•Well-ordered Env trimers conjugated to liposomes maintain the desired antigenic profile•Superior B cell responses were induced using liposome display of Env trimers•mAbs capable of neutralizing the clade C 16055 virus (tier 2) were isolated•The mAbs target the glycan-shrouded V2 cap with a unique angle of approachThe development of soluble envelope glycoprotein (Env) mimetics displaying ordered trimeric symmetry has ushered in a new era in HIV-1 vaccination. The recently reported native, flexibly linked (NFL) design allows the generation of native-like trimers from clinical isolates at high yields and homogeneity. As the majority of infections world-wide are of the clade C subtype, we examined responses in non-human primates to well-ordered subtype C 16055 trimers administered in soluble or high-density liposomal formats. We detected superior germinal center formation and enhanced autologous neutralizing antibodies against the neutralization-resistant (tier 2) 16055 virus following inoculation of liposome-arrayed trimers. Epitope mapping of the neutralizing monoclonal antibodies (mAbs) indicated major contacts with the V2 apex, and 3D electron microscopy reconstructions of Fab-trimer complexes revealed a horizontal binding angle to the Env spike. These vaccine-elicited mAbs target the V2 cap, demonstrating a means to accomplish tier 2 virus neutralization by penetrating the dense N-glycan shield.Download high-res image (179KB)Download full-size image

•Structure-guided design generates an HIV clade C Env crystal structure at 3.9 Å•Cross-clade Env comparison discloses overall structural and N-glycan conservation•The NFL structure reveals stabilizing TD contacts and the 201C-A433C disulfide CC•NFL Env redesign permits the generation of immunogens derived from clades A, B, and CAdvances in HIV-1 envelope glycoprotein (Env) design generate native-like trimers and high-resolution clade A, B, and G structures and elicit neutralizing antibodies. However, a high-resolution clade C structure is critical, as this subtype accounts for the majority of HIV infections worldwide, but well-ordered clade C Env trimers are more challenging to produce due to their instability. Based on targeted glycine substitutions in the Env fusion machinery, we defined a general approach that disfavors helical transitions leading to post-fusion conformations, thereby favoring the pre-fusion state. We generated a stabilized, soluble clade C Env (16055 NFL) and determined its crystal structure at 3.9 Å. Its overall conformation is similar to SOSIP.664 and native Env trimers but includes a covalent linker between gp120 and gp41, an engineered 201-433 disulfide bond, and density corresponding to 22 N-glycans. Env-structure-guided design strategies resulted in multiple homogeneous cross-clade immunogens with the potential to advance HIV vaccine development.Download high-res image (243KB)Download full-size image

HIV-1 neutralization requires Ab accessibility to the functional envelope glycoprotein (Env) spike. We recently reported the isolation of previously unidentified vaccine-elicited, CD4 binding site (CD4bs)-directed mAbs from rhesus macaques immunized with soluble Env trimers, indicating that this region is immunogenic in the context of subunit vaccination. To elucidate the interaction of the trimer-elicited mAbs with gp120 and their insufficient interaction with the HIV-1 primary isolate spike, we crystallized the Fab fragments of two mAbs, GE136 and GE148. Alanine scanning of their complementarity-determining regions, coupled with epitope scanning of their epitopes on gp120, revealed putative contact residues at the Ab/gp120 interface. Docking of the GE136 and GE148 Fabs to gp120, coupled with EM reconstructions of these nonbroadly neutralizing mAbs (non-bNAbs) binding to gp120 monomers and EM modeling to well-ordered trimers, suggested Ab approach to the CD4bs by a vertical angle of access relative to the more lateral mode of interaction used by the CD4bs-directed bNAbs VRC01 and PGV04. Fitting the structures into the available cryo-EM native spike density indicated clashes between these two vaccine-elicited mAbs and the topside variable region spike cap, whereas the bNAbs duck under this quaternary shield to access the CD4bs effectively on primary HIV isolates. These results provide a structural basis for the limited neutralizing breadth observed by current vaccine-induced, CD4bs-directed Abs and highlight the need for better ordered trimer immunogens. The analysis presented here therefore provides valuable information to guide HIV-1 vaccine immunogen redesign.

Vaccine-elicited mAbs bind the HIV-1 Env CD4bs differently from broadly neutralizing infection-induced mAbs.