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1 Immunogenicity and safety of early vaccination with two doses of a combined measles-mumps-rubella-varicella vaccine in healthy Indian children from months of age: a phase III, randomized, non-inferiority trial. Journal: Manuscript ID: bmjopen Article Type: Research Date Submitted by the Author: -Nov-0 Complete List of Authors: Lalwani, Sanjay; Bharati Vidyapeeth Medical College, Pediatrics Chatterjee, Sukanta; Medical College Kolkata, Department of Pediatrics Balasubramanian, Sundaram; Kanchi Kamakoti Childs Trust Hospital, Bavdekar, Ashish; KEM Hospital, Mehta, Shailesh; GSK Pharmaceuticals, Datta, Sanjoy; GSK Vaccines, Povey, Michael; GSK Vaccines, Henry, Ouzama; GSK Vaccines, <b>primary Subject Heading</b>: Paediatrics Secondary Subject Heading: Paediatrics Keywords: immunogenicity, India, measles-mumps-rubella-varicella vaccine, safety, vaccination schedule : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

2 Page of Title page Title: Immunogenicity and safety of early vaccination with two doses of a combined measlesmumps-rubella-varicella vaccine in healthy Indian children from months of age: a phase III, randomized, non-inferiority trial. Authors: Sanjay Lalwani, Vice Principal, Professor and Head of Department a, Sukanta Chatterjee, Professor of Pediatrics b, Sundaram Balasubramanian, Senior Pediatric Consultant c, Ashish Bavdekar, Associate Professor and Consultant Pediatric Gastroenterologist d, Shailesh Mehta, Area Medical Leader (South Asia) e, Sanjoy Datta, VP, Clinical R&D and Medical Affairs f, Michael Povey, Biostatistician f, Ouzama Nicholson, Clinical Research Development Lead g Affiliations: a Department of Pediatrics, Bharati Vidyapeeth Deemed University, Pune 0, India b Department of Pediatrics, Medical College Kolkata, College street, Kolkata 000, India c Kanchi Kamakoti Childs Trust Hospital, -A, Nageswara Road, Nungambakkam, Chennai 000, India d Department of Pediatrics, KEM Hospital, Rasta Peth, Sardar Moodliar Road, Pune 0, India e GSK Pharmaceuticals Ltd., Dr. Annie Besant Road, Worli, Mumbai 0000, India f GSK Vaccines, Avenue Fleming 0, B-00 Wavre, Belgium g GSK Vaccines, 0 Renaissance Boulevard, RN 00, P.O. Box 0, King of Prussia, PA 0-, USA Correspondence to: Sanjay Lalwani Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

3 Page of Contact Details: + 0 Fax: sanjaylalwani00@rediffmail.com Short title: Combined MMRV vaccine in Indian children Abbreviations ATP according-to-protocol; CCID 0 median cell culture infective dose; CI confidence interval; CRS congenital rubella syndrome; ELISA enzyme-linked immunosorbent assay; GMT geometric mean titer; IAP Indian Academy of Pediatrics; miu/ml milli international unit per milliliter; MMR combined measles-mumps-rubella vaccine; MMR+V coadministration of MMR and V; MMRV combined measles-mumps-rubella-varicella vaccine; pfu plaque forming units; SAE serious adverse event; TVC total vaccinated cohort; V varicella; VZV varicella zostervirus; WHO World Health Organization Keywords: immunogenicity, India, measles-mumps-rubella-varicella vaccine, safety, vaccination schedule Word count: Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

4 Page of Abstract Objective: This study (NCT00) compared the immunogenicity and safety of measlesmumps-varicella (MMR) followed by MMR+varicella (V) vaccines to () two doses of combined MMRV and () MMR followed by MMRV, in Indian children. Design: Phase III, open, randomized, non-inferiority study. Setting: Six tertiary care hospitals located in India. Participants: Healthy subjects aged 0 months not previously vaccinated against/exposed to measles, mumps, rubella and varicella or without a history of these diseases. Interventions: Subjects were randomized (::) to receive two doses of either MMRV (MMRV/MMRV group) or MMR followed by MMRV (MMR/MMRV group) or MMR followed by MMR+V (MMR/MMR+V, control group) at and months of age. Antibody titers against measles, mumps and rubella were measured using ELISA and against varicella using an immunofluorescence assay. Main outcome measures: To demonstrate non-inferiority of the two vaccination regimens vs. the control in terms of seroconversion rates, defined as a group difference with a lower bound of the % confidence interval >-0% for each antigen, -days post-dose-. Parents/guardians recorded solicited local and general symptoms for a -day and -day period after each vaccine dose, respectively. Results: The seroconversion rates post-dose- ranged from.%.% for measles,.%.% for mumps and.% 00% for rubella across the three vaccine groups. Seroconversion rates post-dose- for measles, mumps and rubella was 00% and at least.% for varicella Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

