A dose escalation safety and immunogenicity study of a new live attenuated human rotavirus vaccine (rotavin m1) in vietnamese children

Vaccine 30S (2012) A114–A121 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine A dose-escalation safety and immunogenicity study of a new live attenuated human rotavirus vaccine (Rotavin-M1) in Vietnamese childrenଝ,ଝଝ Dang Duc Anh a,∗ , Nguyen Van Trang a , Vu Dinh Thiem a , Nguyen Thi Hien Anh a , Nguyen Duc Mao b , Yuhuan Wang c , Baoming Jiang c,∗∗ , Nguyen Dang Hien d,1 , Le Thi Luan d,1 , the Rotavin-M1 Vaccine Trial Group2 a The National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam Preventive Medicine Center, Phu Tho Province, Viet Nam c Centers for Disease Control and Prevention, CDC, Atlanta, GA, USA d Center for Research and Production of Vaccines and Biologicals, Hanoi, Viet Nam b a r t i c l e i n f o Article history: Received 13 May 2011 Received in revised form 14 July 2011 Accepted 25 July 2011 Keywords: Rotavirus vaccine Rotavin-M1 Vietnam Safety Immunogenicity Vaccine dose RotarixTM a b s t r a c t We tested a candidate live, oral, rotavirus vaccine (Rotavin-M1TM ) derived from an attenuated G1P [8] strain (KH0118-2003) isolated from a child in Vietnam The vaccine was tested first for safety in 29 healthy adults When deemed safe, it was further tested for safety and immunogenicity in 160 infants (4 groups) aged 6–12 weeks in a dose and schedule ranging study The vaccine was administered in low titer (106.0 FFU/dose) on a 2-dose schedule given months apart (Group 2L) and on a 3-dose schedule given month apart (Group 3L) and in high titer (106.3 FFU/dose) in doses months apart (Group 2H) and in doses month apart (Group 3H) For comparison, 40 children (group RotarixTM ) were given doses of the lyophilized RotarixTM vaccine (106.5 CCID50 /dose) month apart All infants were followed for 30 days after each dose for clinical adverse events including diarrhea, vomiting, fever, abdominal pain, irritability and intussusception Immunogenicity was assessed by IgA seroconversion and viral shedding was monitored for days after administration of each dose Two doses of Rotavin-M1 (106.3 FFU/dose) were well tolerated in adults Among infants (average weeks of age at enrollment), administration of Rotavin-M1 was safe and did not lead to an increased rate of fever, diarrhea, vomiting or irritability compared to RotarixTM , indicating that the candidate vaccine virus had been fully attenuated by serial passages No elevation of levels of serum transaminase, blood urea, or blood cell counts were observed The highest rotavirus IgA seroconversion rate (73%, 95%CI (58–88%)) was achieved in group 2H (2 doses – 106.3 FFU/dose, months apart) The dose schedules performed slightly better than the dose schedules and the higher titer doses performed slightly better than the lower titer doses These rates of seroconversion were similar to that of the RotarixTM group (58%, 95%CI (42–73%)) However more infants who received RotarixTM (65%) shed virus in their stool after the first dose than those who received RotavinM1 (44–48%) (p < 0.05) and the percent shedding decreased after subsequent doses of either vaccine Rotavin-M1 vaccine is safe and immunogenic in Vietnamese infants A trial in progress will assess the safety, immunogenicity and efficacy of Rotavin-M1 (2 doses at 106.3 FFU/dose) in a larger number of infants The trial registration numbers are NCT01375907 and NCT01377571 © 2011 Elsevier Ltd All rights reserved Introduction ଝ Presented in part: 9th International Rotavirus Symposium, June 2010, Johannesburg, South Africa ଝଝ Disclaimer: The findings and conclusions in this report are those of the authors and not necessarily represent the views of the Centers for Disease Control and Prevention ∗ Corresponding author Tel.: +84 439712989; fax: +84 438212660 ∗∗ Corresponding author Tel.: +1 404 639 2861; fax: +1 404 639 3645 E-mail addresses: ducanh@nihe.org.vn, ducanhnihe@hn.vnn.vn (D.D Anh), bxj4@cdc.gov (B Jiang) Drs Hien and Luan are members of the Board of Directors, POLYVAC, Vietnam See Appendix A 0264-410X/$ – see front matter © 2011 Elsevier Ltd All rights reserved doi:10.1016/j.vaccine.2011.07.118 Rotavirus (RV) is the most important cause of acute gastroenteritis in children worldwide In Vietnam rotavirus causes an estimated 122,000–140,000 hospitalizations and 2900–5400 deaths per year among children under years of age [1] Over the past 13 years, sentinel hospital surveillance identified rotavirus in 44–62% of children admitted for the treatment of acute diarrhea in Vietnam [2–4] Such a high burden of disease justified accelerated development of a new and locally manufactured vaccine against rotavirus in Vietnam It is estimated that if a vaccine was introduced in the current childhood immunization schedule, it could D.D Anh et al / Vaccine 30S (2012) A114–A121 reduce severe rotavirus disease by about 60% or more given current vaccine efficacies and coverage [5] The Government of Vietnam has pursued a policy to encourage local vaccine production so the country could be self-reliant with affordable vaccines for its population [6] Over the past decades, several locally produced vaccines for poliomyelitis, cholera, Japanese encephalitis, and Diphtheria–Pertussis–Tetanus have contributed to the reduction in the prevalence of these diseases and to the status of poliomyelitis-free While two commercial rotavirus vaccines, RotarixTM (GSK, Belgium) and RotaTeq® (Merck), have both been tested in Vietnam, only RotarixTM is currently available in private market The liquid formula of Rotarix when tested in two schedules, 1-month and 2-month interval between doses compared with placebo control in 375 children had a seroconversion rate of 63.3% and 81.5%, respectively [7] RotaTeq showed a seroconversion rate of 87.8% and an overall efficacy of 63.9% (72.3% in the first year and 64.6% in the 2nd year followingup) in a phase efficacy trial in Vietnam [8] However, neither of the two vaccines is currently available at an affordable