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.

Fig 1. Key features of candidate Env strains.

17 strains were split into group 1 strains (n = 12) that are naturally sensitive to multiple V2 NAbs and group 2 strains (n = 5) that exhibit high membrane trimer expression. Strain names are abbreviated (see Materials and Methods). An asterisk in total glycans/gp160 protomer indicates overlapping sequons in the CE217 and AC10 strains, only one of which can carry a glycan. Glycan holes are listed whenever a ≥80% conserved glycan is absent. V2 sensitivity features are shown, including glycans involved in NAb binding or clashes and loop lengths. A double asterisk for the CNE58 V1 loop denotes a possible internal hairpin disulfide loop (S1 Fig). Rare glycans N49 and N674 are shown. Strand C sequence (AA166-171) is shown with basic residues in blue and acidic residues in red, along with its charge. The residue at position 500 may influence gp120/gp41 processing (gray highlights non-lysine or arginine residues). PV IC50s for V2 NAbs (CF2 assay) (see Fig 2A). JR-FL neutralization data is for the E168K+N189A mutant. Total Env expression, as judged by SDS-PAGE-Western blot (Fig 3C and 3D).

Fig 2. V2 NAb sensitivity of candidate strains.

A) MAb IC50s against candidate PVs bearing full-length wild-type (WT) gp160 spikes, except for WITO, AC10, 6101, KNH1144 and sc422, that were gp160ΔCT WT. For CH01 and VRC38, UCA sensitivities are shown. CH01 and PG9 NAb IC50s were measured against PVs bearing Envs with engineered glycans: GnT1- and B4GalT1+ST6Gal1 (abbreviated as B4G+ST6), respectively. GnT1- PV of strains BB201 and CNE58 strains were not infectious. B) CH01 IC30s and % maximum CH01 neutralization saturation in unmodified (left) and GnT1- (right) formats.

Fig 3. Gp160ΔCT and SOS mutations consistently improve trimer expression.

A) VLP trimer expression with or without gp41 truncation (gp160ΔCT) and SOS mutations, probed with anti-gp120 and anti-gp41 MAb cocktail. SOS gp160ΔCT trimer expression of candidate strains visualized by B) BN-PAGE-Western blot and by SDS-PAGE-Western blot, probing with anti-gp120 (C) or anti-gp41 (D) MAb cocktails. All Envs were expressed using robust plasmids (pVRC8400 or pCDNA3.1), except for Q23 in part A lanes 6–8 and BB201 in part B lane 10, for which pCR3.1 was used. pVRC8400 was used to express Q23 in part B, lane 2.

Fig 4. Effect of mutations on JR-FL SOS gp160ΔCT trimer expression, infectivity and MAb sensitivity.

Effect of mutations on A) JR-FL gp160ΔCT SOS trimer infectivity, and total Env expression (quantified by SDS-PAGE-Western blot), and B) MAb sensitivity. The best mutant is highlighted in red (lane 35). The V3 consensus (V3 cons) mutant in lane 33 included H310R+R315Q+T319A+E322D mutations.

Fig 5. Effects of mutants on JR-FL membrane trimer glycan maturation and occupation.

Related to S1 Data and analysis and Figs S5 and S6. A) In a trimer model (pdb: 6MYY), each glycan is numbered according to the prototype HXB2 strain (see S1 Fig) and is given a maturation score, derived from LC-MS analysis of parent JR-FL SOS E168K+N189A VLPs (S1 Data and analysis). Glycans are colored in shades of green (high mannose) or magenta (complex), according to their score. Untrimmed high mannose glycans are dark green. Trimmed high mannose glycans are shown in lighter hues of green. Heavy complex glycans are shown in dark magenta, whereas smaller complex glycans are shown in lighter hues of magenta. Some glycans, rendered in gray, were not resolved in the JR-FL parent and therefore have no score (not done; n.d.). Glycans at positions N49 and N197 are modeled as blue translucent masses. B) Glycan identity and scores at each sequon in JR-FL SOS E168K+N189A VLPs determined by LC-MS. Glycans were assigned scores by their degree of maturation (S5 Fig). C) Changes in glycan scores at each position between sample pairs. A negative score implies a shift to less mature glycan and vice versa. Data are only shown at positions where a glycan was detected in >10% of the equivalent peptides of both samples in each pair. Score difference calculations are shown in S1 Data and analysis and are modeled in S6 Fig. D) Sequon skipping and core glycans.

Fig 6. Comparison of pNL-Luc and pQC-Fluc assays for HIV pseudovirus infectivity and neutralization sensitivity.

A) The infectivities of JR-FL E168K+N189A, Q23 D49N+N611A, WITO and T250 SOS gp160ΔCT PVs, produced using pNL-Luc or pQC-Fluc plasmid sets were compared in CF2.CD4 CCR5 cells. The dotted line marks an arbitrary cutoff for infection sufficient to measure neutralization. B) Comparative PG9 sensitivity of the same PV in both assays.

Fig 7. Effects of Q23 and WITO SOS gp160ΔCT mutations on trimer expression, infectivity and MAb sensitivity.

Effect of mutations on A) Q23 and C) WITO gp160ΔCT SOS trimer infectivity (by the pQC-Fluc assay) and total Env expression (by SDS-PAGE-Western blot). B) and D) MAb sensitivities of mutants.

Fig 8. Effects of T250 and CE217 SOS gp160ΔCT mutations on trimer expression, infectivity and MAb sensitivity.

Effect of mutations on A) T250 and C) CE217 gp160ΔCT SOS trimer infectivity (by pQC-Fluc and pNL-Luc assays, respectively) and expression (by SDS-PAGE-Western blot). B) and D) MAb sensitivities of mutants.

Fig 9. Effects of group 2 strain AC10, sc422 and KNH1144 SOS gp160ΔCT mutations on trimer expression, infectivity and MAb sensitivity.

Effect of mutants on A) AC10, C) sc422 and E) KNH1144 gp160ΔCT SOS trimer infectivity (by pQC-Fluc assay) and expression (by SDS-PAGE-Western blot). B), D) and F) MAb sensitivities of mutants.

Fig 10. A quarter of particles from transfections using Env plasmid carry surface Env.

293T cells were transfected with WITO SOS gp160ΔCT and/or MuLV Gag, as indicated in part A). Supernatants were precleared, filtered and 1,000-fold concentrated. Samples were probed by A) SDS-PAGE-Western blot, B) negative stain EM (scale bars are 50nm, white arrows point to candidate Env trimers and green arrows point to possible dissociated spikes, and C) Single vesicle flow cytometry. Upper panels show particle diameters and fluorescence intensities after staining with Alexa-647-labeled PGT121. In the lower panel, we show total particle counts versus Alexa-647 fluorescence. D) Total particle and Env+ particle counts per μl of samples are indicated (left) and % Env+ particles as a proportion of total particles (right). Raw vFC data files and data analysis layouts have been deposited in Flowrepository (flowrepository.org; see S6 Text).

Fig 11. Summary of efforts to develop immunofocusing trimers.

The 17 strains are partitioned into groups 1 and 2. The effects of modifications on expression, infectivity and sensitivity to V2 and/or V3 MAbs are indicated. Desired features are depicted in green boxes. Unwanted features are depicted in red boxes. Features of selected clones are shown in the lower rows along with their V3-sensitivities and potential use in vaccine regimens. Asterisks indicate modifications that were made in combination with others, so any effects observed cannot be unambiguously linked to any one modification.