5 Page of across the three vaccine groups. Non-inferiority of MMRV/MMRV and MMR/MMRV to MMR/MMR+V was achieved for all antigens, -days post-dose-. The three vaccination regimens were generally well-tolerated in terms of solicited local and general symptoms. Conclusion: The immune responses elicited by the MMRV/MMRV and MMR/MMRV vaccination regimens were non-inferior to those elicited by the MMR/MMR+V regimen for all antigens. The three vaccination schedules also exhibited an acceptable safety profile in Indian children. Abstract word count: /00 Trial registration: (NCT00) Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

6 Page of Article Summary Measles, mumps, rubella (MMR) and varicella are highly infectious vaccine-preventable childhood diseases with high mortality rates in India. A combined MMR-varicella vaccine may potentially facilitate the control of all four diseases which is as immunogenic as separate MMR and varicella vaccinations. Strengths and limitations of this study This Indian study provides first time data on: A combined MMRV vaccine in a highly endemic measles setting. MMRV administered to children at months of age which aligns with the expanded programme of immunization schedule of measles vaccine administered at this age. Prevaccination serostatus that offers epidemiological indicators on the early disease burden for measles, mumps, rubella and varicella This study had no limitations. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

7 Page of Introduction Measles, mumps, rubella and varicella are highly infectious vaccine-preventable childhood diseases that continue to pose a significant public health problem in India and beyond. - In 00, global measles mortality was estimated at,000 (,00,00) deaths, % of which was estimated to have occurred in India. In 0, large measles outbreaks were reported in India (, cases), Pakistan (, cases), Nigeria (, cases) and other countries. Although measles elimination was declared in the United States in 000, the importation of the disease led to the highest number of cases in 0 (0 cases) since while cases were reported in 0 by states. In the European Union, the Dutch authorites reported 0 measles cases since May 0 and in Germany, the reported number of cases is nearly 0 times higher than the total cases in 0. During the 0 0 outbreak, there were over 000 confirmed cases of measles in England and Wales. 0 Thus, even developed settings may be prone to epidemics if coverage wanes. A dramatic decrease in the worldwide mumps disease burden has been observed since the implementation of large-scale immunization in. However, the true incidence in India is difficult to ascertain due to limited baseline epidemiological data. Data from 00 revealed that.% children aged between and years, and.% aged between 0 and years are susceptible to rubella in the Indian sub-continent. Although congenital rubella syndrome (CRS) has been reported in most parts of India, no measures have been undertaken to control this crippling disease and presently, reliable data on CRS in India is limited. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

8 Page of Epidemiological data on varicella-zoster virus (VZV) are also scarce in India as chickenpox was not a notifiable disease in India until 00 and due to the locally-perceived self-limiting and relatively less severe nature, the disease is under-reported. Globally, routine and effective vaccination has been identified as a critical approach towards achieving high and sustained vaccination coverage rates and to strategically deal with the burden of these four diseases. Consequently, the Indian Academy of Pediatrics (IAP) has recommended the inclusion of a combined measles-mumps-rubella vaccine (MMR) in the national immunization schedule to provide protection against CRS and also to reduce the disease burden of measles and mumps. In countries with ongoing measles transmission, the World Health Organization (WHO) recommends a first dose of measles vaccine at months of age to afford early protection and second dose at months with a minimum interval of one month between the two doses. In India, the observed high morbidity and mortality due to measles has necessitated the administration of the measles vaccine at months of age (by which time most children will have lost their maternal antibodies to measles) followed by MMR at months of age. The IAP also recommends two doses of a varicella vaccine, with the first dose administered at months of age. The second dose may be administered three months after the first, but is usually given at years. There is increasing global evidence in many settings that the high economic burden of varicella would be beneficially alleviated with the inclusion of varicella vaccine. - GSK s MMR and varicella vaccines are available in over 00 countries and 0 countries, respectively. These vaccines are currently not included in the Indian national (governmentprovided) immunization program; however, they are available via private practitioners. Based Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