price for the national program (e.g Rotarix in the private market costs US $35 per dose) Therefore, the candidate vaccine, Rotavin-M1, was developed in order to fill this need for a more affordable vaccine for Vietnamese children [6] This vaccine is similar to RotarixTM , and was developed by selecting a common G1P [8] strain and attenuating it through serial passages and plaque purification in qualified Vero cells under GLP conditions In this study, we sought to evaluate the safety and immunogenicity of Rotavin-M1 produced by the Center for Research and Production of Vaccines and Biologicals (POLYVAC) in adult volunteers and in infants in Vietnam In addition, we evaluated different dosages and schedules to determine the best regimen to test in a clinical trial Materials and methods 2.1 Vaccine The vaccine, Rotavin-M1, manufactured by POLYVAC-Vietnam, was developed from a G1P [8] strain recovered in 2003 from a child hospitalized for the treatment of acute gastroenteritis in Nha Trang city (KH0118-2003) [6] The master and working seeds of this vaccine were produced under GLP conditions using qualified Vero cells and reagents at the US Centers for Disease Control and Prevention (CDC) Pilot vaccine lot, passage 48, was produced by one passage in Vero cells from the working seed, which was provided by the Japanese Polio Research Institute and approved for vaccine production by WHO These cells have been used for oral poliomyelitis vaccine production at POLYVAC The master virus seed for RotavinM1 was tested for porcine circovirus using real-time RT-PCR at the US CDC and appeared to be free of porcine circovirus DNA The test for porcine circovirus in pilot vaccine lot was not done 2.2 Participants and study design The trials were planned in two stages, the first – a Phase trial for safety in adult volunteers of a high titer preparation of the vaccine (106.3 FFU/dose) When results of this trial were evaluated by the Data Safety and Monitoring Committee and the vaccine was deemed to be safe for further study in infants, a Phase and adaptive trial was conducted This trial assessed the safety and immunogenicity of two different preparations of vaccine, one of low titer (106.0 FFU/dose) and the second with high titer (106.3 FFU/dose) that was administered in either a vs dose schedules to infants 6–12 weeks of age A comparison group was included of infants who received the lyophilized RotarixTM A115 vaccine, an established rotavirus vaccine of GSK that was licensed to be used in Vietnam The study was conducted according to Good Clinical Practice and in accordance with the Declaration of Helsinki, as amended in Somerset West, Republic of South Africa, in October 1996 The protocol and consent form was reviewed and approved by the Ethical and Scientific Committees of the National Institute of Hygiene and Epidemiology (NIHE) and of the Ministry of Health, Government of Vietnam, prior to initiating the study 2.2.1 Phase study The Phase study was conducted in a Career Training School, Thanh Son district, Phu Tho province with a total of 29 healthy adult volunteers 18–49 years of age Following receipt of informed consent, each of the volunteers was screened by a physician to ensure they were healthy with no active medical problems and asked to provide a blood specimen to test for blood counts and levels of blood urea nitrogen (BUN) and transaminase The volunteers then each received doses of the high titer vaccine, 106.3 focus-forming units [FFU], at 1-month interval After administration of each dose of the vaccine, the volunteers were followed daily for 10 days for adverse events and for fecal sample collection During the next 20 days, the volunteers were followed by phone to ensure they had no sequelae (e.g diarrhea, vomiting and intussusception) Serum samples taken before and 30 days after the 1st and 2nd doses were tested for blood counts, BUN concentration and serum transaminase levels Data on the volunteers were reviewed by the Data Safety Monitoring Board (DSMB) No adverse events or changes in blood counts, BUN or transaminase were reported The DSMB judged the vaccine to be safe permitting the studies to continue in infants 2.2.2 Phase study Phase was a dose and schedule ranging study, conducted at 12 medical centers in Thanh Son district, Phu Tho provinces from November 2009 through April 2010 Infants 6–12 weeks of age were eligible for inclusion in the study if they were born at full term (38 weeks) and were free of obvious health problem Infants were excluded if they were immunocompromised, had a history of allergic reaction to any vaccine components or had received vaccines against rotavirus or were involved in any other vaccine trials at the same time Infants (n = 200) were randomly assigned to groups (40 infants/group) (Fig 1) Two groups received oral doses of Rotavin-M1 in of titers – 106.0 or 106.3 FFU at 6–12 weeks of age (for the first dose) and months later for the second dose (groups 2L and 2H), respectively These vaccine titers were also given to infants on a 3-dose schedule, beginning at 6–12 weeks of age for the first dose and month and months later for the 2nd and 3rd doses (groups 3L and 3H, respectively) RotarixTM was used as the vaccine control and was given to 40 infants at 6–12 weeks of age and month later (Group RotarixTM ) GSK recommends that the first dose of RotarixTM be started between and 14 weeks of age and that the second dose be separated by at least month The vaccine recipients, the parents/guardians, the laboratory staff, the field teams and working doctors did not know the coding assignment of these groups Other vaccines (BCG, oral polio vaccine, Diphtheria–Tetanus–Pertussis and hepatitis B) used in the country’s Expanded Program of Immunization (EPI) were administered normally to these infants on different days (10–20 days before or after rotavirus vaccine was administered) 2.3 Assessment of immunogenicity Serum samples were obtained for testing levels of anti-rotavirus IgA and IgG antibody on the day that the first dose was