A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers

Article

A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers Graphical Abstract

Authors Lucy Rutten, Yen-Ting Lai, Sven Blokland, ..., Hanneke Schuitemaker, Peter D. Kwong, Johannes P.M. Langedijk

Correspondence [email protected] (P.D.K.), [email protected] (J.P.M.L.)

In Brief Rutten et al. describe a universal repair and stabilize approach that corrects rare mutations and stabilizes refolding regions to obtain high-quality HIV Envs with high yields. The crystal structure shows how the optimization of the trimer interface between a9, a6, and the intersubunit b-sheet stabilizes the membraneproximal base.

Highlights d

High-quality HIV-1 Env proteins by universal repair and stabilize strategy

d

Crystal structure of stabilized clade C trimer explaining stabilization strategy

d

Identification of ten novel stabilizing substitutions, of which seven are at the base

d

a9-helix and intersubunit b sheet at the Env base is critical for trimer stability

Rutten et al., 2018, Cell Reports 23, 584–595 April 10, 2018 ª 2018 The Authors. https://doi.org/10.1016/j.celrep.2018.03.061

Data and Software Availability 6CK9

Cell Reports

Article A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers Lucy Rutten,1 Yen-Ting Lai,2 Sven Blokland,1 Daphne Truan,1 Ilona J.M. Bisschop,1 Nika M. Strokappe,1 Annemart Koornneef,1 Danielle van Manen,1 Gwo-Yu Chuang,2 S. Katie Farney,2 Hanneke Schuitemaker,1 Peter D. Kwong,2,* and Johannes P.M. Langedijk1,3,* 1Janssen

Vaccines & Prevention, Archimedesweg 4-6, Leiden 2333, the Netherlands Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA 3Lead Contact *Correspondence: [email protected] (P.D.K.), [email protected] (J.P.M.L.) https://doi.org/10.1016/j.celrep.2018.03.061 2Vaccine

SUMMARY

The heavily glycosylated native-like envelope (Env) trimer of HIV-1 is expected to have low immunogenicity, whereas misfolded forms are often highly immunogenic. High-quality correctly folded Envs may therefore be critical for developing a vaccine that induces broadly neutralizing antibodies. Moreover, the high variability of Env may require immunizations with multiple Envs. Here, we report a universal strategy that provides for correctly folded Env trimers of high quality and yield through a repair-and-stabilize approach. In the repair stage, we utilized a consensus strategy that substituted rare strain-specific residues with more prevalent ones. The stabilization stage involved structure-based design and experimental assessment confirmed by crystallographic feedback. Regions important for the refolding of Env were targeted for stabilization. Notably, the a9-helix and an intersubunit b sheet proved to be critical for trimer stability. Our approach provides a means to produce prefusion-closed Env trimers from diverse HIV-1 strains, a substantial advance for vaccine development. INTRODUCTION The mature HIV-1 envelope (Env) spike is a trimer of gp120 and gp41 heterodimeric subunits. The gp120 subunit initiates fusion by binding to CD4 receptors and CCR5/CXCR4 coreceptors and transmitting this binding through conformational changes to the transmembrane gp41 subunit. The HIV-1 fusion machinery is sequestered in the prefusion conformation of gp41, which refolds into a postfusion conformation (Melikyan et al., 2000). In the prefusion conformation, Env trimers spontaneously sample distinct conformational states (Munro et al., 2014). Ligands can alter the proportion of molecules in these conformational states, with broadly neutralizing antibodies generally stabilizing a closed state and with CD4 stabilizing an open state (Liu et al., 2008; Munro et al., 2014; Ozorowski et al., 2017; Wang et al., 2016).

