Biology subjects

Binley, J. M.

Publications and source records attributed to Binley, J. M..

3 recordsLinked to original sources

bioRxiv2024

Impact of Glycan Depletion, Glycan Debranching and Increased Glycan Charge on HIV-1 Neutralization Sensitivity and Immunogenicity

Modifying HIV-1s envelope glycoprotein glycans can impact its neutralization sensitivity. The use of the knock out cell line GnT1-prevents the elaboration of complex-type glycans, and opens up the glycan shield, increasing bNAb vulnerability. Some bNAb precursors can bind to GnT1-trimers, supporting their use in vaccine priming. However, GnT1-trimers express poorly and exhibit very low infectious counts. Here, we describe two other potentially vaccine-relevant glycoengineered trimers, 1) To truncate complex glycans, we used of a cocktail of glycosidases, termed "NGAF3" (Neuraminidase, {beta}-Galactosidase, N-Acetylglucosaminidase and endoglycosidase F3). Like GnT1-trimers, NGAF3 reduced glycan clashes and increased bNAb potency while retaining a closed trimer conformation; 2) Modified by {beta}-1,4-galactosyltransferase 1 (B4GalT1) and {beta}-galactoside -2,6 sialyltransferase 1 (ST6Gal1) during Env biosynthesis. Glycan mass spectrometry revealed that NGAF3 removed glycan heads of 3 of 7 positions on the trimer that are largely occupied by complex glycans. It also revealed a novel B4GalT1 activity to favor glycan precursor conversion to hybrid glycans rather than complex glycans. A comparison to monomeric gp120 revealed that B4GalT1s new activity depends on tight glycan spacing. B4GalT1 affected more glycans than NGAF3 (6 out of 7 glycans), perhaps due to greater accessibility during Env folding rather than after folding. Surprisingly, the N611 glycan was unaffected by either modification. B4GalT1 and ST6Gal1 cooperatively increased the abundance of hybrid glycans and -2,6 hypersialylated termini. This occurred largely by amplifying the abundance of a limited number of hybrid glycan structures that are also present on unmodified trimers. In rabbit vaccinations, B4GalT1+ST6GalT1-modified virus-like particles reduced the frequency and titers of serum NAbs that showed a modest preference for modified glycans. Conversely, chronically HIV-1-infected donor plasma neutralizing antibody titers were 1.7- to 10.8- fold higher against B4GalT1+ST6GalT1-modified pseudovirus. Overall, our data provide tools for heterologous prime, boost and polishing vaccine regimens using modified glycans. AUTHOR SUMMARYAn HIV-1 vaccine remains one of the most significant biomedical challenges today. Vaccines often work by triggering virus fighting antibodies to stave off infection. For HIV-1, this has been exceedingly difficult because the target, called the HIV-1 envelope glycoprotein (Env) is extremely variable and carries a thick sugar coat, protecting it from all but a few rare broadly reactive antibodies (termed bNAbs) that sometimes develop during natural HIV-1 infection. Given these challenges, it is reasonable to propose that any successful vaccine will need to be rationally designed to trigger the rare bNAbs. To meet this goal might require more 3 different vaccine components. First, a vaccine "prime" to trigger rare bNAb proliferation, followed by heterologous "boosts" and "polishing" immunogens to select for bNAbs. In this study, we evaluated two potential immunogens. In one approach, we used cocktails of enzymes to strip Envs sugar coat, in the hope of reducing the barrier to stimulate bNAb precursors. In another, we used excess enzymes to build particular structures on Envs coat. We checked how well these two Env variants were recognized by antibodies. We also checked in fine detail how the sugars were changed at the molecular level. Finally, we immunized rabbits. Our data enrich the number of strategies available to further explore the concept of priming, boosting and polishing vaccine shots.

immunology↗

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↗