Impact of stabilizing mutations on the antigenic profile and glycosylation of membrane-expressed HIV-1 envelope glycoprotein

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 nucleic acid-based membrane Env vaccines. Recent HIV-1 vaccine development has centered on "near native" soluble envelope glycoprotein (Env) trimers that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced 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 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these 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. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use.