The relative proportion of these prefusion-conformational states is strain-dependent (Munro et al., 2014), with typical tier 1 (neutralization sensitive) strains adopting a higher proportion of the open state compared to a typical tier 2 (neutralization resistant) virus (Guttman et al., 2014; Liu et al., 2008; Munro et al., 2014; Ozorowski et al., 2017) . Trimer instability has been a substantial barrier to the development of an HIV vaccine that aims to induce broadly neutralizing antibodies (Feng et al., 2016; Guttman et al., 2015; Kwon et al., 2015; Sanders et al., 2013, 2016). Moreover, low expression levels decrease prospects for successful protein subunit development or for the application of a vector based approach. Therefore, stable Env trimers are needed that fold efficiently into a prefusion-closed state and that fully arrest conformational changes to more open states. The introduction of the so-called SOSIP substitutions was an important step to obtain native trimers (Binley et al., 2000; Sanders et al., 2002), but the fraction of prefusion-closed trimer remains less than optimal (Julien et al., 2015) and/or the resultant trimers open in the presence of CD4 (de Taeye et al., 2015). Although elucidation of the trimer structure facilitated substantial progress in recent years to stabilize the conformation of Env based on disfavoring the postfusion conformation and to prevent breathing by introducing disulfide bridges, optimizing packing, utilizing stabilizing substitutions known from other Envs (Binley et al., 2000; Chuang et al., 2017; de Taeye et al., 2015; Dey et al., 2009; Garces et al., 2015; Guenaga et al., 2015, 2017; Julien et al., 2013; Kesavardhana and Varadarajan, 2014; Kong et al., 2016; Kulp et al., 2017; Kwon et al., 2015; Lyumkis et al., 2013; Pancera et al., 2014; Sanders et al., 2002; Steichen et al., 2016; Stewart-Jones et al., 2016; Sullivan et al., 2017; Torrents de la Pen˜a et al., 2017) or by using of a chimeric strategy (Joyce et al., 2017), a general approach to produce prefusion-closed Env trimers of high quality and yield, universally applicable to most HIV-1 strains, would be an important advancement for the field. As sequential immunizations with heterologous stable highquality Envs may be needed to induce broadly neutralizing antibodies, we sought a universal approach to deliver high expression of diverse high-quality prefusion-closed trimers. We hypothesized that (1) a consensus or mosaic sequence would provide a superior starting template for universal adaptation,

584 Cell Reports 23, 584–595, April 10, 2018 ª 2018 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Consensus clade C Env

◊

@

B

mAU (280nm)

*°

mAU (280 nm)

A

Mosaic clade C Env

*° 1 9 1

C

D M

+

- DTT

191 kDa 97 kDa

9 7

gp140 gp120

6 4

5 1

64 kDa 51 kDa

(A and B) SEC profiles of GN lectin-purified HIV Env SOSIPs of (A) clade C consensus (ConC_SOSIP) and closest wild-type Env sequences indicated with GenBank numbers. The trimer peak is indicated with an asterisk, gp140 monomer peak is indicated with a degree symbol, gp120 monomer is indicated with a diamond, and aggregates are indicated with an at symbol. (B) clade C mosaic SOSIP 201C-433C (DS_sC4_SOSIP) and closest wild-type Env sequences indicated with the GenBank numbers. The trimer peak is indicated with an asterisk and gp140 monomer peak with a degree symbol. (C) Analysis of GN lectin-purified ConC_SOSIP on SDS-PAGE under reducing (+DTT) and nonreducing ( DTT) conditions compared with markers (M). (D) Negatively stained EM of ConC_SOSIP trimers with 2D-averaged classes (right).

