The gp140 produced in this fashion generally binds mAbs and undergoes CD4-induced conformational changes similarly to native viral Env. However, since the majority of the gp140 molecules were not trimeric, there is concern that epitopes important for bNab may not be well presented by the non-native forms of the protein. The present study was designed and conducted to confirm the immunogenicity of the R2 gp140 in GSK adjuvant in rabbits, to address considerations regarding purity of the Env immunogen, and to develop further understanding of the responses induced in this rabbit model. The gp140 used in the present study was produced using stably transformed cell lines,FG-4592 such that the level of contamination with cell proteins after purification was dramatically reduced com-pared to the vaccinia-derived protein used in previous studies. The multimeric state of the protein was characterized by biophysical methods, and antigenic reactivity characterized using various mAbs. The gp140 was produced as four different forms. One form was of the same sequence as that used previously, and was purified so that the monomeric, dimeric, and trimeric forms were retained in the immunogen, as previously. A second form was produced as trimeric protein by virtue of in-frame fusion of the R2 gp140 coding sequence to a modified GCN4 multimerization domain . The third form was similar to the second, but had a flexible linker sequence introduced between the gp120 and gp41 coding sequences of R2 Env Foretinib. The rationale for the linker was that the flexibility that it allows might permit the trimeric proteins to assume more native state than would be possible for the uncleaved gp140. The fourth form consisted of gp140 with the linker sequence but no trimerization domain. All immunizations were administered in adjuvant provided by GSK. The results confirm the induction of an Ab response to Env that neutralizes otherwise resistant primary viruses cross-reactively. This study was conducted to confirm the induction of broadly cross-reactive neutralizing antibodies in the rabbit HIV-1 immu-nization model, compare relative antigenic reactivity, function, and immunogenicity of different forms of a gp140 Env, develop additional features of the rabbit model, and evaluate the epitope specificity of the neutralizing response.