonen declares to have no conflict of interest; Dr. Bouckenooghe A was employed by GSK Biologicals at the time of the study; Drs. Smolenov I, Han HH and Mr. Suryakiran PV are employed by GSK Biologicals; Drs. Smolenov I and Han HH also have stock ownership with the commercial entity that sponsored the study; Dr. Han HH also has a personal relationship with other people or organizations that could bias the submitted work. Sources of support: GlaxoSmithKline Biologicals was the funding source and was involved in all stages of the study conduct and analysis. GSK Biologicals also funded all costs associated with the development and the publishing of the present manuscript. All authors had full access to the data and agreed with the submission of the publication.
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[10] Bernstein DI, Sack DA, Reisinger K, Rothstein E, Ward RL. Second year follow-up evaluation of live attenuated human rotavirus vaccine 89-12 in healthy infants. J Infect Dis 2002;186:1487–9. [11] Vesikari T, Karvonen A, Korhonen T, Espo M, Lebacq E, Forster J, et al. Safety and immunogenicity of RIX4414 live attenuated human rotavirus vaccine in adults, toddlers and previously uninfected infants. Vaccine 2004;22:2836–42. [12] Vesikari T, Karvonen A, Puustinen L, Zeng SQ, Szakal ED, Delem A, et al. Efficacy of RIX4414 live attenuated human rotavirus vaccine in Finnish infants. Pediatr Infect Dis J 2004;23:937–43. [13] Linhares AC, Velázquez FR, Pérez-Schael I, Sáez-Llorens X, Abate H, Espinoza F, et al. Efficacy and safety of an oral live attenuated human rotavirus vaccine against rotavirus gastroenteritis during the first 2 years of life in Latin American infants: a randomised, double-blind, placebo-controlled phase III study. Lancet 2008;371:1181–9. [14] van Doorn LJ, Kleter B, Hoefnagel E, Stainier I, Poliszczak A, Colau B, et al. Detection and genotyping of human rotavirus VP4 and VP7 genes by reverse transcriptase PCR and reverse hybridization. J Clin Microbiol 2009;47:2704–12. [15] Ward RL. Mechanisms of protection against rotavirus in humans and mice. J Infect Dis 1996;174:S1–8. [16] Franco MA, Angel J, Greenberg HB. Immunity and correlates of protection for rotavirus vaccines. Vaccine 2006;24:2718–31. [17] Velazquez FR, Matson DO, Guerrero ML, Shults J, Calva JJ, Morrow AL, et al. Serum antibody as a marker of protection against natural rotavirus infection and disease. J Infect Dis 2000;182:1602–9.
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Immunogenicity, reactogenicity and safety of the human rotavirus vaccine RIX4414 oral suspension (liquid formulation) in Finnish infants夽 T. Vesikari a,∗ , A. Karvonen a , A. Bouckenooghe b,1 , P.V. Suryakiran c , I. Smolenov b , H.H. Han d a
Vaccine Research Center, Medical School, University of Tampere, FIN-33014 Tampere, Finland GlaxoSmithKline Biologicals, Wavre, Belgium GlaxoSmithKline Pharmaceuticals Ltd., Bangalore, India d GlaxoSmithKline Biologicals, King of Prussia, USA b c
a r t i c l e
i n f o
Article history: Received 14 October 2010 Received in revised form 30 December 2010 Accepted 5 January 2011 Available online 14 January 2011 Keywords: RIX4414 Rotavirus vaccine Liquid formulation Finnish
