Research paper
Human Vaccines 7:1, 74-80; January 2011; © 2011 Landes Bioscience
Immunogenicity of a human rotavirus vaccine (RIX4414) after storage at 37°C for seven days Angkool Kerdpanich,1 Kulkanya Chokephaibulkit,2,* Veerachai Watanaveeradej,1 Nirun Vanprapar,2 Sriluck Simasathien,1 Nopaorn Phavichitr,1 Hans L. Bock,3 Silvia Damaso,3 Yanee Hutagalung3 and Htay-Htay Han3 Pediatric Infectious Disease Unit; Phramongkutklao Hospital; 2 Pediatric Infectious Unit; Faculty of Medicine Siriraj Hospital; Bangkok, Thailand; 3GlaxoSmithKline Biologicals; Wavre, Belgium
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Key words: stability, cold chain, rotavirus, Thailand, immunogenicity, infants 6–12 weeks Abbreviations: CCID50, cell culture infectious dose 50; DTPa, diphtheria, tetanus and acellular pertussis vaccine; HBV, hepatitis B vaccine; Hib, Haemophilus influenzae type b; ELISA, Enzyme-Linked Immunosorbent Assay; SAS, statistical analysis system; GE, gastroenteritis; GMC, geometric mean concentration; ATP, according-to-protocol; CI, confidence interval; GSK, GlaxoSmithKline; IgA, immunoglobulin A; Group RIX4414_control, the group of subjects who received vaccine with buffer stored at 2–8°C; Group RIX4414_water, the group of subjects who received vaccine with water; Group Placebo, the group of subjects who received placebo with buffer; Group Placebo_water, the group of subjects who received placebo with water; Group RIX4414_37°C, the group of subjects who received vaccine with buffer stored for seven days at 37°C
Aim: The lyophilized formulation of the human rotavirus vaccine, RIX4414 (RotarixTM), is recommended to be stored at 2°C–8°C for optimal immunogenicity. In some settings with inadequate infrastructure for vaccine storage, unforeseen circumstances may cause cold chain breakage, resulting in the vaccine to be left at ambient temperatures. This study evaluated the heat stability of lyophilized RIX4414 vaccine in terms of immunogenicity when stored at tropical room temperature (37°C) for 7 days before reconstitution. Results: There was no statistically significant difference detected between RIX4414 vaccine stored at 2°C–8°C (Group RIX4414_control, n = 171) and that stored at 37°C for seven days (Group RIX4414_37°C, n = 47) in terms of seroconversion rate and vaccine take. The anti-rotavirus IgA seroconversion rate two months post-Dose 2 was 84.7% (95% CI: 78.1%–90%) and 87.8% (95% CI: 73.8%–95.9%) in Groups RIX4414_control and RIX4414_37°C, respectively. None of the 25 infants in placebo group seroconverted. The vaccine take in the respective vaccine groups were 88% (95% CI: 82.1%–92.5%) and 93.5% (95% CI: 82.1%–98.6%) and Geometric Mean Concentrations (GMCs) were 134.4 U/mL (95% CI: 104.5–172.9) and 163.7 U/mL (95% CI: 98.9–271.1). Methods: Healthy infants aged 6–12 weeks, received two oral doses of either the RIX4414 vaccine stored at 2°C–8°C, RIX4414 vaccine stored at 37°C for seven days or placebo, according to a 0, 2 month schedule. Seroconversion rates in terms of anti-rotavirus IgA antibody levels (cut off: ≥20 U/mL by ELISA), anti-rotavirus IgA antibody GMCs and vaccine take were calculated two months post-Dose 2. Conclusion: Lyophilized RIX4414 vaccine stored at 37°C for seven days before reconstitution has similar immunogenicity as the vaccine stored at 2°C–8°C. These results supported the use of RIX4414 in settings where the vaccine might be exposed to higher than the recommended storage temperatures.
©201 1L andesBi os c i enc e. Donotdi s t r i but e.
