In a previous study we had demonstrated the induction of broadly cross-reactive HIV-1 neutralization in rabbits immunized with the particular strain of HIV-1 gp140 used here, designated R2, in proprietary adjuvant. Limitations of that study were that the R2gp140 used was produced by infection of cells with recombinant vaccinia virus such that the gp140 could only be partially purified from contaminating host cell proteins and anti-human antibody responses developed in response to immunization, and the gp140 that was used was a mixture of higher order forms, mostly dimer. The gp140 Gemifloxacin mesylate itself was constructed to include the gp120 AZ191 sequence fused to the gp41 ectodomain as a result of elimination of the natural protease cleavage sequences that are used for processing to mature Env in virus. In this study different forms of highly purified gp140 were obtained from stably transformed cell lines for production of the various recombinant proteins. In an effort to mimic the native tertiary structure of Env, trimeric gp140 was constructed by fusion to a GCN4 trimerization domain, and the effect of insertion of a flexible linker sequence between the gp120 and gp41 sequences was also evaluated. The cell lineproduced, uncleaved gp140 again was found to be a mixture of dimer and trimer, mainly dimer, while the gp140s with or without the linker but with the GCN4 tail were all trimer. The different forms of gp140 reacted similarly with various mAbs, including direct binding to CD4i mAbs in the absence of CD4, and with CCR5. The comparative immunogenicity with respect to induction of neutralizing antibodies was also similar among the different forms of gp140, although this comparative part of the study might have been more discriminatory if it had been continued for additional immunizations. Broadly cross reactive neutralization was elicited in rabbits that were continued to completion of the immunization protocol. Epitope specificity studies revealed evidence of targeting that did not resemble that of known broadly cross-neutralizing mAbs. Studies of trimeric Env-specific antibody producing cells in spleen and lymph nodes demonstrated robust responses.