a b s t r a c t The lyophilized formulation of a human rotavirus vaccine, RotarixTM (RIX4414) is highly immunogenic. In order to comply with the World Health Organization’s (WHO) recommendation, a liquid formulation of the vaccine that does not require reconstitution was developed. The immunogenicity, reactogenicity and safety of the liquid formulation were compared with lyophilized formulation in two Finnish studies. In Study A infants aged 6–12 weeks received two doses of the lyophilized or liquid formulation of the vaccine or placebo following a 0,1 month schedule. In Study B, infants aged 10–17 weeks received two doses of either liquid or lyophilized formulation of the vaccine. In both studies, anti-rotavirus IgA antibodies were assessed pre-vaccination and one month post-Dose 2. In Study A, the anti-rotavirus seroconversion rate was 90% (95% CI: 81.2–95.6%) and 83.7% (95% CI: 74.2–90.8%) in the groups that received the liquid and the lyophilized formulation of RIX4414, respectively; the respective anti-rotavirus IgA seroconversion rates in Study B were 88.6% (95% CI: 86.1–90.8%) and 90.5% (95% CI: 86.2–93.8%). Reactogenicity and safety profiles of the two vaccine formulations were similar. Liquid formulation of the rotavirus vaccine allows greater flexibility in supply and reduces logistical costs. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Worldwide, rotavirus is the most common cause of severe dehydrating gastroenteritis among children <5 years of age [1,2]. It has been estimated that annually rotavirus accounts for more than 25 million clinic visits, 2 million hospitalizations and 475,000–580,000 deaths among children aged <5 years [3]. Implementation of rotavirus vaccination is the only practical intervention for reducing the disease burden [4–7].
Abbreviations: WHO, World Health Organization; UNICEF, United Nations International Children’s Emergency Fund; GSK, GlaxoSmithKline; CCID, cell culture infective dose; DTPa-HBV-IPV-Hib, diphtheria toxoid, tetanus toxoid, acellular pertussis, hepatitis B, inactivated polio and Haemophilus influenzae type B; CI, confidence interval; ATP, according-to-protocol; GMC, geometric mean concentrations. 夽 ClinicalTrials.gov Identifier: 104480/NCT00137930 (Study A); 107876/NCT00382772 (Study B). ∗ Corresponding author. Tel.: +358 3 3551 8444; fax: +358 3 3551 8450. E-mail addresses: timo.vesikari@uta.fi (T. Vesikari), aino.karvonen@uta.fi (A. Karvonen), [email protected] (A. Bouckenooghe), [email protected] (P.V. Suryakiran), [email protected] (I. Smolenov), [email protected] (H.H. Han). 1 Current address: Sanofi Pasteur, Research and Development, Singapore. 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.01.004
RotarixTM (RIX4414, GlaxoSmithKline Biologicals), an oral human rotavirus vaccine containing G1[P8] strain is derived from the parent 89-12 strain [8–10]; the current vaccine was obtained by cloning at passage 43 and further passaging in cell culture which resulted in greater attenuation [11]. A lyophilized formulation of RotarixTM vaccine is currently licensed in over 110 countries. In pre-licensure studies, the lyophilized vaccine was found to be highly immunogenic, well tolerated and efficacious against severe rotavirus gastroenteritis and rotavirus gastroenteritis related hospitalizations [11–13]. The lyophilized formulation has to be reconstituted with a liquid calcium carbonate buffer prior to oral administration, which adds to complexity of vaccine administration. Furthermore, vaccine and buffer come in separate vials which make it bulky and consume cold storage space. Based on the recommendations of World Health Organization (WHO) and United Nations International Children’s Emergency Fund (UNICEF), a liquid formulation of RIX4414 vaccine that does not require reconstitution has been developed. The immunogenicity, reactogenicity and safety of the liquid formulation of RIX4414 were first evaluated in a feasibility study in Finland. Following positive experience, a larger non-inferiority trial was conducted. The results of the two Finnish trials are reported here.
