The correlates study was preceded by pilot studies from November 2009 through July 201120 (Fig. S1 in the Supplementary Appendix , available with the full text of this article at NEJM.org). Pilot assays were performed on samples taken at baseline and week 26 from 50 to 100 uninfected RV144 participants (80% of whom were vaccine recipients and 20% of whom were placebo recipients) and scored according to four statistical criteria: a low false positive rate on the basis of samples from placebo and vaccine recipients at baseline, a large dynamic range of vaccine-induced immune responses, nonredundancy of responses (low correlations), and high reproducibility.
Of the 32 types of antibody, T-cell, and innate immunity assays evaluated in pilot studies, 17 met these criteria, from which 6 primary variables were chosen for assessment as correlates of infection risk. The purpose was to restrict the primary analysis to a limited number of variables in order to optimize the statistical power for showing a correlation of risk between vaccinated persons who acquired versus those who did not acquire HIV-1. The primary variables included 5 Env-specific antibody responses and 1 cellular response: the binding of plasma IgA antibodies to Env, the avidity of IgG antibodies for Env, antibody-dependent cellular cytotoxicity, HIV-1 neutralizing antibodies, the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of the gp120 Env, and the level of Env-specific CD4+ T cells (for details, see theSupplementary Appendix ). All 17 types of immune assays and their 152 component variables were also included in the secondary correlates analyses (Tables S1 and S2 in the Supplementary Appendix ).
Secondary variables were drawn from the remaining 152 assays selected from pilot assay studies; they were evaluated to help interpret the results of the primary analysis and to generate additional hypotheses (Table S1 in the Supplementary Appendix ). For the sensitivity analysis, immune-response variables that were closely related to the six primary variables (within the same assay type) were substituted for each of the primary variables into the multivariable model (eight variables, with three individual variables paired to the primary variable of neutralizing antibodies) (Table S2 in the Supplementary Appendix ). All assays were performed by personnel who were unaware of treatment assignments and case–control status.
Of the 32 types of antibody, T-cell, and innate immunity assays evaluated in pilot studies, 17 met these criteria, from which 6 primary variables were chosen for assessment as correlates of infection risk. The purpose was to restrict the primary analysis to a limited number of variables in order to optimize the statistical power for showing a correlation of risk between vaccinated persons who acquired versus those who did not acquire HIV-1. The primary variables included 5 Env-specific antibody responses and 1 cellular response: the binding of plasma IgA antibodies to Env, the avidity of IgG antibodies for Env, antibody-dependent cellular cytotoxicity, HIV-1 neutralizing antibodies, the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of the gp120 Env, and the level of Env-specific CD4+ T cells (for details, see the
Secondary variables were drawn from the remaining 152 assays selected from pilot assay studies; they were evaluated to help interpret the results of the primary analysis and to generate additional hypotheses (