9 Page of on commercially available formulations, a combined MMR-varicella vaccine (MMRV) has been developed to not only realize the benefits of vaccination against measles, mumps and rubella, but also to facilitate the potential inclusion of varicella into national immunization programs. - The new vaccine is as immunogenic as separate MMR and varicella vaccinations. -0 This study evaluated the non-inferiority of two different vaccination regimens of the new MMRV vaccine to the control regimen of separate injections when the vaccines were administered at and months of age to healthy Indian children. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

10 Page of Methods Study design and subjects This phase IIIb, open, randomized, controlled study (NCT00) was conducted at six tertiary care centers (Supplementary table) in India between November 00 and February 0. Healthy subjects aged 0 months were randomized (::) to receive either two doses of the MMRV vaccine (MMRV/MMRV group) or MMR followed by MMRV (MMR/MMRV group), or MMR followed by MMR+V (MMR/MMR+V group; control) at and months of age. The control regimen largely reflects the optimum standard of care available in India under the IAP recommendations. Among the six centers, the center in Bangalore did not enroll subjects according to the randomization scheme and enrollment ceased at a small number of subjects because the investigator was transferred (Supplementary table). Subjects were excluded from the study if they had received any investigational drug/vaccine 0 days before the study vaccine or immunosuppressants/immune-modifying drugs/blood products six months before the study. Subjects previously vaccinated against/exposed to measles, mumps, rubella and varicella or with a history of these diseases could not participate. A history of allergy likely to be aggravated by any of the vaccine components, neurological disease/seizures, chronic illness or family history of immunodeficiency, or symptoms of acute illness at the time of enrollment were other reasons for exclusion. Vaccination was postponed for subjects with a rectal temperature.0 C/an axillary temperature. C. Subjects were also excluded if they lived in a household with newborn infants or pregnant women who have not contracted chickenpox previously or immunocompromised individuals. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

11 Page 0 of The study adhered to Good Clinical Practice, the Declaration of Helsinki, and all applicable regulations. The participating centers Institutional Ethics Committees/Institutional Review Boards reviewed and approved the protocol. Parents/guardians provided written informed consent before performing any study-related procedures. Study Vaccines All study vaccines: MMR (Priorix ), varicella (Varilrix ) and MMRV (Vammrix [same as Priorix- Tetra ]) were manufactured by GSK, Belgium. The minimum expected potencies for measles, rubella and varicella were identical between the MMR+V and MMRV vaccines. 0 The minimum expected potency for the mumps content was higher in the MMRV vaccine ( 0. median cell culture infective dose [CCID 0 ]) than in the MMR vaccine ( 0. CCID 0 ).The vaccines supplied in monodose vials contained a freeze-dried pellet which was reconstituted with the diluent (provided in a pre-filled syringe) before subcutaneous injection into the anterolateral thigh. Immunogenicity assessment Blood samples were collected at pre-vaccination and days after doses one and two. Antibody titers were measured using a commercial enzyme-linked immunosorbent assay (ELISA; Enzygnost, Dade Behring, Marburg, Germany) with cut-off values of 0 miu/ml (measles), U/mL (mumps), and IU/mL (rubella). For varicella, antibody titers were measured using an immunofluorescence assay (Virgo, Hemagen Diagnostics, Columbia, MD, USA) (assay cutoff value of dilution - ). Safety/Reactogenicity assessment Parents/guardians used diary cards to record the occurrence of solicited local symptoms (pain, redness and swelling at the injection site) for days after each dose and solicited general Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