3 9

39 kDa 28 kDa

Figure 1. HIV-1 Env Selection for Stabilization

gp41

2 8

and (2) the stabilization of specific subunit and protomer interfaces that move between closed and open conformations would limit breathing and prevent irreversible refolding. We showed our two-pronged ‘‘repair and stabilize’’ procedure to yield prefusion-closed HIV-1 Env trimers with high expression, stability, and desired antigenicity for Envs—for which SOSIP mutations yielded only unstable, low expressing Envs with poor antigenicity. RESULTS Selection of Clade C Env Because HIV-1 clade C comprises the most common circulating isolates, we evaluated expression and folding of a consensus clade C-based Env stabilized with SOSIP mutations. This so-called ConC_SOSIP Env showed higher trimer content compared to the closest matched wild-type (WT) Envs in the database (Figure 1A) suggesting a superior folding for ConC_SOSIP. Similarly, the mosaic clade C Env (DS_sC4_ SOSIP), based on a compilation of Env sequences with a broad coverage of T cell epitopes (Fischer et al., 2007), showed higher trimer content compared to its closest wild-type sequence matches (Figure 1B). The mosaic Env did not bind to the trimer-specific apex-binding antibodies PGT145 and PGDM1400 (Sok et al., 2014), whereas ConC_SOSIP showed slightly weaker PGDM1400 but otherwise comparable binding to these monoclonal antibodies (mAbs) relative to that of BG505_SOSIP (data not shown). Furthermore, ConC_SOSIP was almost fully cleaved (Figure 1C) and formed homogeneous trimeric population as assessed by negatively stained electron microscopy (Figure 1D). Therefore, the consensus C-based Env was selected as a template for further stabilization.

Design Considerations for Stabilizing Substitutions Knowledge of the correct folding of Env and a detailed understanding of the interactions that underlie the refolding mechanism can provide guidance for design of prefusion-closed Env trimers. To stabilize the Env trimer, we took into consideration the overall fusion process in which Env shows reversible breathing to different states. At a critical point in the successive conformational changes, a tipping point is reached in which folding to the postfusion state is irreversible. In refolding region 1 of gp41 (Figure 2A), the loop between prefusion a6-a7 helices refolds into a single helix, which is assembled on the base helix a7 together with a6 and the N-terminal fusion peptide making one long extended trimeric helical coiled coil with the other protomers of the trimer. Subsequently, additional gp41:gp120 interactions are lost and helices a8 and a9 refold and dock against the coiled coil region to complete the six-helix bundle of the postfusion gp41. For a further understanding of the relation of Env instability and viral fusion kinetics, directed evolution for increased (Leaman and Zwick, 2013) or decreased stability allows for the identification of essential contact regions that modulate irreversible refolding; K574R and I535M are associated with increased Env stability in different clades (Dey et al., 2008; Leaman and Zwick, 2013) and Q590E and K601E are predicted to accelerate refolding (Eggink et al., 2009). Here, we observed that mutations Q590E and K601E did indeed decrease Env stability (Figure S1A). Residue Q590 in the base of the a7 helix is directed toward the trimer axis between three a7 chains; if mutated to a Glu, it likely causes charge repulsion between trimer protomers. In most published crystal structures, no side chain density is visible for residue 601, but in PDB: 5FYJ (Stewart-Jones et al., 2016) clear side chain density is visible for R601, which makes ionic interactions with E654 and E657 in a9 of a neighboring protomer. The large reduction in trimer formation upon replacing K601 with Glu is likely due to charge repulsion and concomitant loss of

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A

B gp41 Cα movement >5Å L129I/F

gp120 Cα movement >5Å Residues not present in 5VN3

E172I/V; A174I; F176W L193I/F; N195I/L/F N302F/W I423F T316W; Y318W

M154F/L S199V; T200W/A; I201F/C; T202F/W

S199V; T200W/A; I201F/C; T202F/W

T123F

T161I/F; T162I/E; E164D

P313I/F/W; Q315W/F

A204I/F; P206D; V208F D211W; I213F/W; I215F/W A60P/R; Y61R/W; E62R/P;K63R/P/K; L288F E64K; H66R; N67I/L

E429R/G; V430F; R432F/W; A433C

K117W

Y395W

Q114W;S115I/F V570I/F/W/D I573F/W

S56P; A58P; K59F/W

I271M

S110I/F

T51L

I583L; Y586F/K; K588E/M/Q/V/I/L/F/W D589V/I/L/A ;Q590F/W/E;L592F D640E

Q540I ; Q543I/F/R/N I535N/Q/M; T536V/I; T538N G521P/F; G527F/W G514P/F; G516P/F

E647F/I

E106F

D102R Q550W Q551W Q552W S553W N554W L556P/W/K R557F/I/W A558W/P E560K/R Q567K

N651F/I/W/L/A E654V K655I/F/W/A K658V

Q619L K500F/W/R

S599W/Y/F K601E K492E K500F/W/R R504Y/W/Q

D

C

2.5

I573F K655I

2.0 1.5 1.0

D589V I535N

L556P A558P

N651F E647F K588E A204I ConC

Detector voltage (V)

