The results indicate areas of opportunity for future HIV-1 vaccine studies. The present study was conducted to further evaluate the rabbit model for induction of cross-reactive HIV-1 neutralizing antibod-ies using R2 Env in AS02A adjuvant that we reported previously. With respect to immunogen design there were two changes planned,GANT61 producing R2 gp140 using a stably transformed cell line instead of cells acutely infected with recombinant vaccinia virus, and testing different forms of the protein. We were able to produce substantial amounts of the different forms of gp140 using stably transformed 293T cells, and to purify the proteins to high levels. The different forms of gp140 we compared allowed us to evaluate the effect of trimerization and of the presence of a flexible linker sequence between gp120 and gp41 ectodomain sequences on antigenic reactivity. Neither of these types of changes appeared to have major effects on antigenic reactivity as measured by binding to cross-neutralizing human mAbs, binding to CD4i mAbs in the presence or absence of sCD4,GDC-0449 or binding to CCR5 in the presence or absence of CD4. Thus, we did not find in vitro evidence that one or another form was likely to be a better immunogen. De novo induction of potent, broadly cross-reactive HIV-1 neutralizing antibodies in animal models has not been achieved. The use of various adjuvants that ligate TOLL-like receptors as components of Env immunogens has been a common approach to overcome this challenge. In a previous study we employed an adjuvant, AS02A, which is an emulsion of monophosphorylated lipid A and QS21, as an adjuvant for soluble R2 Env immunization, and achieved a cross-reactive, low potency response. When planning to confirm and extend that study by conducting the present study, a different adjuvant, designated AS01, was used. This adjuvant is a liposomal formulation including MPL and QS21, and had produced superior results to AS02 in comparative studies of malaria antigen immunization. Thus, the first four immunizations in the present study utilized AS01.