The purpose of the study and the procedures were explained and signed informed consent was obtained from the parents or legal guardians. Enrolled children were randomized to receive three or five doses at six, 10 and

14 weeks or at six, 10, 14, 18 and 22 weeks respectively, along with scheduled childhood vaccines, based on randomization codes provided by a biostatistician not connected with the study as serially numbered opaque sealed envelopes. All routine vaccines were administered as per the National Immunization Schedule or the Indian Academy of Paediatrics Schedule at six-10-14 weeks of age (i.e., DTPw/DTap, Haemophilus influenza type b, OPV/IPV and, Hepatitis B) [21], followed

by the Rotarix vaccination at six, 10 and 14 weeks, and in the five dose arm two additional doses at 18 and 22 weeks. Two blood samples learn more of 3.5 ml were collected from all infants, the first prior to the administration of the first dose of Rotarix vaccine and the second 28 days after the last (third or fifth) dose of vaccine administration. Each sample was analyzed for rotavirus specific IgA by an antibody-sandwich enzyme immunoassay which has been validated by the same laboratory that carried out pre-licensure vaccine evaluations for several vaccines [22]. Briefly, 96 well microtiter plates were coated the overnight with serum from rabbits hyper-immunized with purified double-layered SA11 derived rotavirus particles. buy KU-55933 The next day, partially purified cell culture lysates derived from G1P8 (RIX4414) infected or mock-infected MA 104 cells were added. Dilutions of a standard pool of human serum assigned an arbitrary value of 1000 U or test sera were added followed by the addition of biotinylated rabbit anti-human IgA, peroxidase-conjugated avidin-biotin, and substrate (orthophenylenediamine/H2O2).

After 30 min, the reaction was stopped with 1 M H2SO4, and absorbance was read at 492 nm. The IgA titer was determined by comparing the optical density values from sample wells with the standard curve based on derived units of IgA arbitrarily assigned to a pooled human serum samples, as previously described [22]. Seropositivity was defined as an anti-rotavirus IgA concentration ≥20 U/ml. Seroconversion was considered as the presence of ≥20 U/ml anti-rotavirus IgA in infants who were negative for anti-RV IgA prior to vaccination. A cut-off of 20 RV-IgA units [11], or at least twofold changes in case of a higher baseline values. Seroconversion rate and geometric mean concentrations (GMCs) were assessed at one month after dose three or dose five of Rotarix administration.