To the extent that this molecule resembles the authentic, functional Env spike, antibodies that bind to the trimer are likely to be neutralizing. ELISpot assays were conducted to measure numbers of antibody secreting plasma cells in spleens. Both total Ig secreting cells and R2 gp140 trimer binding Ig secreting cells were markedly increased by immunization, and the majority of Ig secreting cells appeared to be specific for the gp140 trimer. By flow cytometry analyses,ICG-001 total numbers of B cells displaying surface Ig that bound to gp140-GCN4-L trimer were also substantially increased. The results demonstrate that preimmune B cells were effectively induced to produce immunogen-specific Ig and to differentiate into plasma cells. The results do not clarify the reasons for the lack of potency or durability of the neutralizing responses. Possibly, some of the trimer-binding antibodies were non-neutralizing or the initial vigorous responses observed were not sustained and did not evolve into mature B memory and long term plasma cell responses. Additional studies will be needed to resolve explain the problems with durability of the responses. Further,INCB18424 the availability of a small animal model for induction of neutralizing antibody responses against HIV-1 would be a useful advancement for the field of HIV vaccine development. Here we demonstrate the biophysical and antigenic characteristics of several different forms of highly purified R2 gp140, and demonstrate the ability of gp140-GCN4-L, gp140 trimer with a flexible linker between the gp120 and gp41 ectodomain sequences, to induce a broadly cross-reactive neutralizing response in outbred NZW rabbits. The breadth of the neutralizing cross-reactivity was similar to that reported previously in study of a less purified form of R2 gp140. Studies of the specificity of the neutralization did not indicate that the epitope targeted corresponded to well-known humAbs with broad neutralizing cross-reactivity; better definition of the specificity of the response may depend upon development of neutralizing mAbs from rabbits with such responses.