Introduction Rotavirus is the major causative agent of childhood diarrhea and diarrhea-related hospitalization worldwide.1 Annually, rotavirus has been estimated to cause 527,000 deaths (range 475,000– 580,000), approximately two million hospitalizations and greater than 25 million clinic visits worldwide in children younger than five years of age.2,3 In Thailand, rotavirus accounts for 30% – 36% of diarrheal disease in hospitalized cases and remains the leading cause of diarrhea contributing to childhood morbidity and mortality.4,5
Preventing rotavirus disease through effective vaccination strategy is considered to be the best method to curtail the rotavirus disease burden in both industrialized and developing countries as the disease burden does not seem to be alleviated by improvement in sanitation and water supply.6 GlaxoSmithKline (GSK) Biologicals has developed a lyophilized, oral live attenuated human rotavirus vaccine, RIX4414 (Rotarix TM) from the parent 89-12 strain.7,8 Two oral doses of the human rotavirus vaccine, RIX4414 has been found to be highly efficacious, immunogenic with no safety concerns in studies conducted across Latin America, Europe and Asia.9-11
*Correspondence to: Kulkanya Chokephaibulkit; Email:
[email protected] Submitted: 07/05/10; Revised: 08/11/10; Accepted: 08/25/10 DOI: 10.4161/hv.7.1.13412 74
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research paper
Research paper
©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 1. Study profile.
The lyophilized preparation of RIX4414 vaccine has to be stored at the recommended temperature of 2°C–8°C for the vaccine to possess optimal immunogenicity.12 In tropical resourcelimited settings, uncertainty in power supply, improper storage facilities, old refrigeration equipment, non-compliance with cold chain procedures and inadequate monitoring can lead to a break in the vaccine cold chain.13 This may result in a situation where in the vaccine may be stored at ambient temperatures for a period before the cold chain is restored. Although it is extremely crucial to maintain the cold chain for the vaccines at the recommended temperature during transportation and storage, it is important to evaluate the effect of high temperature on rotavirus vaccine stability in unforeseen circumstances when the cold chain is broken. A study was conducted to evaluate the effect on the RIX4414 vaccine when the vaccine was stored or reconstituted in circumstances different from the recommendations: i.e., when not reconstituted with a buffer or when stored for seven days at 37°C before reconstitution. This paper presents the part of the study data on the heat stability of lyophilized RIX4414 vaccine in terms of immunogenicity when stored prior to administration at tropical room temperature (37°C) for seven days at GSK, Biologicals, Rixensart as compared to vaccine stored at 2°C–8°C. The effect of buffer on the
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immunogenicity of RIX4414, which is another objective of this study, has been presented elsewhere.14 Results Study population. The study was conducted between March 2005 and December 2005 at Siriraj Hospital, Mahidol University and Phramongkutklao Hospital in Thailand. A total of 450 infants (Group RIX4414_control = 174, Group RIX4414_37°C = 50, Group Placebo = 26, Group RIX4414_water = 174, Group Placebo_water = 26) were enrolled. Data from 250 infants who belonged to Groups RIX4414_control, RIX4414_37°C and Placebo is presented in this manuscript. The number of infants included in each group is presented in Figure 1. Demographic profiles in terms of age, gender and race were similar across the three study groups presented in this paper (total vaccinated cohort). The mean age of infants at the time of Dose 1 of RIX4414 vaccine/placebo in the three groups ranged between 8.6–9 weeks; there were 50.6%, 46% and 57.7% females in the Groups RIX4414_control, RIX4414_37°C and Placebo, respectively. All participating infants were Thais. At the time of Dose 1 of RIX4414 vaccine/placebo, 28.7% (49/171) of infants in the RIX4414_control and 34% (16/47) of infants in the
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Figure 2. Seroconversion rate, vaccine take and GMCs 2 months post-Dose 2 (ATP cohort for immunogenicity).
©201 1L andesBi os c i enc e. Donotdi s t r i but e.
