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. 2023 Aug 7;19(8):e1011452.
doi: 10.1371/journal.ppat.1011452. eCollection 2023 Aug.

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

Tommy Tong  1 , Alessio D'Addabbo  2 , Jiamin Xu  1 , Himanshi Chawla  2 , Albert Nguyen  1 , Paola Ochoa  1 , Max Crispin  2 , James M Binley  1
Affiliations

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

Tommy Tong et al. PLoS Pathog. .

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. HIV-1 gp160 mutants.
A schematic of gp160 showing gp120 (green) variable (V1-V5) domains, constant domains (C1-C5), and gp41 (salmon), including the transmembrane domain (TM). Mutants are shown in different colors that we also use consistently hereafter for clarity. Amino acids are numbered according to HxB2 subtype B reference strain. Gp41 was truncated from position 709 onwards, leaving three amino acids after the TM domain in gp160ΔCT. Most of our JR-FL mutants are based on gp160ΔCT SOS E168K+N189A, unless otherwise stated. NFL is “native flexibly linked” with Gly-Gly-Gly-Gly-Ser sequence repeated twice, which replaces REKR at position 508–511. EK is an “enterokinase” digestion site with sequence Asp-Asp-Asp-Asp-Lys and is added downstream of NFL. UFO is “uncleaved prefusion-optimized” consists of the sequence Gly-Ser repeated four times replacing residues 548–568. The UNC consists of K510S+R511S. The NT1-5 mutant consists of M535I+L543Q+N553S+Q567K+G588R.
Fig 2
Fig 2. SDS-PAGE-Western blot of gp160ΔCT mutants.
VLPs expressing various JR-FL gp160ΔCT E168K+N189A mutant combinations were analyzed by reducing SDS-PAGE-Western blot. Duplicate blots were probed with A) anti-gp120, B) anti-gp41 or C) anti-gp120+gp41 MAb cocktails. Env species are indicated by colored dots.
Fig 3
Fig 3. Endoglycosidase H digestion of I559P, DS and I559P+DS gp160ΔCT mutants.
VLPs were lysed and boiled in SDS/DTT, then treated with endo H or PBS. Samples were then analyzed in duplicate SDS-PAGE-Western blots probed with anti-gp120+gp41 MAb cocktail (Lanes 1–8) or anti-gp41 MAb cocktail (Lanes 9–16).
Fig 4
Fig 4. Enterokinase processing and endoglycosidase H digestion of gp160ΔCT SOS NFL mutants into gp120/gp41.
VLPs were digested with enterokinase or PBS for 30h at 37°C, then lysed, incubated with endo H or PBS then analyzed in reducing SDS-PAGE-Western blot and probed with A) anti-gp120 or B) anti-gp41 MAb cocktails. The asterisk (*) marks an ~80kDa band detected by anti-gp41 MAb cocktail in parent (non-I559P) samples, that suggests non-specific digestion, perhaps of the V3 loop, even in the absence of EK enzyme. The black dot detected in the anti-gp120 MAb cocktail (Lane 7) represents the non-specific digestion of gp160m by the EK enzyme.
Fig 5
Fig 5. BN-PAGE-Western blot of gp160ΔCT mutants.
A) The same mutants in Fig 2 were analyzed by BN-PAGE-Western blot, detecting with anti-gp120+gp41 MAb cocktail. Densitometry analysis of trimer, dimer and monomer band is shown in S1 Fig) BN-PAGE analysis of enterokinase-treated samples from Fig 4. Env species are indicated by colored dots.
Fig 6
Fig 6. Effect of lysis on gp160ΔCT trimer stability in the presence of BS3 crosslinker.
VLPs corresponding to lanes 1, 2, 3, 11, 17 and 18 of Figs 2 and 5 were crosslinked with and without BS3, then analyzed by BN-PAGE-Western blot, detecting with anti-gp120+gp41 MAb cocktail. Crosslinking efficiency was checked by boiling crosslinked or PBS-treated SOS VLPs in SDS and DTT (lanes 13 and 14). The asterisk (*) marks monomer shifted upwards with BS3 crosslinking. Env species are indicated by colored dots.
Fig 7
Fig 7. MAb binding to JR-FL gp160ΔCT E168K+N189A in BN-PAGE “shifts”.
MAbs were mixed with SOS VLP (A and B) or SOS NFL I559P VLP (C and D) and were incubated at 37°C for 1h, then washed, lysed and analyzed by BN-PAGE-Western blot, probing with anti-gp120+gp41 MAb cocktail, followed by anti-human IgG AP conjugate (A and C) or just the anti-human IgG AP conjugate (B and D). The asterisk (*) marks a ~220kDa observed with b12 and 15e, possibility arise from decreased lateral stability during MAb binding.
