Protective HIV-1 vaccines may need to induce antibodies that neutralize diverse HIV-1. A major target for cross-reacting neutralizing antibodies (nabs) is the CD4 binding site (CD4bs) on the envelope glycoprotein trimer (Env). There is an urgent need to identify novel Env trimers where the CD4bs is optimally displayed without exposing immunogenic sites that induce non-neutralizing antibodies or strain specific nabs. We used a novel saturation mutagenesis approach, EMPIRIC (Exceedingly Meticulous and Parallel Investigation of Randomized Individual Codons) to investigate effects of the CD4 binding loop on trimer structure and identify Env mutants with enhanced exposure of the CD4bs. This region of Env contains (1) CD4 contact sites, (2) residues that modulate trimer opening and (3) sites that influence macrophage-tropism. Here, we describe Env mutants that carry a more exposed CD4bs yet differ in the exposure of the V3 loop at the trimer apex.
Using EMPIRIC, we prepared plasmid libraries for all possible mutations covering the CD4 binding loop region (aas 361-380). Libraries were cloned into LN40 env (from an AIDS patient lymph node) incorporated in a replication competent pNL4.3 chimera. LN40/NL4.3 libraries were transfected into 293T cells and progeny virus rescued. This virus was used to infect PBMCs and the replication fitness of each mutant estimated by deep sequencing progeny virus. Env mutants that conferred wt replication or higher were investigated for their properties via Env+ pseudoviruses using Env mabs via neutralization and a trimer binding assay.
Env mutants were identified that carried an exposed CD4bs and a modified trimer apex. These included mutants with substitutions at 373, 375, 377 and 380. Several substitutions at residue 380 resulted in a more exposed CD4bs and a modified trimer apex with an exposed V3 loop. In contrast, while substitutions at residue 375 also resulted in enhanced Env:CD4 interactions, the V3 loop remained occluded. The same observations were made when these substitutions were introduced into another clade B Env and a clade C Env.
We identified specific residues in the CD4 binding loop that expose the CD4bs, yet differ in their effects on the trimer apex and V3 loop in clade B and C Envs. Env mutants that enhance the exposure of the CD4bs yet maintain a closed trimer structure with the V3 loop occluded may be desired immunogens for inclusion in vaccines aimed at eliciting CD4bs nabs.