Abstract
Soluble HIV-1 envelope glycoprotein (Env) trimers are under active investigation as vaccine candidates in relevant pre-clinical models. Like SOSIPs, the cleavage-independent native flexibly linked (NFL) trimers are faithful mimics of the Env spike. Here, we analyzed multiple new designs to explore alternative modifications, informing tertiary interactions, while maintaining NFL trimer homogeneity and integrity. Accordingly, we performed a proline (P) substitution screen in the gp41 heptad repeat 1 region, identifying other trimer-enhancing Ps, including L555P. This P improved trimer integrity compared to I559P in selected properties. Next, we screened 15 structure-guided potential cysteine pairs in gp140 and found that A501C-L663C (“CC2”) forms an inter-protomer disulfide bond that demonstrably increased NFL trimer thermostability. We combined these two approaches with trimer-derived substitutions, coupled with glycine substitutions at helix-to-coil transitions, developed by our group. To increase the exposure of the fusion peptide (FP) N-terminus, we engineered an enterokinase (EK) cleavage site upstream of the FP for controlled post-expression cleavage. In combination, the redesigns resulted in highly stable and homogeneous NFL mimics derived from different clades. Following recombinant EK cleavage, the NFL trimers retained covalent linkage, maintaining a native-like structure while displaying enhanced stability and favorable antigenic features. These trimers also displayed increased exposure of neutralizing epitopes in the FP and gp120/gp41 interface, while retaining other neutralizing epitopes and occluding non-neutralizing elements. This array of Env-structure-guided designs reveals additional interactive regions in the prefusion state of the HIV Env spike, affording the development of novel antigens and immunogens.
Highlights
Despite many attempts, an effective HIV vaccine remains a daun ting scientific challenge
We introduced single-site proline (P) substitutions in heptad repeat 1 (HR1) to identify other positions in envelope glycoprotein (Env) that might efficiently form well-ordered trimers. This screen had added interest to probe the plasticity of Env and the native flexibly linked (NFL) platform in terms of accepting other P substitution in HR1 that were compatible with the pre-fusion state
Comparable antigenicity profiles were detected for BG505 NFL TD 2CC+ D4K L555P and I559P trim ers (Figures S6F,G in Supplementary Material), consistent with trimer integrity. These analyses demonstrated that the combi nation of L555P, CC2, TD CC+, and engineered post-expression cleavage site, preserve the pre-fusion state of the NFL trimers with improved trimer formation, biophysical properties, and antigenicity
Summary
An effective HIV vaccine remains a daun ting scientific challenge. The SOSIPs are proteolytically cleaved by cellular furins to gp120 and gp subunits, covalently linked by an engineered intra-protomer disulfide bond A501C-T605C (SOS) These trimers contain a I559P substitution in the gp heptad repeat 1 (HR1) region to generate well-ordered oligomers and, as well, require expression of exogenous furin for con formational integrity [3, 8,9,10,11,12,13,14,15,16,17]. In the past several years, we developed an improved native-like trimer design, generating well-ordered soluble Env mimics that are completely cleavageindependent and termed native flexibly linked (NFL) trimers. The original NFL design is relatively inefficient in generating high yields of trimers derived from clade C strains, such as 16055 [19]
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