RIX4414_37°C groups were exclusively breast-fed (ATP cohort for immunogenicity). Immunogenicity. The anti-rotavirus IgA seroconversion rate two months post-Dose 2 was 84.7% (95% confidence interval [CI]: 78.1% –90%) and 87.8% (95% CI: 73.8% –95.9%) in the Group RIX4414_control and Group RIX4414_37°C, respectively. The respective Geometric Mean Concentrations (GMCs) were 134.4 U/mL (95% CI: 104.5–172.9) and 163.7 U/mL (95% CI: 98.9–271.1) (Fig. 2). None of the infants in Group placebo seroconverted two months post-Dose 2. No statistically significant difference was found between the Group RIX4414_control and Group RIX4414_37°C since the two-sided asymptotic standardized 95% CI for the difference in seroconversion rate between the groups contained the value 0 (Table 1A). This result was further confirmed as the two-sided 95% CI for GMC ratio between the two groups contained the value 1 (Table 1B). The vaccine take at two months post-Dose 2 in Group RIX4414_control was 88% (95% CI: 82.1% –92.5%) and in Group RIX4414_37°C was 93.5% (95% CI: 82.1% –98.6%). A statistically significant decrease in vaccine take rate was not detected between the Group RIX4414_37°C as compared to the Group RIX4414_control: p-value >0.05 (Table 1A). Gastroenteritis episodes. Gastroenteritis episodes were reported in 14.4% (95% CI: 9.5% –20.5%) of infants in RIX4414_control group, 12% (95% CI: 4.5% –24.3%) of infants in RIX4414_37°C group and 19.2% (95% CI: 6.6% – 39.4%) in the placebo group between Dose 1 of RIX4414 vaccine/placebo and two months post-Dose 2. Rotavirus antigen was detected from one infant in the RIX4414_control group (unknown rotavirus type) and two infants in the RIX4414_37°C group (one each of G1P[8] vaccine strain and
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unknown rotavirus type). No infants in the placebo group had rotavirus gastroenteritis. Reactogenicity and safety. During the 15 day post-vaccination follow-up period, irritability was the most commonly reported solicited general symptom in all the three groups (reported in up to 59.6% of doses) (Fig. 3). Grade 3 symptoms following vaccination recorded at a very low frequency in all groups and did not exceed 4.1% of doses. The percentage of infants reporting at least one unsolicited symptom within 31 days follow-up period did not exceed 29% of doses in the three groups. No fatal serious adverse events (SAEs) were reported. Eight infants reported non-fatal SAEs during the study period: four infants from the RIX4414_control (gastroenteritis, urinary tract infection, neonatal hypertension, Escherichia urinary tract infection in one infant each), two infants from the RIX4414_37°C (convulsion, bronchiolitis in one infant each) and two infants from the placebo group (gastroenteritis in one infant, Escherichia urinary tract infection and febrile convulsions in one infant). Discussion
In developing countries, implementation of vaccination programs is of paramount importance to reduce the morbidity and mortality caused by vaccine-preventable diseases. The success of a vaccination program can be hindered in tropical developing countries due to the lack of adequate infrastructure in ensuring vaccine cold chain, which is very critical, especially for live attenuated vaccines. Despite rotavirus being a major cause of healthcare utilization,15 rotavirus vaccine is currently not included in the Expanded Program of Immunization (EPI) in many developing countries, including Thailand. As the rotavirus vaccine may potentially be included in EPI in many resource-limited settings, it is important
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Table 1A. Difference between groups in terms of seroconversion rate for anti-rotavirus IgA antibody and vaccine take at 2 months post-dose 2 (ATP cohort for immunogenicity) RIX4414_control
Criteria
RIX4414_37°C
Difference (RIX4414_control-RIX4414_37°C)
p-value4
N
%
N
%
% (95% CI3)
Seroconversion rate1
157
84.7
41
87.8
-3.09 (-12.79, 11.08)
NC5
Vaccine take2
167
88
46
93.5
-5.45 (-12.97, 6.17)
0.854
For the criteria of seroconversion rate, N = number of infants with available results; % = percentage of subjects who seroconverted at two months after the second dose. 2For the criteria of vaccine take, N = number of subject with available anti-rotavirus IgA results at two months post-Dose 2 or with vaccine virus (rotavirus in stools collected at pre-determined time points or vaccine virus in stools collected in case of GE episode) in stools collected after Dose 1 up to two months after the second dose; % = percentage of subject who seroconverted at two months after the second dose or with vaccine virus (rotavirus in stools collected at pre-determined time points or vaccine virus in stools collected in case of GE episode) in stools collected after Dose one up to two months after the second dose. 395% CI = asymptotic standardized 95% confidence interval. 4p-value = one-sided asymptotic standardized p-value for the null hypothesis that there is no decrease in proportion with the RIX4414_37°C group as compared to RIX4414_control group. 5NC = Not computed. 1
Table 1B. GMC ratios for anti-rotavirus IgA antibodies (ATP cohort for immunogenicity)
Criteria
GMC
RIX4414_control
RIX4414_37°C
GMC Ratio (95% CI) (RIX4414_control/ RIX4414_37°C)
N
GMC
N
GMC
GMC (95% CI)
157
134.4
41
163.7
0.82 (0.47, 1.43)
is a relatively heat-tolerant virus in nature and can survive in feces for more than two months at ambient tropical temperatures.17 Moreover, the stabilizer used in the RIX4414 may further increase stability of the vaccine virus. The efficacy of this vaccine can be expected in cases of inadvertent breaks in the cold chain for up to seven days. It must be emphasized, however, that the vaccine is recommended to be stored at 2°C–8°C and cold chain be maintained throughout the storage period for the vaccine to possess optimal immunogenicity and to avoid vaccine wastage. Furthermore, it is recommended that RIX4414 vaccine after reconstitution in buffer be stored at 2°C–8°C and used within 24 h.18
©201 1L andesBi os c i enc e. Donotdi s t r i but e.