12 Page of symptoms (fever[axillary temperature. C/rectal temperature C], rash/exanthema, parotid/salivary gland swelling and any suspected signs of meningeal irritation, including febrile convulsions) for days after each dose. Body temperature was measured daily via the rectal/axillary route for days after each vaccination. Between days and, the presence of fever was monitored using a temperature-sensitive pad, and if fever was suspected, temperature was accurately measured with a thermometer. There were two follow-up visits with the investigator at each study center, one vist days following each vaccine administration. During these visits, diary cards were returned to the investigator for assessment. Unsolicited symptoms were recorded for days after each dose and the occurrence of serious adverse events (SAEs) was recorded throughout the study. Intensity of symptoms was graded on a scale of 0. Grade solicited symptoms were defined as: pain: child cried when limb was moved or a spontaneously painful limb; redness and swelling: injection site surface diameter >0 mm; fever: axillary temperature > C/rectal temperature >. C. Unsolicited symptoms (including SAEs) were defined as grade when they prevented normal daily activity. Statistical Analyses All statistical analyses were performed using SAS version., and % confidence intervals (CI) were calculated using Proc StatXact.. The sample size was estimated taking into consideration the co-primary objectives of non-inferiority. Non-inferiority was achieved if the lower limit of the two-sided standardized asymptotic % CI for the difference in seroconversion rates between the two treatment groups and control group (MMRV/MMRV minus MMR/MMR+V; MMR/MMRV minus MMR/MMR+V) was -0% for each vaccine antigen, Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

13 Page of days post-dose-. Similarly, the secondary non-inferiority objective was achieved if the lower limit of the two-sided standardized asymptotic % CI for the difference in seroconversion rates between the MMRV/MMRV group and pooled MMR results from the MMR/MMRV and MMR/MMR+V groups, days post-dose- was -0% for measles, mumps and rubella. A sample size of 0 evaluable subjects in each treatment group and evaluable subjects in the control group was planned, which gave a power of at least.% to meet the co-primary objectives. A central randomization system using a minimization algorithm provided each child with a unique treatment number. A randomization (::) blocking scheme ensured that the balance between treatments was maintained by providing a unique treatment number that identified the vaccine dose to be administered to the subjects. Furthermore, given different physical characteristics of the study vaccines and the number of injections between study groups, the study was conducted in an open manner where in the treatment allocation of subjects was known to the investigators and the parents/guardians. Immunogenicity analysis was performed on the according-to-protocol (ATP) cohort which included all subjects for whom pre- and post-vaccination serology results were available and who complied with study procedures. Seroconversion rates (defined as the appearance of antibodies [i.e. antibody concentration/titer cut-off value] in the serum of subjects who were seronegative before vaccination) and geometric mean titers (GMTs) were calculated with exact % CIs for antibodies against each vaccine antigen after each dose. The % CIs for the GMTs were obtained by exponential transformation of the % CI for the mean of log-transformed titer. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

14 Page of Safety analysis was performed on the total vaccinated cohort (TVC) which included all vaccinated subjects. Solicited and unsolicited symptoms reported for the subjects during the respective post-vaccination periods were calculated with exact % CIs. All SAEs reported during the entire conduct of the study were described. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

15 Page of Results Demographics All 0 subjects enrolled in the study were vaccinated and included in the TVC: MMRV/MMRV (n=0); MMR/MMRV (n=0) and MMR/MMR+V (n=0). Of these, subjects were included in the ATP cohort for immunogenicity: MMRV/MMRV (n=); MMR/MMRV (n=) and MMR/MMR+V (n=) (Figure ). The median age of subjects in the ATP was months (range: 0 months);.% were male and all subjects were Indian. No demographic variations were observed between the study groups (Supplementary table). Immunogenicity The proportion of initially seropositive subjects for measles, mumps and rubella was <.% in all three groups. For varicella,.% subjects in the MMRV/MMRV group,.% in the MMR/MMRV group and.% in the MMR/MMR+V group were initially seropositive. After dose-, the seroconversion rates ranged from.%.% for measles,.%.% for mumps and.% 00% for rubella (Table ). Post-dose- seroconversion rates were 00% for measles, mumps and rubella and were at least.% for varicella. Across the three vaccination groups, the observed GMTs to measles, mumps and rubella increased between doses one and two (Table ). The co-primary objectives of non-inferiority with respect to seroconversion rates days after dose- were achieved for all vaccine antigens, i.e. the lower bounds of the % CIs for the difference in seroconversion rates between groups (MMRV/MMRV vs MMR/MMR+V; MMR/MMRV vs MMR/MMR+V) was -0%. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

