The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp41 is an important vaccine target as it mediates fusion between viral and host membranes. However, the design of gp41 immunogen has been difficult. In this study, a combination of molecular genetics, structural modeling and biochemical approaches was used to produce soluble near full-length gp41 trimers that mimic the prehairpin structure, an intermediate potentially vulnerable to neutralizing antibody interactions that can block virus entry. The two long heptad repeat helices, HR1 and HR2, of gp41 ectodomain were mutated to disrupt the intra-molecular HR1-HR2, but not the inter-molecular HR1-HR1 interactions. Attachment of a 27-residue “foldon” containing a trimerization motif to the C-terminus and slow refolding channeled gp41 into trimers. The trimers were stabilized in a prehairpin-like structure, as evidenced by binding of a HR2 peptide to the exposed HR1 grooves, lack of binding to six-helix bundle-specific mAb NC-1, and inhibition of virus neutralization by the broadly neutralizing antibodies 2F5 and 4E10. By fusing with the bacteriophage T4 small outer capsid protein, Soc, and displaying on the capsid, the gp41 trimers were converted into nanoparticle-arrayed immunogens. Various gp41 formulations, including a powerful T4-based vaccine delivery platform that simultaneously delivers both DNA and protein were tested for immunogenicity in mouse and rabbit models. The results showed that the specificity of antibody responses depended on the conformation of the immunogen and the vaccine formulation. The DNA vaccines induced robust gp41 antibody titers with broad specificity, including those for neutralization epitopes in the membrane proximal external region, whereas the protein vaccines induced antibodies with greater specificity to the prehairpin structure. These results provided insights into the design of gp41 immunogens and generated novel delivery tools that in future may lead to efficacious HIV-1 vaccine formulations.