administered and month after the second or third dose In addition, serum samples were also obtained from groups that received doses of vaccine (groups 3L and 3H) immediately before the A116 D.D Anh et al / Vaccine 30S (2012) A114–A121 assurance, an anonymized subset of serum specimens (52 samples) were also shipped and tested at CDC Agreement between two laboratories (antibody titers within 2-fold dilution of the samples) was >90% 2.4 Assessment of reactogenicity and safety For 30 days following each vaccine administration, parents or guardians were asked to note general symptoms (cough, running nose, diarrhea, irritability, loss of appetite, fever and vomiting) on a daily diary card Daily temperature was recorded and a temperature >38 ◦ C was considered as fever Any severe unsolicited symptoms and serious adverse events were reported throughout the study period (90 days for each child) Aliquots of blood from each child at each time point were also assayed for serum transaminase and BUN 2.5 Viral shedding and strain characterization Fig Study design including vaccination and sampling schedules for different vaccination groups Arrows in figure indicate times for each vaccination dose (wide arrows) and for collection of samples of blood (thin solid arrows) and stool (dotted arrows) In 3-dose regimen (a), groups 3L and 3H received doses of Rotavin-M1 106.0 FFU/dose and 106.3 FFU/dose, respectively In 2-dose regimen (b), groups 2L and 2H received doses of Rotavin-M1, 106.0 FFU/dose and 106.3 FFU/dose, respectively (n = 40/group) Group RotarixTM received doses of RotarixTM and only had serum samples and according to schedule (c) 3rd dose (Fig 1) Each blood sample from a child was collected in tubes, one with anti-coagulant (EDTA) (whole blood) and one without anti-coagulant (serum) Serum and whole blood samples were immediately transferred to the provincial hospital for analysis of blood cell counts (red blood cells, white blood cells and platelet), transaminase levels (aspartate aminotransferase, AST and alanine aminotransferase, ALT) and BUN within h after collection Aliquots of serum samples for antibody detection were stored at −20 ◦ C at the District Preventive Medicine Center, Thanh Son district, Phu Tho Province until they were shipped to the laboratory at NIHE The immunogenicity of the vaccine was evaluated at the Vaccine Immunology Laboratory, NIHE, by measuring seroconversion of rotavirus IgA antibody, using an end-point ELISA [9] Briefly, 96-well microtiter plates (NUNC, Langenselbold, Germany) were coated with rabbit-anti RRV hyperimmune serum (obtained from Dr Baoming Jiang, CDC) Virus (RRV) and mock-infected supernatant were added to the plates in alternate wells Serum samples in 2-fold serial dilutions starting at 1:10 were added to these virus/mock wells Biotinylated anti-human IgA (␣) (Kirkegaard and Perry Laboratory, Gaithersburg, Maryland) and peroxidase labelled extravidin (Sigma–Aldrich, Inc, St Louis, MO) were added for the detection of RV specific IgA antibody Positive and negative control sera were tested in the same manner Antibody titers in serum were calculated as the reciprocal of the highest dilution that gave a mean optical density greater than the cut-off value (mean + standard deviations of the negative control and blotto wells) An IgA titer of 20 or higher was considered positive Seroconversion was defined as a rise in anti-rotavirus IgA titer from undetectable (≤10) in prevaccination serum to ≥20 in post-vaccination serum or a ≥4-fold rise from pre-vaccination to post-vaccination serum For quality We attempted to collect daily stool samples during the days following each dose to assess virus shedding In addition, stool samples were also collected at every episode of diarrhea during the study period and tested for rotavirus antigen by ELISA (ProSpecT, Oxoid, UK) All rotavirus positive specimens were G and P-typed by RT-PCR [3,10] To distinguish vaccine from wild viruses, we sequenced the VP7 gene of the G1P [8] samples from diarrhea cases and selected G1P [8] samples collected within days of vaccine administration (non-diarrheal samples), using an ABI Prism BigDye Terminator Cycle Sequencing (Applied Biosystems, Foster City, CA) and compared the sequences with the corresponding gene sequences of Rotavin-M1 and RotarixTM 2.6 Statistical analysis Data was managed using Microsoft Visual Foxpro 7.0 software (Microsoft) and analysed using the Stata 11.1 program The mean blood cell counts and concentrations of BUN and transaminase were compared between groups with a Paired Student’s t-Test Between groups, the percentages of children with adverse events were compared using Fisher’s Exact Test The analysis for reactogenicity was performed on the intentionto-treat population (including all children who received at least dose of vaccine) The number of children with general symptoms was determined for each group after administration of each vaccine dose and compared between groups The analysis of immunogenicity was also performed for both the per protocol and intention-to-treat populations (at least doses of vaccine were required) The IgA seroconversion rate (with 95%CI) was calculated for each group to evaluate the immune responses induced by the vaccines and geometric mean antibody titers (GMT) were calculated for those individuals who seroconverted Viral shedding was calculated as the percentage of children shedding virus each day post-vaccination when stool samples were available In addition, the percent of children who shed at least once during the 7-day observation period after each dose was also calculated Results 3.1 Safety in adult volunteers We first tested the safety of doses of the higher titer vaccine (106.3 FFU/dose) in 29 adult volunteers aged 18–40 years During the 30 days post-vaccination of each dose, no diarrhea or severe adverse reaction was reported by any of the volunteers One month after each dose, neither blood cell counts nor BUN concentration increased Serum transaminase levels stayed below D.D Anh et al / Vaccine 30S (2012) A114–A121 A117 A total of 200 subjects (119 boys and 81 girls) were enrolled in the infant study Their mean age (±SD) was 8.7 ± 1.6 weeks at the time they received the first dose and 17.2 (±1.6) weeks at the time of 2nd dose for groups 2L and 2H For groups 3L and 3H (the 3-dose group), the mean age was 13 (±1.6) weeks at the time of 2nd dose and 17.9 (±1.6) at the 3rd dose After each vaccine dose, the children gained weight and height normally and we found no difference between vaccination groups The blood cell counts, serum transaminase levels and BUN were normal and no significant increase was observed over the range of normal healthy infants after administration of each vaccine dose of RotarixTM or Rotavin-M1 but none of the differences between groups reached significance Of special notes, within days after receiving the first dose, children from group 3L (7.5%), from group 2H (7.5%), from group 3H (2.5%) and from group RotarixTM (2.5%) exhibited mild diarrhea Given the small numbers, this difference was not statistically significant and suggested that the vaccine virus had been adequately attenuated (Table 1) Rotavirus antigen was isolated in fecal specimens from case in each of the groups RotarixTM , 3H and 2H during this period From days 8–30, diarrhea episodes were reported only in groups RotarixTM and 3H (1 and cases, respectively), of which only one case in group 3H was positive for rotavirus While a few infants had mild diarrhea after administration of dose or 3, only case in group 3H (within days after dose 2) and case in group 3L (within days after dose 3) were identified as rotavirus G1P [8] Sequences of VP7 gene of these samples revealed that they were 100% homologous with the sequence of Rotavin-M1 or RotarixTM (in respective groups) Of note, RotarixTM and Rotavin-M1 share 93.6% homology in the 793 nucleotide sequence of VP7 gene and 94.7% homology in the 263 amino acid sequence of the encoded protein 3.3 Reactogenicity and safety of infant study 3.4 Immunogenicity During the entire observation period (90 days after the first dose), no serious adverse events that required hospitalization and no cases of intussusception were recorded We observed all infants for acute reactions for 30 following vaccination and parents observed their infants for 30 days after each dose to note adverse events including diarrhea, fever, vomiting, loss of appetite, cough, allergy, abdominal pain and irritability These symptoms following vaccination were grouped into time periods: immediate reactions (i.e within 30 min), short term reactions (within days post-vaccination) and longer term reactions (from through 30 days post-vaccination) (Table 1) After each dose, no immediate reactions were observed After any dose fewer children reported any symptoms within days compared to the 3-week period from to 30 days past vaccination Fewer children reported any symptoms after dose and dose 3, compared with dose Irritability and fever were the most frequently reported symptoms following administration any dose Serum samples were analysed at NIHE and anonymized results were confirmed at CDC Most infants (94.5%) did not have detectable RV-IgA before vaccination and all children with one prevaccination serum and at least one post-vaccination serum samples were included in the analysis of immunogenicity One of the children who was seropositive before vaccination seroconverted (group 3H, data not shown) One month after the 2nd dose of vaccine, the rate of seroconversion to Rotavin-M1 vaccine was 61% (95%CI (45%, 76%)) for group 2L (106.0 FFU) and 73% (95%CI (58%, 88%)) for group 2H (106.3 FFU) (Table 2) The IgA-GMT, ranging from 76 (group 2H) to 89 (group 2L), did not differ between these two groups For groups receiving doses of vaccines (groups 3L and 3H), anti-RV-IgA seroconversion rates at month after doses of vaccine were 51% (95%CI (36%, 67%)) for group 3L (106.0 FFU) and 61% (95%CI (45%, 77%)) for group 3H (106.3 FFU) The IgA seroconversion rates month after dose increased to 56% (95%CI (39%, 71%)) in group 3L and 63% (95%CI (46%, 79%)) in group 3H The IgA-GMT did not increase significantly in group 3H (from 61 post dose to 83 40 IU/ml for >85% of volunteers or slightly elevated (42–56 IU/ml) in 10% of volunteers after doses of vaccination One individual had elevated levels of both SGOT and SGPT (71 and 48 IU/ml, respectively) before vaccination and the levels remained in this range after doses of vaccine No shedding of the vaccine virus occurred in these adults following vaccination Thus the Ethical Review Committees allowed the vaccine to be tested further in healthy infants 3.2 Infant study Table Number of infants with adverse events after vaccination with Rotavin-M1 or RotarixTM during 30 days after each dose Diarrhea is defined as having more than loose stools per day Diarrhea in these children is not accompanied with vomiting or fever (>37.5 ◦ C) In the majority of cases, diarrhea lasted 1–4 days Difference between groups was not significant *Figure in bracket indicates the number of samples positive for Rotavin-M1 or Rotarix vaccine Symptoms 2L (106.0 FFU) Days 0–7 After 1st dose Diarrhea Irritability Fever Vomit Abdominal pain After 2nd dose Diarrhea Irritability Fever Vomit Abdominal pain After 3rd dose Diarrhea Irritability Fever Vomit Abdominal pain 40 0 38 0 2H (106.3 FFU) 8–30 10 0 0 Days 0–7 40 3(1)* 0 36 0 3L (106.0 FFU) 8–30 14 0 0 Days 0–7 40 1 40 2 39 1(1)* 0 3H (106.3 FFU) 8–30 12 1 7 0 Days 0–7 40 1(1)* 1 39 1(1)* 0 35 1 0 RotarixTM 8–30 4(1)* 12 0 6 0 0 Days 0–7 40 1(1)* 0 40 0 8–30 0 10 0 A118 D.D Anh et al / Vaccine 30S (2012) A114–A121 Table Anti-RV-IgA antibody responses after administration of Rotavin-M1 vaccine (groups 2L, 2H, 3L and 3H, compared to RotarixTM ) Group 2L 2H 3L (post dose 2) 3L (post dose 3) 3H (post dose 2) 3H (post dose 3) RotarixTM Vaccine load (FFU) Doses 106.0 106.3 106.0 2 106.3 Na GMTe Seroconversion (%) 38a,b 33a,b 39a 36b 36a 32b 40a,b c d % (95%CI) GMT (95%CI) 61 73 51 56 61 63 58 (45, 76) (58, 