16 Page of Table Seroconversion rates and Antibody GMTs of the MMRV/MMRV, MMR/MMRV and MMR/MMR+V groups days postvaccination (ATP cohort) Antigen MMRV/MMRV group (N=) MMR/MMRV group (N=) MMR/MMR+V group (N=) 0 Post-dose-. (.;.) 0. (.;.). (.0;.) 0. (.0;.). (.;.) 00.0 (.;.) Post-dose- 00 (.; 00). (.; 0.) 00 (.; 00). (0.;.) 00 (.0; 00).0 (0.; 0.) 0 Mumps Pre-dose Post-dose- Nov 0 0 Dose N a SC b (%) c (% CI) GMT d (% CI) N a SC b (%) c (% CI) GMT d (% CI) N a SC b (%) c (% CI) GMT d (% CI) Measles Pre-dose (.;.). (.; 00.). (.;.). (.0;.). (.;.). (00.;.) on April 0 by guest. Protected by copyright. - : first published as 0./bmjopen on September 0. Downloaded from

17 Page of Post-dose-. (.;.). (.;.). (.; 00). (.;.) 00 (.; 00).0 (.;.) 0 Post-dose (.; 00). (.;.) 00 (.; 00). (.;.) 00 (.; 00).0 (.0;.) Varicella Pre-dose Post-dose- Post-dose- Nov 0 Post-dose- 00 (.; 00).0 (.;.) 00 (.; 00) 00. (.; 0.0) 00 (.0; 00). (00.;.) 0Rubella Pre-dose (.;.) 0. (0.; 0.0). (0.;.). (.0;.). (0.0;.). (.;.) 00 (.; 00). (.;.). (.0;.).0 (.;.). (.;.).0 (.;.) a Number of children initially seronegative with available results b Seroconversion on April 0 by guest. Protected by copyright. - : first published as 0./bmjopen on September 0. Downloaded from

18 Page of c Percentage of children that seroconverted for each antigen d Geometric mean titers The secondary objective of non-inferiority of the MMRV/MMRV group and pooled MMR results from the MMR/MMRV and MMR/MMR+V groups in terms of seroconversion rates -days post-dose- for measles, mumps and rubella was also achieved (Table ). Table Post-dose- seroconversion rates between the MMRV/MMRV group and pooled MMR/MMRV + MMR/MMR+V groups (ATP cohort) MMRV MMR Antibody (MMRV/MMRV group) (Pooled [MMR/MMRV] + [MMR/MMR+V] groups) Difference in percentage (MMRV minus MMR) N a % b N a % b % c (% CI) Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

19 Page of MMRV MMR Antibody (MMRV/MMRV group) (Pooled [MMR/MMRV] + Difference in percentage (MMRV minus MMR) [MMR/MMR+V] groups) N a % b N a % b % c (% CI) Measles..0. (-.0;.) Mumps... (-.;.) Rubella (-.;.) Varicella a Number of children initially seronegative with available results b Percentage of children that seroconverted for each antigen Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

20 Page of c Difference in percentage of children that seroconverted for each antigen between the MMMR/MMRV group and the pooled MMR results from the (MMR/MMRV + MMR/MMR+V) groups Safety and Reactogenicity During the -day post-vaccination period, the occurrence of solicited and unsolicited symptoms ranged between.%.% after dose- and.%.% after dose- across the three vaccine groups. During the -day post-dose- follow-up period, injection site pain was the most commonly reported solicited local symptom: MMRV/MMRV (.%), MMR/MMRV (%) and MMR/MMR+V (0.%). Post-dose-, injection site symptoms were reported by fewer than.% of subjects (Table ). Redness was the grade local symptom reported by subjects (.%) in the MMRV/MMRV group, post-dose-. Fever was the most commonly reported solicited general symptom in all three vaccine groups during the -day post-vaccination follow-up period after each dose (Table ). The observed incidence of fever across all vaccine groups decreased between doses one and two during the -day and -day post-vaccination follow-up periods (Table ). Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

21 Page 0 of Table Incidence of solicited local symptoms (during the -day) and fever (during the -day and -day) post-vaccination period (Total vaccinated cohort) MMRV/MMRV group MMR/MMRV group MMR/MMR+V group Dose- N a =; Dose- N a = Dose- N a =; Dose- N a = Dose- N a =; Dose- N a = % b (% CI) % b (% CI) % b (% CI) Pain Post-dose-. (.;.).0 (.;.) 0. (.0;.) Post-dose-. (.; 0.). (.;.). (0.; 0.) Redness Post-dose-. (.;.). (.0;.0). (0.; 0.) Post-dose-. (.;.). (.;.0) 0.0 (0.0;.) Swelling Post-dose-. (.0;.). (.0;.). (0.; 0.) Post-dose-. (.; 0.). (.;.0) 0.0 (0.0;.) Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