Fig 8
Fig 8. Dissecting mutant contributions to antigenic profiles by BN-PAGE mAb “shifts”.
MAb shifts were performed with various mutants and resolved on BN-PAGE, detecting with anti-gp120+gp41 MAb cocktail. Mutants are all JR-FL gp160ΔCT E168K+N189A, except for some full length (FL) gp160 clones. (A) SOS NFL (lanes 1–7), WT NFL I559P (lanes 8–14), SOS I559P (lanes 15–21), SOS D197N (lanes 22–28); (B) SOS DS (lanes 1–7), SOS UNC (Lanes 8–14), SOS D197N NT1-5 (lanes 15–21), SOS D197N+A328G (lanes 22–28); (C) SOS FL–Wash out (lanes 1–7), WT FL–Wash out (lanes 8–14), WT (lanes 15–21); (D) SOS FL–Leave in (lanes 1–7), WT FL–Leave in (lane 8–14). For VLPs bearing FL Env, excess MAbs are either removed by washing in PBS (Wash out) or not removed (Leave in). UNC mutant is gp160ΔCT SOS E168K+K510S+R511S.
Fig 9
Fig 9. Antigenic profile of VLPs carrying different mutations assessed by virus capture assay.
Capture of various JR-FL mutant VLPs by a subset of MAbs. For each mutant, data was normalized by 2G12 capture which approximates Env expression. This allows mutants to be compared regardless of expression differences. Virus capture assays were performed in quadruplicate and repeated at least three times. Error bars represent the standard deviation of the mean. UNC is gp160ΔCT SOS E168K+K510S+R511S. Kruskal-Wallis test was used to analyze for significant difference of different VLPs captured by a MAb, relative to SOS ΔCT VLP. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 10
Fig 10. JR-FL gp160ΔCT E168K+N189A VLP binding titer of human HIV+ plasma probed by VLP ELISA.
(A) Plasma binding titer of human HIV+ plasma (n = 22) to JR-FL gp160ΔCT WT, SOS or SOS I559P VLP analyzed by ELISA. Plasma binding titers were calculated as the dilution where its binding OD was 0.5. Geometric mean binding titer (GMT) against each VLP is indicated as horizontal line. One-way ANOVA Dunn’s multiple comparison test revealed a significant difference of plasma binding titer between WT, SOS and SOS I559P VLPs. **p < 0.01, ****p < 0.0001. (B) Correlation analysis of HIV+ plasma neutralization titers (ID50) against JR-FL WT gp160ΔCT PV and binding titer ratio SOS:SOS I559P VLP. Correlation was significantly dependent on the highly neutralizing N308 plasma (p < 0.0001). The correlation was not significant when this plasma was excluded. Plasma NAb specificities, where known, are indicated in parentheses.
Fig 11
Fig 11. Effects of NFL, I559P and A433P mutants on gp160ΔCT glycan maturation and occupation.
Related to S13–S15 Figs and S1 Main glycan analysis and S1 File. Each glycan on the trimer models (pdf: 6MYY) is numbered according to HxB2 strain and is given a maturation score derived from LC-MS analysis. Glycan score differences between two clones are colored in shades of red (i.e., a negative score indicates decrease in processing), white (no difference in glycan processing) or blue (i.e., positive score indicates increase in processing). Data are only shown at positions where a glycan was detected in >10% of the equivalent peptides of both samples in each pair. Some glycans, rendered in gray, were not resolved in the sample, and therefore have no score (not done; n.d.). Glycan score difference calculations between sample pairs are shown in S1 Main glycan analysis, and are modeled here as follow: (A) Viral gp120 band vs viral gp160 bands; (B) Parent average vs SOS NFL VLP gp160; (C) Parent average vs SOS NFL I559P VLP gp160; (D) Parent average vs SOS NFL A433P VLP gp160; (E) SOS NFL VLP gp160 vs SOS NFL I559P VLP gp160; (F) SOS NFL VLP gp160 vs SOS NFL A433P VLP gp160. *Parent average is the glycan score average of “total VLPs” preparation from 2020, 2021 and 2023 (S14F Fig).
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