88) (36, 67) (39, 71) (45, 77) (46, 79) (42, 73) 89 76 94 71 61 83 82 (58, 138) (44, 126) (56, 162) (35, 141) (43, 87) (55, 126) (52, 135) post dose 3), while the GMT did not increase in group 3L The RV-IgA seroconversion rate in group RotarixTM was 58% (95%CI (42%, 73%)) The IgA-GMT among seropositive children did not differ between groups (Table 2) For children receiving doses of vaccine (groups 3L and 3H), serum samples were collected month after dose and month after dose to determine whether a third dose might improved the seroresponse The 3rd dose induced seroconversion in and more children in group 3L and 3H, respectively, who had failed to seroconvert after the first doses The majority of children (14 in group 3L and 16 in group 3H) converted after second dose and did not further convert after the third dose Three children (7.5%) from each group (3L and 3H) seroconverted after both dose and dose 3.5 Viral shedding after each dose We examined the kinetics of rotavirus shedding in vaccinated children (Figs and 3) The prevalence of children shedding virus was greater in the group of children who received RotarixTM (65% after the 1st dose) vs any group that received Rotavin-M1 (44–48% after the 1st dose) (Fig 2) Furthermore, after the first dose, shedding of RotarixTM peaked or days earlier than shedding of Rotavin-M1 (Fig 3) Nonetheless, we observed little difference in Percent of infants with virus shed in stool Note: a Number of subjects for immunogenicity analysis (intention-to-treat) b Number of subject for immunogenicity analysis (per protocol) c Percentage of subjects seroconverted for RV-IgA d 95%CI, exact 95% confidence interval e GMT: geometric mean titers (and 95%CI) calculated for seropositive samples only 60% 2L 3L 50% Rotarix 3H 2H 40% 30% 20% 10% 0% Days after dose 1 Days after dose 2 Days after dose Fig Shedding of vaccine virus after each dose Percent of infant shed virus at any time during days after each dose was illustrated Asterisk: group RotarixTM ; opened triangle: Group 2L, doses, 106.0 FFU; opened circle: Group 2H: doses, 106.3 FFU/dose; closed triangle: Group 3L, doses, 106.0 FFU/dose; and closed circle: Group 3H, doses, 106.3 FFU/dose the pattern of shedding between the groups received RotavinM1 Viral shedding reduced significantly in any group after dose (6–20%) (Fig 2) Interestingly after dose 3, 30–37% of children shed the virus at day post-vaccination in both 3L and 3H groups Discussion Fig Dynamics of viral shedding within days post-vaccination after each dose Percent of children shed virus in stool per day after each vaccination dose was illustrated The ratio was calculated per number of stool available each day because not all children could produce stool every day for days Stool samples were considered positive for RV if it is positive in ELISA (ProSpecT) and confirmed by G and P genotyping All the samples collected during days after each dose are typed as G1P [8] Asterisk: group RotarixTM ; opened triangle: Group 2L, doses, 106.0 FFU; opened circle: Group 2H: doses, 106.3 FFU/dose; closed triangle: Group 3L, doses, 106.0 FFU/dose; and closed circle: Group 3H, doses, 106.3 FFU/dose This report documents the first Phase and Phase clinical studies of a new candidate rotavirus vaccine developed in Vietnam, Rotavin-M1 The live oral vaccine, which has been described previously, is derived from the most common strain of Rotavirus, genotype G1P [8], obtained from a Vietnamese child with diarrhea, attenuated by cell passage (>30×), plaque purification, and prepared in Vero cells for human studies [6] A Phase trial in 29 adult volunteers demonstrated that the vaccine administered orally in a titer of 106.3 FFU/dose was not associated with symptoms, adverse events or laboratory changes in blood counts or selected chemistry and little virus shedding, similar to that reported for RotarixTM [11] The DSMB reviewed the data and approved the continuation of studies in infants In the Phase 1–2 adaptive study, the candidate vaccine administered in either a low (106.0 FFU/dose) or high (106.3 FFU/dose) titer on a 2- or 3-dose schedule to infants 6–12 weeks of age did not cause significant or more diarrhea than that associated with the licensed vaccine, RotarixTM , demonstrating that the candidate strain had been successfully attenuated In this study, the number of children with diarrhea episodes was low, mainly after the 1st dose and there was no difference between Rotavin-M1 and RotarixTM These diarrhea episodes were mild since they were not accompanied by vomiting and fever However higher numbers of diarrhea cases occurred in the group receiving 106.3 FFU/dose even though yet vaccine virus was only found in diarrhea cases cumulatively D.D Anh et al / Vaccine 30S (2012) A114–A121 in Rotavin-M1 groups 3H and 2H and for case in RotarixTM group, suggesting that diet or bacterial and protozoal infections might be the cause of diarrhea in these children In another RotarixTM trial in Vietnam, the percentage of children with diarrhea after each vaccination dose was 3.1–6.1%, equivalent to what was found in this study [7] RotarixTM at 105.6 –106.8 CCID also caused 8.5–11% diarrhea case among children in the US and Canada [12] The detection of vaccine virus in diarrhea cases is not an uncommon phenomenon in trials using attenuated vaccine In a dose-escalation study of 116E rotavirus vaccine in India, virus vaccine was also isolated in out of 19 diarrhea cases and out of 17 diarrhea cases after the 1st dose of 104 FFU and 105 FFU, respectively [13] Thus, the rate of diarrhea observed in our study is comparable to similar studies of RotarixTM and other live attenuated rotavirus vaccines and it is unlikely that the vaccine causes significant numbers of diarrhea cases in our children Nonetheless, further investigation is in progress in a larger group of infants to determine if the 106.3 FFU dose can cause an increase in diarrhea cases among vaccinees The safety profile of Rotavin-M1 is also featured in that the 160 infants who received the