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2. Materials and methods
2.3. Assessment of immunogenicity
2.1. Subjects and study design
In both studies, serum samples were collected prior to Dose 1 and one month post-Dose 2 of vaccine/placebo to measure antirotavirus IgA antibody concentration using ELISA at GSK Biologicals, Belgium, adapting a previously described method [8,9]. The assay cut-off was 20 Units/milliliter (U/ml). Stool samples were collected from all infants in Study A at Day 0 (or Day 0 minus 1 day), Day 7 ± 1 day and 15 ± 1 day after each dose and one month post-Dose 2 (or one month post-Dose 2 minus 1 day) of vaccine/placebo. Stool samples were tested for rotavirus using ELISA at Dr. R. Ward’s laboratory at the Children’s Hospital Medical Center, Cincinnati, OH, United States. Presence of vaccine strain rotavirus in any of the stool samples collected at pre-determined time point indicated vaccine take. In study A, vaccine take was defined as appearance of serum IgA antibodies to rotavirus in post-vaccination sera at a concentration of ≥20 U/ml and/or presence of vaccine virus in any stool sample collected from Dose 1 of vaccine/placebo to one month post-Dose 2 in infants who were negative for rotavirus IgA antibody prior to Dose 1. In study B, vaccine take was determined by seroconversion only, as appearance of rotavirus IgA antibodies in infants who were rotavirus IgA negative prior to Dose 1.
The phase II feasibility study (Study A) and the phase III noninferiority study (Study B) were multi-center studies conducted in Finland. The phase II study was conducted in five centers between August 2005 and November 2005 and the phase III study was conducted in 12 centers from November 2006 to April 2007. The studies were performed following the Good Clinical Practice guidelines and the 1996 version of Declaration of Helsinki. Each study was approved by the Ethics Committee of the Pirkanmaa hospital district. Parents/guardians of infants provided written informed consent prior to enrolment. Healthy infants aged 6–12 weeks (Study A) and 10–17 weeks (Study B) with a birth weight >2 kg were enrolled. In Study A, infants were randomized into four groups (4:1:4:1) to receive two oral doses of either the liquid formulation of RIX4414/placebo or the lyophilized formulation of RIX4414 (RotarixTM )/placebo following a 0,1 month schedule. The study was double-blind with respect to each of the vaccine formulation and their respective placebo; however, blinding between the two vaccine formulations was not technically possible because of the difference in appearance of the vaccines. Infants in Study B were randomized into four groups (1:1:1:1) to receive two oral doses of either the liquid formulation (any of the three lots) or lyophilized formulation of RIX4414 vaccine concomitantly with routine childhood vaccines. The three lots of the liquid formulation of the vaccine were pooled to form a single pooled liquid group. The study was doubleblind with respect to the three lots of the liquid formulation and open label with respect to the liquid and the lyophilized formulation. The vaccine/placebo was administered following a 0,1 month schedule. A standard SAS® program was used for generating the randomization list and a block randomization was used in order to ensure that the balance between the treatment arms were maintained. A unique treatment number identified the vaccine/placebo doses that were to be administered to the infants. Infants were excluded from participating in the studies if they had received any other investigational drug or vaccine 30 days prior to the administration of the first dose of the study vaccine or had a history of allergy or rotavirus gastroenteritis. Infants with acute illness at the time of enrolment also could not receive the vaccine until the condition was resolved.
2.2. Vaccine The study vaccines were provided by GlaxoSmithKline (GSK) Biologicals, Belgium. Each dose of RIX4414 oral suspension (liquid formulation) (RIX4414 liq; 1.5 ml) and RIX4414 (RotarixTM ) lyophilized formulation (RIX4414 lyo; 1 ml after reconstitution with calcium carbonate buffer) contained at least 106 median cell culture infective dose (CCID50 ) of live attenuated RIX4414 human rotavirus strain. The liquid formulation of RIX4414 contained sucrose as excipient and the content of sucrose in the liquid formulation is higher than one in the lyophilized formulation. The placebo preparation had the same constituents as the active vaccines but it did not contain the viral strain. In study A, the minimum time interval between study vaccine/placebo and routine infant vaccine administration was 14 days. Infants in Study B concomitantly received a combined diphtheria toxoid, tetanus toxoid, acellular pertussis, hepatitis B, inactivated polio and Haemophilus influenzae type B vaccine (DTPaHBV-IPV-Hib, Infanrix hexaTM ).
