PD03.03
Background: Our previous study suggests that YU2 gp120 trimers, deleted of their major variable regions and partially stabilized in the CD4-bound state display better elicitation of CD4 binding site (CD4bs)-directed neutralizing antibodies compared to core and full-length trimers. The elicited neutralizing antibodies target elements exposed only on Env of sensitive viruses and cannot neutralize more resistant isolates. Structural studies reveal a “layered” gp120 architecture in which three topologically separate and structurally plastic layers act as a shape-changing spacer, facilitating movement between the outer domain and gp41 to allow movement among alternative conformations required for virus entry and immune evasion. A recent report demonstrates that disulfide bond (C65-C115) inserted between layers can lock gp120 in a CD4 bound state.
Methods: Here, we integrated the disulfide bonds C109-C428 (inner and outer domain), C65-C115 (layers 1 and 2) and C95-C484 (layers 2 and 3), into the YU2 gp120 trimers.
Results: These stabilized Envs displayed improved stability and antigenic profiles. The substituted cysteines eliminated binding of most non-neutralizing CD4bs antibodies while retaining recognition by most broadly neutralizing CD4bs antibodies. Armed with this promising antigenic profile, we immunized guinea pigs and non-human primates with these disulfide-stabilized Envs with or without conjugation onto liposome particles. The parental trimers once again elicited Tier 1 neutralization, however, introduction of the C109-C428 rendered antibodies unable to neutralize Tier 1 viruses. However, the stabilized Envs elicited improved binding antibodies toward the CD4bs and, in addition, conjugation onto liposomes shifted the immune responses to the CD4bs.
Conclusions: These results suggest that structure-guided stabilization can be used to better expose conserved epitopes of gp120 and the immune responses can be shifted onto the conserved epitopes by particulate display or by other stabilization strategies.