vaccine in either of the or doses did not have any severe adverse events, any significant excess of symptoms of diarrhea, vomiting, fever or irritability, or alterations in blood count or selected blood chemistries compared to the group that received the licensed vaccine Adverse effects mainly occurred after the 1st dose and decreased considerably after the 2nd and 3rd doses, similar to adverse events observed during in RotarixTM trials in Vietnam or in other countries [7] As a comparison, when the liquid form RotarixTM was tested, approximately 50–65% children developed fever during the observation period [7] In Singapore, fever rate after vaccination reached 25–30% after each dose of this licensed vaccine [14] Once safety was established, the Phase study examined the immune response and shedding from both a low and a high titer formulation of the vaccine and both a 2-dose (8 and 16 weeks) and a 3-dose (8, 12 and 16 weeks) schedule These results were compared with a group that received the licensed vaccine, RotarixTM , in its standard 2-dose schedule Overall, the immune response measured as a 4-fold rise in IgA titers to rotavirus ranged from 51% to 73%, a range surrounding the response observed for RotarixTM (58%) While the higher titer formulation performed slightly better than the low titer preparation, the addition of a third dose to the schedule (i.e 8, 12 and 16 weeks) did not dramatically improve the immune response to the vaccine This might be explained by the observation that high titers of the remaining transplacental antibody against rotavirus can inhibit the immune response to the 2nd dose of vaccine in the 8-12-16-week schedule Steele found that doses of RotarixTM given at 10 and 14 weeks performed as well as doses given at 6, 10, and 14 weeks but better than doses given at and 10 weeks [15] In other words, the older the infant was when he received the vaccine, the lower was the initial titer of transplacental antibody and the better the immune response to the vaccine [16] In both the and the dose schedules in our study, last dose was administered when the infant was the same age, i.e 18 weeks (95%CI (16.6–19.2)), unlike studies with the RotarixTM vaccine where a third dose was added to the schedule at 14 weeks Therefore, the immune response to doses of the high titer RotavinM1 vaccine at 2-month interval yielded the most robust immune response Of the same notes, an interval of months between doses was more efficient in inducing immune response compared to a 1month interval in both low and higher titer formulation Similar observations were documented when the liquid form RotarixTM was tested in Vietnamese children [7] In that study, doses of RotarixTM , delivered month apart gave a seroconversion rate of 63.3% at month after the 2nd vaccine dose The same dose vaccine however, when delivered months apart gave a A119 seroconversion rate of 81.5% Application of this 2-month interval between doses of RotarixTM in European countries such as Spain, Italy and Finland led to high seroconversion rates of 92.3–94.6% [17] Thus again, the higher immune response with this 2-month schedule might be associated with the slightly older children who are immunologically more mature compared to those with the 1month schedule [7] The immune responses induced by Rotavin-M1 are comparable to those seen in the RotarixTM group in this study and in a previous study that employed the liquid form of the vaccine with a similar schedule (58–63.3%) [7] It is noted that the pattern of IgA response to rotavirus vaccine in Vietnam seems to follow the trend of developing countries In particular, the IgA responses to RotarixTM in Brazil, Mexico, Venezuela and Vietnam were reported at 61–65%, which are lower than those in USA, Canada, Europe and Singapore (78.2–88.3%) [18–21] and higher than those in Malawi and South Africa [22] In particular, when RotarixTM is introduced in the expanded immunization program of European countries such as France, Germany, Spain and Czech republic, the IgA response rates were very high, 82–94.6% [17] In Singapore the response was 76–91% depending on the vaccine titers [23,24] Thus in Vietnam, as in other developing countries, the vaccine is facing the possible influence of high rotavirus disease burden, malnutrition, concurrent infections, damaged gut mucosal layers, maternal antibodies and age of first administration as previously described [9,25,26] The age at which the children was administered the first dose might play an important role in determining seroconversion rates In this study and the study with RotarixTM in Vietnam the average age of first dose administration was weeks In comparison, the average age for the first dose in the US is 9–11 weeks and 11–17 weeks in Singapore [23,24] In Finland and Italy, vaccine has been used at even older age (3 months) [17] It is generally believed that vaccination at older age induces better immune responses possibly due to a more mature immune system of the child and declining maternal antibody titers in breast milk or from placental transmission This notion is also supported by a study of RotarixTM in the Philippines in which children were 5.5 weeks of age at the first dose and the seroconversion rate was lower compared to that in Vietnamese children As vaccines, Rotavin-M1 is very similar to RotarixTM in that both are derived from common G1P [8] strains attenuated by serial passage and prepared in Vero cells Like RotarixTM , the majority of children shed after the 1st dose of Rotavin-M1, whereas this proportion declined considerably after 2nd dose, similar to other studies [24] Shedding of RotarixTM in different studies worldwide is 35–80%, corresponding to the shedding rate of this vaccine found in our study [27] One interesting difference between the behavior of the two vaccines is the increased shedding observed for RotarixTM (65%) compared to Rotavin-M1 (44–48%) after the 1st dose although this was not accompanied by an increased immune response Another difference between the two vaccines is