N = number of subjects with available results. 95% CI = 95% confidence interval for the GMC ratio (Anova model).
to understand the stability of the vaccine when exposed to room temperature due to cold chain breakage. In the current study, the stability of the rotavirus vaccine, RIX4414, was assessed by evaluating the impact of high temperature on vaccine immunogenicity. The results showed that RIX4414 vaccine stored at 37°C for seven days had similar immunogenicity in terms of anti-rotavirus antibody seroconversion rate, GMCs and vaccine take when compared to control vaccine, which was stored at the recommended 2°C–8°C. The safety and reactogenicity of the vaccine was also similar in all the three groups assessed in this study. Two months after the second vaccine dose, at least 84% of infants in both groups showed vaccine take and had anti-rotavirus antibody concentration ≥20 U/mL. Additionally, there was no statistically significant difference detected between the Group RIX4414_control and Group RIX4414_37°C in terms of seroconversion rates and GMCs. This clearly indicated that RIX4414 was stable at 37°C for up to seven days and showed similar immunogenicity to the vaccine stored at the recommended temperature. Cold chain is crucial for effective vaccine delivery. Live attenuated vaccines have been known to be heat sensitive. Oral poliovirus vaccine (OPV) is an example of a live vaccine that is extremely heat-sensitive which rapidly loses its ability to generate optimal immune response when stored at high temperatures.16 Thermostability studies with OPV demonstrated that the vaccine was stable at -20°C for an extended period and was stable for over six months at 2°C–8°C. However, if the cold chain was broken accidentally, the vaccine was stable only for two days at 37°C.12 In contrast to OPV, the results from this study suggested that rotavirus vaccine RIX4414 was much more heat stable. Rotavirus
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Materials and Methods
Subjects and study design. In this partially blind, phase IIIb, placebo-controlled study (Rota-039; e-Track study number: 103477; ClinicalTrials.gov study Identifier: NCT00169455), healthy infants aged 6–12 weeks who had received BCG and two doses of hepatitis B vaccine were enrolled to receive two doses of the study vaccine or placebo according to a 0, 2 month schedule. The study was performed in accordance with Good Clinical Practice and was approved by the institutional ethics committee. The parents or guardians of participating infants provided their consent by signing the written informed consent form prior to initiation of any study-related procedures. Infants were randomly (7:7:1:1:2 ratio) allocated to one of the following groups: Group RIX4414_control (vaccine with buffer stored at 2–8°C), Group RIX4414_water (vaccine with water), Group Placebo (placebo with buffer), Group Placebo_water (placebo with water) and Group RIX4414_37°C (vaccine with buffer stored for seven days at 37°C). The data from Groups RIX4414_ control, RIX4414_37°C and Placebo are presented and discussed in this manuscript; the data from the other groups has been presented elsewhere.14 Infants who had received any other investigational drug or vaccine or had a history of gastrointestinal disease or rotavirus gastroenteritis, were allergic to any of the vaccine components or had a history of immunosuppressive or immunodeficient condition were excluded from participating in the study. Vaccine. Infants in Groups RIX4414_control and RIX4414_37°C received the lyophilized formulation of the
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©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 3. Solicited general symptoms with exact 95% CI during the 15 day post-vaccination follow up period, for all doses (Total vaccinated cohort).
