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 (α) (Kirkegaard and Perry Laboratory, selleckchem 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 + 3 standard deviations of the see more 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 pre-vaccination serum to ≥20 in post-vaccination serum or a ≥4-fold rise from pre-vaccination to post-vaccination serum. For quality 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%. For 30 days following

each vaccine administration, parents or guardians were asked to Oxalosuccinic acid 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. We attempted to collect daily stool samples during the 7 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] and [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 7 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 Rotarix™. Data was managed using Microsoft Visual Foxpro 7.0 software (Microsoft) and analysed using the Stata 11.1 program.