These results indicate that the altered epitopes in the gp41 MPER, rather than those in the gp41 6HB core, elicit neutralizing TWS119 antibodies in vaccinated animals. The precise mechanism by which these mutations exposed neutralization epitopes remained unclear, since chemical crosslinking assay, together with antibodies binding assays by ELISA and SPR, indicated no significant difference between NCM and mutants. However, several studies have demonstrated that strictly conserved T569 and I675 residues play important roles in viral entry. T569 residue is located in the inner face of NHR trimer and falls within the hydrophobic pocket which is critical for interaction with CHR. Therefore, T569A mutation may partially disrupt the gp41 6HB core conformation, resulting in change of the overall conformation of gp41. It was reported the I675V mutation could affect some physicochemical properties of MPER which contribute to increased immunogenicity. However, these changes could not fully explain the strong immunogenicity of MPER in NCM, since single-mutated NCM did not elicit high titers of MPER-specific antibodies. Therefore, we considered the effect of another interesting finding that NCM showed lower capture level to membranes. Many studies reported that the membrane plays a crucial role in defining the structure of the MPER and, consequently, the formation of the 2F5 and 4E10 epitopes. The long CDR-H3s of these antibodies were involved in the interaction with lipids. However, the immersion of MPER in membrane may have a negative effect on immunogenicity of MPER because of the steric restrictions imposed by the viral membrane which may hamper the generation of antibodies. Although the rabbits were immunized with NCM and its mutants in the absence of liposomes, the immunogen could diffuse and nonspecifically interact with membrane systems in multiple immune processes after subcutaneous injections. This interaction may result in the decreased exposure of the epitopes on MPER, rendering a correspondingly poorer immunogenicity. On the contrary, decreasing the immersion depth of epitopes in membrane could contribute to an increase of immunogenicity. In fact, the 4E10 epitope was immersed in the polar/apolar interfacial region of the lipid bilayer, whereas 2F5 epitope was more solvent exposable. Thus the exposure of 4E10 epitope might be more sensitive to immersion depth. I675 residue was reported to be one of the rare residues which immersed deeply both before and after 4E10 binding. Therefore, a shorter side chain of Valine in I675V mutant may Life Science Reagents facilitate the decreasing immersion depth of MPER, especially the 4E10 epitope. Still, how T569A and I675V mutations synergistically affect the capture level of NCM remains to be further studied.