D'Addabbo, A. | bioRxiv Science

bioRxiv2023

Impact of Common Modifications on the Antigenic Profile and Glycosylation of Membrane-Expressed HIV-1 Envelope Glycoprotein.

Recent HIV-1 vaccine development has centered on "near native" soluble envelope glycoprotein (Env) trimers. These trimers are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These same stabilizing mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their precise effects in this context are unclear. To address this question, we investigated the effects of Env mutations expressed on virus-like particle (VLP) in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 (NFL) neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed processing post-expression, resulting in increased trimer stability. Gp41 N-helix mutations I559P and NT1-5 both imparted lateral trimer stability, but concomitantly reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ donor plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common effect of mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. We observed a minor species of high mannose glycan only gp160 in particle preparations. This was unaffected by any mutations and instead bypasses normal folding and glycan maturation processes. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and increased the proportion of high mannose gp160. Remarkably, NAbs were unable to bind to full-length Env trimers. Overall, our findings suggest caution in leveraging mutations to ensure they impart valuable membrane trimer phenotypes for vaccine use. AUTHOR SUMMARYA vaccine that induces virus-fighting antibodies to block HIV-1 infection remains elusive. To ablate HIV-1 infection, antibodies must bind to authentic envelope (Env) glycoprotein on the virus surface. However, Env can exist in various forms, many of which are relatively easy targets for non-effective antibodies. Therefore, a key challenge of vaccine design is to create pure authentic Env that is unfettered by these other forms of Env. Vaccine research to date has focused largely on stabilizing soluble Env trimers, as this format simplifies purification and translational studies. However, incomplete Env authenticity may blunt the efficacy of this approach. By comparison, the manufacture of particle-based vaccines that express Env in situ on membranes is cumbersome. In an alternative approach, lipid particles can deliver mRNA vaccines encoding membrane trimers, bypassing the manufacturing challenges of previous methods. Stabilizing mutations derived from soluble trimers are now being leveraged for membrane trimers. Here, we evaluated the effects of these mutations. Our results show that some mutations alter Env conformation, and therefore might best be omitted from membrane Env vaccines.

immunology↗

bioRxiv2021

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

HIV-1 vaccine immunofocusing strategies have the potential to induce broadly reactive nAbs. Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets of neutralizing antibodies (NAbs), 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 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 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-neutralizing antibodies, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the V2 loops C-strand. Notably, a 167N mutation improved V2-sensitivity. 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 other glycans in some cases had local and global "ripple" effects on glycan maturation and sequon occupation in the gp120 outer domain and gp41. V2 mAb CH01 selectively bound trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a N49 glycan perturbs gp41 glycans via a distal glycan network effect, 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. Overall, we identified 7 diverse trimers with a range of sensitivities to two targets that should enable rigorous testing of immunofocusing vaccine concepts. Author SummaryDespite almost 40 years of innovation, an HIV vaccine to induce antibodies that block virus infection remains elusive. Challenges include the unparalleled sequence diversity of HIVs surface spikes and its dense sugar coat that limits antibody access. However, a growing number of monoclonal antibodies from HIV infected donors provide vaccine blueprints. To date, these kinds of antibodies have been difficult to induce by vaccination. However, two antibody targets, one at the spike apex and another at the side of the spikes are more forgiving in their demands for unusual antibodies. Here, we made a diverse panel of HIV spikes vulnerable at these two sites for later use as vaccines to try to focus antibodies on these targets. Our selection criteria for these spikes were: i) that the spikes, when expressed on particles, are infectious, allowing us to appraise our vaccine designs in an ideal manner; ii) that spikes are easy to produce by cells in quantities sufficient for vaccine use. Ultimately, we selected 7 trimers that will allow us to explore concepts that could bring us closer to an HIV vaccine.

immunology↗