2.4. Assessment of reactogenicity and safety Parents/guardians of infants were provided diary cards to record solicited general symptoms (loss of appetite, fussiness/irritability, fever, diarrhea, vomiting, and cough/runny nose) during a 15-day (Study A) and 8-day (Study B) post-vaccination follow-up period. The intensity of each adverse event was assessed using a 4-point scale where “0” refers to ‘absent’ and “3” refers to ‘severe’. Grade 3 solicited general symptoms were defined as not eating at all (loss of appetite), preventing normal activities (fussiness/irritability, cough/runny nose), axillary temperature >39.0 ◦ C (fever), ≥6 looser than normal stools per day (diarrhea) and ≥3 episodes of vomiting per day (vomiting). Unsolicited symptoms were recorded for the 31-day follow-up period after each dose and serious adverse events (SAEs) were recorded throughout the study period. 2.5. Assessment of gastroenteritis Gastroenteritis was defined as diarrhea (≥3 looser than normal stools) with or without vomiting. Parents/guardians of infants were advised by the investigator to collect stool samples as soon as possible during each gastroenteritis episode between Dose 1 and one-month post-Dose 2. Gastroenteritis stool samples were tested for rotavirus by ELISA (Ward laboratory for Study A and GSK laboratory for Study B). Rotavirus positive stool samples were further tested to determine the G and P type using reverse transcriptase polymerase chain reaction (RT-PCR) at Delft Diagnostic Laboratory, Delft, the Netherlands [14]. Wild-type G1 rotavirus was differentiated from the vaccine G1 strain by sequencing. 2.6. Statistical analyses All statistical analyses were performed using SAS 8.2 and 95% confidence interval (CI) calculated using Proc StatXact-5. A sample size of 250 infants (100 infants each in RIX4414 liq and RIX4414 lyo groups and 25 infants each in the respective placebo groups) in Study A was planned to have at least 190 evaluable infants and a power of 80% to detect a difference in vaccine take between each of the vaccine formulations and the placebo groups (˛ = 5%, one-sided standardized asymptotic score test). In Study B, a sample size of 1200 infants (900 infants in the RIX4414 liq group and 300 infants in the RIX4414 lyo group) was
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Fig. 1. Number of infants enrolled and included in the ATP immunogenicity cohort (Study A).
planned to have at least 960 evaluable infants and 94% power to rule out that the seroconversion rate in the RIX4414 liq group was decreased by more than 10% as compared to the RIX4414 lyo group (one-sided test, ˛ = 2.5%, Bonferroni adjustment of beta). Immunogenicity analysis was performed on the according-toprotocol (ATP) cohort which included infants who complied with the protocol and for whom immunogenicity data was available at pre-vaccination and one month after the completion of vaccination. Safety analysis was performed on the total vaccinated cohort that included infants who received at least one vaccine/placebo dose.
In Study A, the percentage of infants with vaccine take after combined doses was calculated with their exact 95% CI. In Study B, non-inferiority of RIX4414 liq to RIX4414 lyo in terms of antirotavirus IgA seroconversion rate one month post-Dose 2 was established if the upper limit of the two-sided asymptotic standardized 95% CI for the difference in seroconversion rates between the RIX4414 lyo and (minus) the RIX4414 liq group was ≤10%. In both studies, seroconversion rates and geometric mean concentrations (GMCs) one month post-Dose 2 were tabulated with 95% CI. Percentage of doses followed by each of the solicited general symptom including those graded 3 in intensity during the 15-
Fig. 2. Number of infants enrolled and included in the ATP immunogenicity cohort (Study B).