RIX4414 vaccine reconstituted with liquid calcium carbonate buffer (calcium carbonate 60 mg/mL and xanthane 0.25% in 1 mL of water for injection). Each dose of RIX4414 vaccine contained at least 106.0 Cell Culture Infective Dose 50 (CCID50) of the RIX4414 strain. The placebo was identical in appearance and composition to the active vaccine but it did not contain the vaccine viral strain. During the study period, participating infants were offered commercially available GSK Biologicals’ diphtheria toxoid, tetanus toxoid, acellular pertussis, inactivated polio and H. influenzae type b combination vaccine (Infanrix TM-IPV/Hib) at two and four months of age and diphtheria toxoid, tetanus toxoid, acellular pertussis, hepatitis B, inactivated polio and H. influenzae type b combination vaccine (Infanrix hexa TM) at six months of age. Assessment of immunogenicity. Serum samples were collected from all infants before vaccination and two months post-Dose 2 to evaluate the anti-rotavirus antibody concentrations at GSK Biologicals’ laboratory using Enzyme-Linked Immunosorbent Assay (ELISA; assay cut-off = 20 U/mL) that was based on the test initially designed by Prof. R.L. Ward.7,8
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Stool samples were collected at pre-determined time points from all infants at Day 0, 7 ± 1 days and 15 ± 1 days after each dose of vaccine or placebo. These stool samples were tested by ELISA to detect rotavirus. Presence of rotavirus in any stool sample collected at pre-determined time points indicated replication of vaccine virus in the gut. Vaccine take was defined as the appearance of serum rotavirus IgA 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 RIX4414 vaccine/placebo up 2 months post-Dose 2 in infants who were negative for rotavirus prior to Dose 1 of RIX4414 vaccine/placebo. During each gastroenteritis episode (defined as diarrhea [three or more looser than normal stools] with or without vomiting) that occurred during the study period, parents/guardians of infants were advised to collect stool samples as soon as possible. These stool samples were tested for rotavirus using ELISA at Dr. R.L. Ward laboratory, Children’s Hospital Medical Center, Cincinnati, OH, USA. Rotavirus positive stool samples were tested further with reverse transcriptase-polymerase chain reaction (RT-PCR) at DDL Diagnostic Laboratory, Delft, the Netherlands to determine the G and P types.19
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Assessment of reactogenicity and safety. Diary cards were provided to the parents/guardians of infants to record the solicited general symptoms occurring during the 15 day follow up period after each vaccine dose. The solicited general symptoms were loss of appetite, fussiness/irritability, fever, diarrhea, vomiting and cough/runny nose. The intensity of each of these symptoms was graded on a 3-point scale where “0” indicates normal and “3” indicates severe. Grade 3 symptoms were defined as not eating at all (loss of appetite), preventing normal activities (fussiness/irritability, cough/runny nose), axillary temperature >39°C (fever), ≥6 looser than normal stools per day (diarrhea) and ≥3 episodes of vomiting per day (vomiting). Unsolicited symptoms within the 31-day post-vaccination follow-up period following each vaccine/placebo dose were recorded. Safety of the vaccine in terms of SAEs was also recorded throughout the study. Statistical analyses. Primary immunogenicity analysis was performed on the according-to-protocol (ATP) cohort. This included infants who complied with all the protocoldefined criteria, and who were initially negative for rotavirus. Immunogenicity was evaluated in terms of seroconversion rate (defined as the appearance of anti-rotavirus IgA antibody concentration ≥20 U/mL) and vaccine take with exact 95% CI. The corresponding GMCs were also tabulated with 95% CI. Two-sided asymptotic standardized 95% CI for difference in anti-rotavirus seroconversion rate and vaccine take between the Group RIX4414_control and Group RIX4414_37°C were computed. The corresponding one-sided asymptotic standardized p-value comparing the vaccine take between Group RIX4414_control and Group RIX4414_37°C was computed. If the p-value was