22 Page of Any. (.;.). (.;.). (.;.) Grade. (.;.). (0.;.0). (0.0;.) Fever ( days (>. C) post-dose-) Related. (.;.). (.;.). (.;.) Medical Advice. (.;.0). (.;.). (0.;.) Any. (.;.). (.;.). (.;.0) Grade. (0.;.). (0.;.) 0.0 (0.0;.) (>. C) Fever ( days post-dose-) Related. (.; 0.0). (.;.0). (.;.0) Medical Advice. (.;.). (0.;.). (0.0;.) Any. (.;.) 0. (.;.). (.;.) Grade. (.;.0). (.0;.). (0.0;.) Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

23 Page of (>. C) Fever ( days post-dose-) Related 0. (.;.). (.;.). (0.;.0) Medical Advice. (.0;.). (.;.). (.;.) Any. (.;.). (.; 0.). (.;.) Grade. (0.;.). (.;.0). (0.;.) Fever ( days (>. C) post-dose-) Related. (.; 0.). (.;.). (.;.0) Medical Advice. (.;.). (.; 0.). (.;.) a Number of children with at least one documented dose b Percentage of children reporting the symptom at least once Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

24 Page of An absence of a clear peak in the prevalence of fever during the -day period after dose- for all three vaccine groups is depicted in Figure (a). Post-dose- fever is depicted in Figure (b). Rash occurred in one subject after the first dose of MMRV in the MMRV/MMRV group as compared to three subjects after the first MMR dose (MMR/MMRV [n=]; MMR/MMR+V [n=]) groups). Only one subject developed rash after dose- in the MMR/MMRV group. There were no reports of meningeal irritation, febrile convulsions or parotid gland swelling during the - day period after each vaccine dose. At least one unsolicited symptom was reported in 0.% subjects after the first dose of MMRV in the MMRV/MMRV group and in.% and 0.0% subjects after the first dose of MMR in the MMR/MMRVand MMR/MMR+V groups, respectively. The most commonly reported symptoms in each group were: upper respiratory tract infection in the MMRV/MMRV group (n=0;.%); cough in the MMR/MMRV group (n=0;.%) and nasopharyngitis, rhinitis and cough in the MMR/MMR+V group (n=;.% for each symptom). Similarly, at least one unsolicited symptom was reported in 0.% subjects after the second dose of MMRV in the MMRV/MMRV group, in 0.0% subjects who received their first dose of MMRV in the MMR/MMRV groups and in.% subjects following dose- of MMR+V in the MMR/MMR+V group. The most commonly reported symptoms in each group were: rhinitis and cough in the MMRV/MMRV group (n=;.% for each symptom) and rhinitis in the MMR/MMRV (n=;.%) and MMR/MMR+V groups (n=;.%). Overall, SAEs occurred in the study. At least one SAE occurred in subjects (MMRV/MMRV [n=]; MMR/MMRV [n=]; MMR/MMR+V [n=0] groups). The most commonly reported SAEs were lower respiratory tract infection (in two subjects) in the MMRV/MMRV group, and Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

25 Page of gastroenteritis (in three subjects) in the MMR/MMRV group. Other SAEs reported were gastroenteritis, pneumonitis, wheezing, viral infection, pneumonia, febrile convulsion, upper respiratory tract inflammation, dehydration and bronchiolitis. All SAEs resolved without sequelae and were considered unrelated to vaccination. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