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day/8-day post-vaccination follow-up period was tabulated with 95% CI. The percentage of infants with gastroenteritis episodes was also tabulated. During the 31-day follow-up period, the percentage of doses followed by unsolicited symptoms was tabulated with 95% CI. All SAEs reported during the entire study period were recorded. 3. Results 3.1. Study population and demography In Study A, 250 infants were enrolled (RIX4414 liq = 100, RIX4414 lyo = 100, Placebo liq = 25 and Placebo lyo = 25; pooled placebo = 50), and in Study B 1200 infants (RIX4414 liq = 900 and RIX4414 lyo = 300) were enrolled. The number of infants included in each of the study cohorts is presented in Figs. 1 and 2. Mean age of infants at Dose 1 in Study A was 9.1 weeks with a standard deviation of 1.94 weeks; in Study B, mean age was 11.6 with a standard deviation of 1.25 weeks (total vaccinated cohort). The distribution of males and females in the study groups was similar and majority (>98%) of the infants were white in both studies. 3.2. Immunogenicity Vaccine take on combined doses in Study A was 93.4% (95% CI: 86.2–97.5%) in RIX4414 liq group (N = 91) and 89.4% (95% CI: 81.3–94.8%) in RIX4414 lyo group (N = 94). By rotavirus IgA seroconversion only, vaccine uptake in RIX4414 liq group was 90% (95% CI: 81.2–95.6%) and in RIX4414 lyo group 83.7% (95% CI: 74.2–90.8%) (Table 1). None of the infants seroconverted in the pooled placebo group (Table 1). Stool samples collected at pre-determined time points showed that rotavirus vaccine shedding peaked on Day 7 after Dose 1 of vaccination (57.5% in the RIX4414 liq group 58.5% and in the RIX4414 lyo group). On day 15 post-Dose 1, 32.2% of the infants in the RIX4414 liq group and 25.6% of infants in the RIX4414 lyo group still shed the vaccine virus. In study B, seroconversion by rotavirus IgA antibody was seen in 90.9% (95% CI: 86.6–94.2%), 90.4% (95% CI: 86.1–93.7%) and 84.4% (95% CI: 79.3–88.7%), respectively, in infants receiving the three lots of liquid vaccine one month after Dose 2. The three groups were pooled for comparison with lyophilized formulation. The seroconversion rate in the group receiving RIX4414 lyo was 90.5% (95% CI: 86.2–93.8%). The GMCs in the recipients of the both vaccine formulation in each study are shown in Table 1. Non-inferiority of the RIX4414 liquid formulation to that of RIX4414 lyophilized formulation in terms of seroconversion rate was demonstrated as the upper limit of the 2-sided standardized asymptotic 95% CI for the group difference was <10% (1.87% [95% CI: −2.85% to 5.83%]).
Fig. 3. Solicited general symptoms-overall/dose during the 15-day post-vaccination follow-up period (Study A) (total vaccinated cohort).
In Study B, vomiting was reported following 13.9% (95% CI: 12.3–15.7%) of combined doses in the RIX4414 liq and 15.4% (95% CI: 12.5–18.6%) of combined doses in the RIX4414 lyo group; diarrhea was reported after 2.6% (95% CI: 1.9–3.5%) of combined doses and 1.8% (95% CI: 0.9–3.2%) of combined doses in the respective groups (Fig. 4). Gastroenteritis episodes were reported in 9.7% (95% CI: 7.8–11.8%) of infants in RIX4414 liq group and 7.3% (95% CI: 4.7–10.9%) of infants in RIX4414 lyo group throughout the study period. Two stool samples collected in infants from RIX4414 liq were positive for rotavirus and the G1P [8] vaccine strain was isolated from both the samples. One infant experienced diarrhea and mild fever during the first seven days after Dose 1. The child simultaneously received a 10-day course of antibiotic for prophylaxis of Streptococcus tonsillitis which started 2 days before onset of diarrhea. Gastroenteritis and fever were assessed to be related to vaccination. The other infant reported gastroenteritis 23 days postDose 2 of vaccination. This episode was assessed to be unrelated to vaccination by the investigator.
