2. Materials and Methods
HIV-uninfected toddlers
DECAY
The primary study was a dose-escalation, randomized, placebo-controlled trial to investigate the safety and immunogenicity of the P2-VP8-P[8] vaccine in healthy, HIV-uninfected toddlers and infants conducted at the Respiratory and Meningeal Pathogens Research Unit, Johannesburg, South Africa during 2014–2015. The infant cohort included term infants (gestation period > 37 weeks) aged from 6 to <8 weeks at time of enrolment and determined to be in good health through medical history, physical examination, and laboratory testing. A parent or legal guardian provided written informed consent for their child’s participation in the trial. In total, 162 infants were enrolled and randomized to receive vaccine (All immunogenicity testing was performed as part of the original study. In brief, IgA and IgG titers directed against P2-VP8-P[8] vaccine antigen (anti-P[8] IgA and IgG) and the whole rotavirus lysate (anti-RV IgA) were measured, as well as neutralizing antibody (NAb) responses against four RV strains: Wa (G1P[8]), 89-12 (G1P[8]), DS-1 (G2P[4]), and 1076 (G2P[6]), as described [Infants who had received three injections of either of the two highest vaccine doses (30 µg or 60 µg) or placebo, had a serum sample collected four weeks after the last injection and provided at least one stool sample following administration of the first dose of Rotarix were included in the analysis. Infants with at least one stool sample testing rotavirus positive by ELISA were considered to be shedders and those with all stool samples testing ELISA negative were considered to be non-shedders. IgA, IgG, and NAb were analyzed in terms of both seroresponse and magnitude of the response. Unadjusted seroresponse was defined as a four-fold or more increase in the magnitude of the antibody response from baseline to one or six months after receiving three doses of P2-VP8-P[8] vaccine or placebo. The magnitude of the antibody response was described by geometric mean titers (GMT). IgG and NAb titers at the one month post-third-dose time point were adjusted for decay in maternal antibodies using the half-life calculated from participants in the placebo group who had detectable baseline titers that were higher than at the post-injection visit This was established separately for each assay. Adjusted seroresponse was defined as a four-fold or more increase in titer from baseline to one month after receiving three doses of P2-VP8-P[8] vaccine or placebo (adjusted titer) in infants with an unadjusted post-injection titer greater than the limit of detection [Continuous variables were assessed for normality and means or geometric means with 95% confidence intervals (CI) described, as appropriate. Wilcoxon Rank-Sum test or Student’s 