After only very weak neutralization responses were obtained, two more immunizations were administered, now using R2 gp140 in AS02, and neutralizing responses were still very weak in all gp140 groups. At that point we elected to discontinue most of the study and give one more immunization to the rabbit group that received the gp140-GCN4-L form of the Env. Surprisingly, this group developed modest potency, cross-reactive neutralizing responses after this last dose. Although we had sought to compare the immunogenicity of the different forms of gp140 tested, we consider it to be unknown whether differential responses would be obtained using an optimal gp140-adjuvant combination. The neutralizing response that developed in the rabbits that received seven doses of the gp140-GCN4-L immunogen cross-reacted extensively among the strains of HIV-1 tested. The neutralization observed included strains of subtypes GDC-0879 as well as strains that were relatively neutralization sensitive and neutralization resistant. There was no neutralization observed against a control virus, VSV. Nor was the neutralizing activity of the sera reduced by extensive absorption of the sera to 293T cells prior to neutralization testing. These data support the interpretation that the neutralizing activity was directed against HIV-1 epitopes and not a non-specific effect on the virus infectivity in the neutralization assay. Evidence to be discussed below showing that markedly different effects of sera were observed in assays using different SF162 strain mutants also demonstrates that the apparent neutralizing activity was not an artifact of the neutralization assay. The results confirm those we obtained previously,GDC-0941 that low to modest potency cross-reactive HIV-1 neutralizing responses can be obtained in the rabbit model. The epitope specificity of the neutralizing responses observed was probed to look for similarity to know broadly cross-reactive neutralizing human mAbs. The neutralizing activity in the rabbit sera was unlike that of the human mAbs VRC01 and VRC03 that target the CD4 binding site on HIV-1in that it was not blocked by the soluble protein RSC3 that inhibits the neutralizing activity of these mAbs.