that Rotavin-M1 vaccine, at the dosage of 106.0 FFU or 106.3 FFU caused delayed in virus shedding compared to RotarixTM at doses of 106 CCID50 (corresponding to 105.5 FFU/dose) These differences between the two vaccines suggest that further research on vaccine formulation, improving the yield of virus so that higher titer candidates could be available which helps advance the development of this locally manufactured vaccine through efficacy trials In this study, the Rotavin-M1 was administered separately from the oral polio virus vaccine (OPV) (10–20 days from the EPI schedule), thus the study was not designed to investigate the effect of other vaccines, in particular OPV on Rotavin-M1 While the coadministration of Rotarix or RotaTeq with OPV seemed to reduce seroconversion rates, antibody titers and vaccine take compared to rotavirus vaccines without OPV, the reductions were not statistically significant [28,29] Thus further study should be designed to A120 D.D Anh et al / Vaccine 30S (2012) A114–A121 investigate whether there is any interference to Rotavin immunogenicity due to concomitant usage of OPV and Rotavin-M1 This study has several limitations which will need to be addressed as development of this vaccine progresses First, we elected to use RotarixTM instead of a placebo for the comparison group so we could well miss important differences in background rates of natural infection over the course of the study that might have altered our results The aim in including Rotarix is to investigate if Rotavin in any schedule or dose shows non-inferiority to Rotarix In addition, since Rotarix (lyophilized form) has been licensed for use in Vietnam in 2007, it is of ethical consideration for children participating in the study to benefit from this vaccine While the placebo group is important, this background of natural infection could be derived from the previous study with the liquid form of Rotarix in Vietnam [7] In addition, the infants were randomized so this would likely have affected the immune responses in the RotarixTM group as well More important is that while we attempted to examine two different titered formulations, 106.0 FFU/dose and 106.3 FFU/dose, the difference in these preparations is not great, perhaps not even within the variability of our titration methods Consequently, while we believe that the higher titer might be superior, we really have not examined the full range of titers to see if by significantly raising the titer, we might improve the immune response This decision is more based upon the ability to raise the titer of the vaccine during production which well could be the limiting step Finally, while we tested a 2- vs 3-dose schedule, we might well improve the immune response to the vaccine substantially if we were to administer the third dose at an older age, say 20 or 28 weeks, when transplacental antibody has waned At the same time, RotarixTM provided substantial efficacy in Vietnamese infants on a similar schedule and if the immune response is at all a predictor of efficacy, Rotavin-M1 might be expected to perform comparably in a clinical trial In conclusion, the Vietnamese rotavirus vaccine, Rotavin-M1 has safety and immunogenicity profile in children, comparable to RotarixTM A multi-center study is in progress to further evaluate this vaccination regimen in a larger number of children Acknowledgements We thank all the medical staffs, the volunteers and the children in Thanh Son, Phu Tho for their participation in this study We deeply thank Dr Roger I Glass (Fogarty International Center, National Institutes of Health), Dr Tetsu Yamashiro (Nagazaki University), Dr Duncan A Steele (PATH) and Dr Jon R Gentsch (US CDC) for critical reading of this manuscript Conflict of interest: Drs Anh, Trang, Thiem, Hien-Anh, Mao, Wang and Jiang have no conflict of interest Financial support: The Ministry of Science and Technology, KC.10.33/06-10, Government of Vietnam Ethical approval: The study and protocol (No 962/CN-BYTSeptember 29, 2009) were approved by the Ethics Committees of the National Institute of Hygiene and Epidemiology and the Ministry of Health, Government of Vietnam Appendix A Members of the Rotavin-M1 Vaccine Trial Group include: NIHE – Vu Thi Bich Hau, Le Thi Kim Anh, Le Thi Hong Nhung, and Le Huy Hoang; POLYVAC – Nguyen Thuy Huong, Ngo Thu Huong, Nguyen Thi Mai Huong, Tran Bich Hanh, and Dang Ngan Ha; Center of Preventive Medicine – Phu Tho: Nguyen Thien An; and Center of Preventive Medicine, Thanh Son – Phu Tho: Nguyen Thi Ly and Le Thi Ngan References [1] Anh DD, Thiem VD, Fischer TK, Canh DG, Minh TT, Tho le H, et al The burden of rotavirus diarrhea in Khanh Hoa Province, Vietnam: baseline assessment for a rotavirus vaccine trial Pediatr Infect Dis J 2006;25(January (1)):37–40 [2] Van Man N, Luan le T, Trach DD, Thanh NT, Van Tu P, Long NT, et al Epidemiological profile and burden of rotavirus diarrhea in Vietnam: years of sentinel hospital surveillance, 1998–2003 J Infect Dis 2005;192(September (Suppl 1)):S127–32 [3] Ngo TC, Nguyen BM, Dang DA, Nguyen HT, Nguyen TT, Tran VN, et al Molecular epidemiology of rotavirus diarrhoea among children in Haiphong, Vietnam: the emergence of G3 rotavirus Vaccine 2009;27(November (Suppl 5)): F75–80 [4] Nguyen TA, Yagyu F, Okame M, Phan TG, Trinh QD, Yan H, et al Diversity of viruses associated with acute gastroenteritis in children hospitalized with diarrhea in Ho Chi Minh City, Vietnam J Med Virol 2007;79(May (5)):582–90 [5] Kim SY, Goldie SJ, Salomon JA Cost-effectiveness of Rotavirus vaccination in Vietnam BMC Public Health 2009;9:29 [6] Luan le T, Trang NV, Phuong NM, Nguyen HT, Ngo HT, Tran HB, et al Development and characterization of candidate rotavirus vaccine strains derived from children with diarrhoea in Vietnam Vaccine 2009;27(November (Suppl 5)):F130–8 [7] Anh DD, Carlos CC, Thiem DV, Hutagalung Y, Gatchalian S, Bock HL, et al Immunogenicity, reactogenicity and safety of the human rotavirus vaccine RIX4414 (Rotarix) oral suspension (liquid formulation) when