3.3. Reactogenicity and safety In study A, during the first 15 days period (overall/dose), vomiting was reported after 14.2% (95% CI: 9.7–19.9%) of doses in the RIX4414 liq group, 21.1% (95% CI: 15.7–27.4%) in the RIX4414 lyo group, and 13.1% (95% CI: 7.2–21.4%) of doses in pooled placebo group. Diarrhea was reported following 2.0% (95% CI: 0.6–5.1%), 5.5% (95% CI: 2.8–9.7%) and 4.0% (95% CI: 1.1–10.0%) of doses in the three groups, respectively (Fig. 3). The percentage of infants with gastroenteritis episodes reported between Dose 1 until one month post-Dose 2 was 6% (95% CI: 2.2–12.6%) in RIX4414 liq, 11% (95% CI: 5.6–18.8%) in RIX4414 lyo and 12% (95% CI: 4.5–24.3%) in the pooled placebo groups in Study A. Of these gastroenteritis episodes, rotavirus was isolated from four cases in RIX4414 lyo group; G1P [8] vaccine strain was isolated from one stool sample and the rotavirus serotype was unknown in the remaining three cases.
Fig. 4. Solicited general symptoms-overall/dose during the 8-day post-vaccination follow-up period (Study B) (total vaccinated cohort).
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Table 1 Anti-rotavirus IgA seroconversion rate and GMCs one month post-Dose 2 (ATP cohort for immunogenicity). N (n) Study A
RIX4414 liq RIX4414 lyo Pooled placebo
Study B
RIX4414 RIX4414 RIX4414 RIX4414 RIX4414
liqA liqB liqC liq* lyo
% (95% CI)
GMC value (95% CI)
80 (72) 86 (72) 44 (0)
90 (81.2–95.6) 83.7 (74.2–90.8) 0 (0–8.8)
301.3 (205.4–442.0) 360.6 (236.4–549.8) <20
242 (220) 260 (235) 244 (206) 746 (661) 252 (228)
90.9 (86.6–94.2) 90.4 (86.1–93.7) 84.4 (79.3–88.7) 88.6 (86.1–90.8) 90.5 (86.2–93.8)
384.4 (309.1–478.2) 418.8 (337.8–519.1) 324.4 (253.4–415.3) 374.7 (328.8–426.9) 331.8 (265.0–415.4)
N, number of infants with available results; n, number of infants with anti-rotavirus antibody concentration above the cut-off; %, percentage of infants with anti-rotavirus antibody concentration above the cut-off; 95% CI, 95% confidence interval; GMC, geometric mean concentration in U/ml; RIX4414 liqA, RIX4414 liqB, RIX4414 liqC: three lots of the RIX4414 liquid vaccine formulation; RIX4414 liq*: the three lots of the liquid formulation of RIX4414 are pooled to form the pooled liquid group.
The percentage of doses followed by at least one unsolicited symptom was similar in the two studies (data not shown). In Study A, two SAEs (bronchiolitis and otitis media) in RIX4414 liq and one SAE (otitis media) in RIX4414 lyo groups were reported. A total of 20 SAEs (mainly pneumonia, otitis media, gastroenteritis, bronchitis and laryngitis) in 18 infants were reported until one month after Dose 2 of vaccination in Study B. One SAE (infantile spasms) required long-time therapy in an infant belonging to RIX4414 lyo group and the infant was withdrawn from the study as a result of this SAE. No cases of intussusception were reported during the entire study period.