26 Page of Discussion This non-inferiority study evaluated the immunogenicity and safety of two vaccination regimens () two-dose MMRV/MMRV () MMR followed by MMRV compared to a control group (MMR followed by MMR+V) when administered to healthy Indian children at and months of age. The co-primary non-inferiority criterion ruling out a 0% difference in seroconversion rates post-dose- of MMRV/MMRV and MMR/MMRV compared to MMR/MMR+V was achieved for all antigens, indicating that the immune responses elicited by the two vaccination regimens were non-inferior to those elicited by the control regimen. Although the two-dose MMRV/MMRV schedule is not included in several immunization programs, this regimen has been established to be non-inferior to the two-dose MMR+V schedule in separate studies in Germany and Singapore. 0 Additionally, on comparing the post-dose- responses, one dose of MMRV in the MMRV/MMRV group elicited non-inferior immune responses against measles, mumps and rubella compared to pooled results of one dose of MMR in the MMR/MMRV and MMR/MMR+V groups. In the three vaccine groups, we observed low baseline seropositivity rates (<.%) for measles, mumps and rubella in subjects at months of age. This finding suggests a possible decline in circulating maternal antibodies and measles infection by this age which would support months as a suitable age for initial vaccination. However, it should be noted that this finding is inconsistent with a notable Indian study conducted approximately a decade ago that suggested the persistence of high circulating maternal antibodies at months of age. Additionally, while lowering the measles vaccination age in low income countries is supported by many, vaccinating at months or earlier may mean that the immune system has not reached Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

27 Page of optimum maturity to mount an effective response and provide effective long-term protection against measles or the other diseases with just a single vaccine dose. The seroconversion rates for all antigens in the MMRV/MMRV group were consistent with previous observations in Singaporean children at months of age by Goh et al. However, with a first dose of MMR, the observed post-dose- seroconversion rates to measles (MMR/MMRV=.%; MMR/MMR+V=.%) and mumps (MMR/MMRV=.%; MMR/MMR+V=.%) in this study were somewhat lower than those reported by Schuster et al, following administration of a first dose of MMR to children aged months in Germany (measles:.%; mumps:.%). 0 A contrast in immune responses between the current and German study may be attributed to the age at vaccination, maturity of the immune system and circulating maternal antibodies. Also, the higher GMTs oberved to measles in the MMRV/MMRV group compared to the MMR/MMRV and MMR/MMR+V groups could translate to more effective protection in a highly endemic measles environment where coverage from a second dose of a measles-containing vaccine remains variable throughout the country. Lower seroconversion rates with other liveattenuated vaccines (such as the oral polio vaccine and the rotavirus vaccine) have also been observed in India and South Asia compared to more industrialized settings. - Nevertheless, we observed high seroconversion rates for all vaccine antigens following administration of second vaccination at months of age, indicating that an early two-dose immunization strategy when the first dose is administered as early as months elicits a satisfactory immune response. An interesting observation was the markedly high GMT against mumps post-dose- in the MMR/MMRV (00.) group compared to the MMR/MMR+V (.) and MMRV/MMRV (.0) groups. This observation has not been reported previously Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

28 Page of despite the higher mumps antigen content in the MMRV vaccine when compared to the MMR vaccine. Studies to further evaluate this finding may be needed in the future. Early administrations of all three vaccination regimens were well-tolerated when administered to young children at and months of age. Similar differences in solicited general symptoms have been observed in studies conducted in the Indian subcontinent with other live attenuated viral vaccines (such as Rotarix ) compared with other countries. Notably, unlike previous studies conducted in other countries, -0 this study did not demonstrate any difference in fever rates between MMRV and MMR when used as a first dose of measles-containing vaccine. Although this may be related to the epidemiological context in India, which differs from developed countries, the reason is unclear. In general, the reporting rate of fever was also lower than that seen in other studies. -0 Again, this may be due to the younger age of children enrolled, or the presence of maternal antibodies, which may have limited measles virus replication post-dose-, resulting in the blunting of immune response and fever response rates; or cross-cultural/geographical differences in the reporting of symptoms. Further data may be needed to determine if there is in fact a difference in the reactogenicity profile between developed and developing countries. It is possible to eliminate measles from a specific region by sustaining high immunization coverage as is evident from Latin America, Finland and the United States 0- ; countries such as Finland have also successfully eliminated mumps and rubella using the MMR vaccine. In view of the ongoing transmission and high mortality risk of measles in India, immunization coverage of % for the first and second dose would be required to ensure prevention of measles virus transmission. Currently, the Indian national immunization schedule s months single dose Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