co-administered with expanded program on immunization (EPI) vaccines in Vietnam and the Philippines in 2006–2007 Vaccine 2011;29(March (11)):F2029–36 [8] Zaman K, Dang DA, Victor JC, Shin S, Yunus M, Dallas MJ, et al Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: a randomised, double-blind, placebo-controlled trial Lancet 2010;376(August (9741)):615–23 [9] Moon SS, Wang Y, Shane AL, Nguyen T, Ray P, Dennehy P, et al Inhibitory effect of breast milk on infectivity of live oral rotavirus vaccines Pediatr Infect Dis J 2010;29(October (10)):919–23 [10] Gentsch JR, Glass RI, Woods P, Gouvea V, Gorziglia M, Flores J, et al Identification of group A rotavirus gene types by polymerase chain reaction J Clin Microbiol 1992;30(June (6)):1365–73 [11] Zibrik L, Dove W, McArdle F, Rustom R, Hart CA, Jackson MJ Lack of shedding of the RIX4414 live attenuated rotavirus vaccine administered to adult volunteers Arch Virol 2007;152(10):1951–4 [12] Dennehy PH, Bertrand HR, Silas PE, Damaso S, Friedland LR, Abu-Elyazeed R Coadministration of RIX4414 oral human rotavirus vaccine does not impact the immune response to antigens contained in routine infant vaccines in the United States Pediatrics 2008;122(November (5)):e1062–6 [13] Bhandari N, Sharma P, Taneja S, Kumar T, Rongsen-Chandola T, Appaiahgari MB, et al A dose-escalation safety and immunogenicity study of live attenuated oral rotavirus vaccine 116E in infants: a randomized, double-blind, placebocontrolled trial J Infect Dis 2009;200(August (3)):421–9 [14] Phua KB, Emmanuel SC, Goh P, Quak SH, Lee BW, Han HH, et al A rotavirus vaccine for infants: the Asian experience Ann Acad Med Singapore 2006;35(January (1)):38–44 [15] Steele AD, Reynders J, Scholtz F, Bos P, de Beer MC, Tumbo J, et al Comparison of different regimens for reactogenicity, safety, and immunogenicity of the live attenuated oral rotavirus vaccine RIX4414 coadministered with oral polio vaccine in South African infants J Infect Dis 2010;202(September (Suppl.)):S93–100 [16] Patel M, Shane AL, Parashar UD, Jiang B, Gentsch JR, Glass RI Oral rotavirus vaccines: how well will they work where they are needed most? J Infect Dis 2009;200(November (Suppl 1)):S39–48 [17] Vesikari T, Karvonen A, Prymula R, Schuster V, Tejedor JC, Thollot F, et al Immunogenicity and safety of the human rotavirus vaccine Rotarix coadministered with routine infant vaccines following the vaccination schedules in Europe Vaccine 2010;28(July (32)):5272–9 [18] Dennehy PH, Brady RC, Halperin SA, Ward RL, Alvey JC, Fischer Jr FH, et al Comparative evaluation of safety and immunogenicity of two dosages of an oral live attenuated human rotavirus vaccine Pediatr Infect Dis J 2005;24(June (6)):481–8 [19] Salinas B, Perez Schael I, Linhares AC, Ruiz Palacios GM, Guerrero ML, Yarzabal JP, et al Evaluation of safety, immunogenicity and efficacy of an attenuated rotavirus vaccine, RIX4414: a randomized, placebo-controlled trial in Latin American infants Pediatr Infect Dis J 2005;24(September (9)): 807–16 [20] De Vos B, Vesikari T, Linhares AC, Salinas B, Perez-Schael I, Ruiz-Palacios GM A rotavirus vaccine for prophylaxis of infants against rotavirus gastroenteritis Pediatr Infect Dis J 2004;23(October (10 Suppl.)):S179–82 [21] Dennehy PH A short report on the highlights of world-wide development of RIX4414: a North American experience comparative evaluation of safety and immunogenicity of two dosages of an oral live attenuated human rotavirus vaccine (RIX4414) in infants in the United States and Canada Vaccine 2006;24(May (18)):3780–1 [22] Madhi SA, Cunliffe NA, Steele D, Witte D, Kirsten M, Louw C, et al Effect of human rotavirus vaccine on severe diarrhea in African infants N Engl J Med 2010;362(January (4)):289–98 [23] Steele AD, Reynders J, Scholtz F, Bos P, de Beer MC, Tumbo J, et al Comparison of different regimens for reactogenicity, safety, and immunogenicity of the live attenuated oral rotavirus vaccine RIX4414 coadministered with D.D Anh et al / Vaccine 30S (2012) A114–A121 oral polio vaccine in South African infants J Infect Dis 2010;(September (202 Suppl.)):S93–100 [24] Phua KB, Quak SH, Lee BW, Emmanuel SC, Goh P, Han HH, et al Evaluation of RIX4414, a live, attenuated rotavirus vaccine, in a randomized, double-blind, placebo-controlled phase trial involving 2464 Singaporean infants J Infect Dis 2005;192(September (Suppl 1)):S6–16 [25] Patel M, Pedreira C, De Oliveira LH, Tate J, Orozco M, Mercado J, et al Association between pentavalent rotavirus vaccine and severe rotavirus diarrhea among children in Nicaragua JAMA 2009;301(June (21)):2243–51 [26] Glass RI, Parashar UD, Bresee JS, Turcios R, Fischer TK, Widdowson MA, et al Rotavirus vaccines: current prospects and future challenges Lancet 2006;368(July (9532)):323–32 A121 [27] Nakagomi T, Nakagomi O A critical review on a globally-licensed, live, orallyadministrable, monovalent human rotavirus vaccine: Rotarix Expert Opin Biol Ther 2009;9(August (8)):1073–86 [28] Zaman K, Sack DA, Yunus M, Arifeen SE, Podder G, Azim T, et al Successful co-administration of a human rotavirus and oral poliovirus vaccines in Bangladeshi infants in a 2-dose schedule at 12 and 16 weeks of age Vaccine 2009;27(February (9)):1333–9 [29] Ciarlet M, Sani-Grosso R, Yuan G, Liu GF, Heaton PM, Gottesdiener KM, et al Concomitant use of the oral pentavalent human-bovine reassortant rotavirus vaccine and oral poliovirus vaccine Pediatr Infect Dis J 2008;27(October (10)):874–80  ... Bhandari N, Sharma P, Taneja S, Kumar T, Rongsen-Chandola T, Appaiahgari MB, et al A dose- escalation safety and immunogenicity study of live attenuated oral rotavirus vaccine 116E in infants: a. .. of vaccine virus in diarrhea cases is not an uncommon phenomenon in trials using attenuated vaccine In a dose- escalation study of 116E rotavirus vaccine in India, virus vaccine was also isolated... Yarzabal JP, et al Evaluation of safety, immunogenicity and efficacy of an attenuated rotavirus vaccine, RIX4414: a randomized, placebo-controlled trial in Latin American infants Pediatr Infect