4. Discussion Higher manufacturing output, easy handling and low logistical costs of the liquid formulation of RIX4414 formed the basis for the manufacturing transition of the RIX4414 rotavirus vaccine from the lyophilized formulation to a liquid formulation. Although the liquid formulation has the same live vaccine virus at the same viral concentration as the licensed lyophilized formulation of RIX4414 (RotarixTM ), there was a need to assess the immunogenicity and safety of the new vaccine formulation in the target population. Immunogenicity was assessed in terms of rotavirus IgA antibody response. Rotavirus IgA antibodies, as measured by ELISA, have been used as an immunogenicity end point in all recent rotavirus vaccine studies and also as a proxy correlate of protection. While many factors may contribute to protection against rotavirus, a high titer of rotavirus serum IgA antibody is generally accepted as a surrogate marker for protective immunity [15–17]. The results of the phase II feasibility (Study A) study suggested that the RIX4414 liquid formulation was highly immunogenic and non-inferior to the RIX4414 lyophilized formulation of the vaccine. In Study A, the anti-rotavirus seroconversion rate was slightly higher in the RIX4414 liq group (90%) when compared to the RIX4414 lyo group (83.7%); the GMCs were higher in the RIX4414 lyo group. However, the difference was smaller when vaccine virus shedding was also considered as an end point. In Study B, the seroconversion was higher in the RIX4414 lyo group (90.5%) when compared to the RIX4414 liq group (88.6%). In earlier immunogenicity studies in Finland with the lyophilized rotavirus vaccine formulation, the seroconversion rate following two doses ranged between 80% and 96%, which is in line with what was observed in both the studies presented in this paper using liquid formulation [11,12]. Including the vaccine virus shedding in the study with lyophilized vaccine, the uptake was 96% [11]. Co-administration of routine vaccines with rotavirus vaccines in Study B did not have an impact on the immune response generated by both the rotavirus vaccine formulations. Even so, the uptake rate might have been higher if the vaccine virus shedding had been included as an end point.
Since the vaccine virus titer of the liquid formulation is similar to that in the lyophilized formulation, a similar safety profile is expected. At most, the different constituents of the two formulations might be associated with different side effects. Nevertheless, no difference in side effects was observed between the infants who received liquid formulation and those that received the lyophilized formulation in the two studies presented here. The percentage of infants reporting solicited and unsolicited symptoms and SAEs were similar between the two vaccine formulations. Keeping the advantages of using a liquid formulation of the vaccine in perspective and considering the high global demand and a higher manufacturing capacity, RIX4414 liquid formulation holds the promise to become a formulation of choice for global use.
Acknowledgements We thank the infants and their families for participating in this trial; all investigators, the study nurses and other staff members for contributing in many ways to this study. We acknowledge DDL Diagnostic Laboratory (the Netherlands) for performing reverse transcriptase polymerase chain reaction (RT-PCR) followed by reverse hybridization to determine rotavirus G and P types. The authors thank Silvia Damaso for her contribution to study design; to Rosalia Calamera and their team for acquisition of data; to Yolanda Guerra and safety team for management of safety information, to Bruno Anspach for global study management, to Satu Sumanen and Markku Pulkkinen for country study management, Geetha Subramanyam, Nancy Van-Driessche and Manjula K for providing technical assistance in the preparation of this manuscript. Rotarix and Infanrix hexa are trademarks of GlaxoSmithKline group of companies. Conflicts of interest statement: Prof. Vesikari T is on advisory boards of GSK, SP-MSD, Merck, Novartis and MedImmune and has received consultation and lecture fees from the same; Dr. Karvonen declares to have no conflict of interest; Dr. Bouckenooghe A was employed by GSK Biologicals at the time of the study; Drs. Smolenov I, Han HH and Mr. Suryakiran PV are employed by GSK Biologicals; Drs. Smolenov I and Han HH also have stock ownership with the commercial entity that sponsored the study; Dr. Han HH also has a personal relationship with other people or organizations that could bias the submitted work. Sources of support: GlaxoSmithKline Biologicals was the funding source and was involved in all stages of the study conduct and analysis. GSK Biologicals also funded all costs associated with the development and the publishing of the present manuscript. All authors had full access to the data and agreed with the submission of the publication.
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