29 Page of immunization coverage is % ; however, as this represents the average, coverage rates in some parts of the country may be even lower. Several European countries, where the first MMR dose is administered to children in the second year of life ( months), continue to face a relatively low immunization coverage rates resulting in measles and mumps outbreaks, as was recently observed even in South Wales. 0 An earlier vaccination schedule was implemented in Germany as a result of such outbreaks, whereby the first dose is now administered at months followed by the second dose at months of age. This revised strategy achieved well-documented successful immunization coverage rates, and highlights the importance of early vaccination with increased compliance and subsequently higher coverage rates. Conclusion This study demonstrates that in an Indian setting, the two-dose vaccination regimens of MMRV/MMRV and MMR/MMRV are non-inferior to the control MMR/MMR+V regimen (i.e. the local standard of care recommended by the IAP) in terms of immunological response. Both vaccination schedules demonstrated an acceptable safety profile when administered to healthy Indian children at and months of age. Introduction of the MMRV vaccine may achieve higher coverage rates in India by increasing compliance for two-dose MMR and varicella vaccines, and thereby improving the limited success of the MMR vaccination program. This will not only facilitate effective population protection against measles, but against three other common childhood viral infectious diseases: mumps, rubella and varicella. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

30 Page of Trademarks Vammrix, Priorix-Tetra, Priorix, Varilrix and Rotarix are registered trademarks of GlaxoSmithKline group of companies. Virgo is a registered trademark of Hemagen Diagnostics, Columbia, MD, USA. Enzygnost is a registered trademark of Dade Behring, Marburg, Germany. Contributors: SL, SB, SC and AB were involved in the recruitment of subjects. SL critically reviewed the protocol and study report and also provided scientific and medical inputs during the development of the study report. SB was involved in providing scientific and medical inputs to the protocol and critically reviewing the protocol. SC coordinated and supervised data collection at one site and carried out the initial analyses. AB provided critical inputs to the study report and reviewed the content of the study report. SM and SD conceptualized and designed the study. SM also supervised data collection at all sites. MP was involved in data analysis and interpretation. SD and ON provided input to the interpretation of clinical data. All authors critically reviewed and revised the manuscript during the development and approved the final version of the manuscript. All authors are guarantors and accept full responsibility for the work and/or conduct of the study, had access to the data, and controlled the decision to publish. Funding: This study was sponsored and funded by GlaxoSmithKline Biologicals SA. GlaxoSmithKline Biologicals SA was involved in all stages of the study conduct and analysis; and also took charge of all costs associated with the development and the publishing of the manuscript. Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

31 Page 0 of Competing interests: All authors have completed the ICMJE uniform disclosure form at SL declares to have received institutional grant (for BVDU Medical college, Dept. of Pediatrics) and travel support for the meeting from GlaxoSmithKline group of companies. He also declares to have received fees for lecture at Pedicon 0. SB declares that The childs trust medical research foundation received grant from GlaxoSmithKline group of companies. AB declares to have received institutional grant for conducting this trial and has pending institutional grants from GlaxoSmithKline group of companies for other vaccine trials. He also declares to have received support for travel/accomodation while participating in investigator meetings and renumeration for participating in a meeting on adjuvants from GlaxoSmithKline group of companies. SM, SD, MP and ON are employees of GlaxoSmithKline group of companies; SD and ON declare to have stock options. MP declares that he received payment/fees for review of statistical analysis and reviewing this manuscript from GlaxoSmithKline group of companies. SC has no conflict of interest. The authors have no financial relationships relevant to this article to disclose. Data sharing: Upon authorization or termination of development of this medicine, anonymized patient-level data underlying this study will be made available to independent researchers, subject to review by an independent panel, at Consent for data sharing was not obtained from the participants, but the presented data are anonymized and risk of identification is low. Acknowledgements The authors thank the study nurses and staff members involved, and the parents and children who participated in the study. The authors acknowledge Dr. Sonali Palkar for her support in Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

32 Page of conducting the clinical trial and Dipti Phatarpekar for coodinating and supervising data collection at two study sites. The authors also thank Ashmita Ravishankar for medical writing and publication coordination (GlaxoSmithKline Vaccines), Jarno Jansen (Keyrus Biopharma for GlaxoSmithKline Vaccines), Geetha Subramanyam (GlaxoSmithKline Vaccines) and Shruti MP (GlaxoSmithKline Vaccines) for editorial assistance and publication coordination. Trial registration: (NCT00) Nov : first published as 0./bmjopen on September 0. Downloaded from on April 0 by guest. Protected by copyright.

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