Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

Abstract

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

Structure-Based Stabilization of SOSIP Env Enhances Recombinant Ectodomain Durability and Yield

“…By enhancing the durability of the prefusion conformation of Env, it may be possible to increase the residence time of Env immunogens in B cell germinal centers, theoretically mimicking the conditions of natural infection where natively expressed Env is continually present ( 15 ). Reliable presentation of the prefusion Env is thought to be strictly required to elicit a polyclonal, broadly neutralizing response because the UCAs of many bNAbs, such as DH270 and CH01, are only capable of recognizing Env in the prefusion conformation ( 28 , 44 ). The enhanced durability observed in our thermostability and forced degradation assays suggests that 2P-stabilized Env proteins may not require the same restrictive cold-chain storage conditions that are necessary for unstabilized Env immunogens.…”

Single-component multilayered self-assembling protein nanoparticles presenting glycan-trimmed uncleaved prefusion optimized envelope trimers as HIV-1 vaccine candidates

“…The tier 2 NAb responses in wildtype mice and NHPs are sporadic and often target glycan-rich regions on the Env that are immunodominant in human infection (67,70,71). Although bNAb responses were occasionally reported (52,58,(139)(140)(141), it is unclear whether these are reproducible outcomes of a general vaccine solution or coincidence. Therefore, despite all advances achieved to date, HIV-1 vaccine development still faces critical challenges posed by the Env, an atypical viral antigen (142).…”

“…Although bNAb responses were occasionally reported (52,58,127,148,149), it is unclear whether these are reproducible outcomes of a general vaccine solution or coincidence. Therefore, despite all advances achieved to date, HIV-1 vaccine development still faces critical challenges posed by the Env, an atypical viral antigen (150).…”

Structure-based stabilization of SOSIP Env enhances recombinant ectodomain durability and yield

“…By enhancing the durability of the prefusion conformation of Env, it may be possible to increase the residence time of Env immunogens in B cell germinal centers, theoretically mimicking the conditions of natural infection where natively expressed Env is continually present (15). Reliable presentation of the prefusion Env is thought to be strictly required in order to elicit a polyclonal, broadly neutralizing response since the UCAs of many bnAbs, such as DH270 and CH01, are only capable of recognizing Env in the prefusion conformation (28,42). The enhanced durability observed in our thermostability and forced degradation assays suggest that 2P-stabilized Envs may not require the same restrictive cold-chain storage conditions that are necessary for unstabilized Env immunogens.…”