Relative PGT145 binding

K658V

3.0

* °

0.5 0.0 0.0

0.5

1.0 1.5 Relative expression

2.0

Figure 2. Single Point Mutations that Enhance Yield and Stability of ConC SOSIP Trimers (A) Schematic definition of regions of HIV-1 Env that move more than 5 A˚ between prefusion closed and CD4-bound forms are shown blue for gp120, green for gp41, and black if not defined in either conformation. Residues 31–511 form the mature gp120. Refolding region 1 encompasses residues 512–570. The base helix is residues 571–595. Refolding region 2 encompasses residues 596–664. Previously defined HR1 (residues 540–590) and HR2 (residues 624–664) are also indicated. (B) Location of structure-based mutation in the prefusion closed Env trimer. Two protomers are shown in surface representation (pink and peach) and the third protomer is shown in ribbon representation, with residues that move more than 5 A˚ between prefusion-closed and CD4-bound conformations in blue (for gp120) or in green (for gp41) and otherwise gray or black (if not defined in either conformation).

(legend continued on next page)

586 Cell Reports 23, 584–595, April 10, 2018

interaction with the a9 helix. Therefore, the interaction of a9 with the remainder of the protein appears to be of importance to the integrity of the entire trimer ectodomain. Regions of interest for stabilizing the prefusion-closed conformation of the Env ectodomain trimer appeared to be the a9-helix (particularly the interactions made by a9 to a6 and to b27) and the a7-helix (particularly the interactions between a7 and a7 on neighboring protomer and between a7 and gp120). Because interprotomer and gp41-gp120 contacts at the membrane-proximal base of the trimer were expected to be essential for stability, a component of our design efforts focused on stabilizing the a9 trimer interface with the intersubunit sheet composed of gp41 and gp120 (b27/b4/b26). Additional design efforts focused on other regions including (1) the disordered hinge loop (a6 a7 loop) in refolding region 1, (2) other parts of the trimer interface, and (3) between layers 2 and 3 of the inner domain of gp120 (Figure 2B). Production and Screening of Stabilized Variants Based on considerations of the fusion process and the prefusion-closed conformation of Env detailed above, a panel of 150 ConC_SOSIP variants with single amino acid substitutions were designed and synthesized (Table S1). Substitutions were introduced at positions of conserved residues at the trimer and gp120/gp41 interfaces that are (1) buried hydrophilic residues without salt bridges or hydrogen bonds, (2) exposed hydrophobic residues, and (3) involved in charge repulsions. Furthermore, hydrophobic packing was optimized at the trimer and gp120/ gp41 interface and at the layer 2-layer 3 interface (Pancera et al., 2010). The panel of single amino acid substitutions was screened for increased expression and trimer content by AlphaLISA using a panel of type-specific and non-neutralizing Abs and a panel of broadly neutralizing antibodies (bNAbs) including the trimer-specific PGT145 to measure the formation of native-like prefusionclosed trimers (Figure 2C), and the performance was compared to several previously published point mutations (Table S1). 25 AlphaLISA hits were selected for expression in 30 mL scale cell culture and purified with lectin and analyzed with SEC-MALS of which top 11 with highest expression and PGT145 binding are shown in Figure 2D. Strongest increase in PGT145 binding and trimer content based on SEC-MALS was observed for substitutions K658V, L556P, E647F, N651F, K655I, I535N, D589V, I573F, and K588E/Q in gp41 and A204I in gp120. Combinations of Stabilizing Substitutions Next, stabilizing substitutions in Env were combined to determine additive effect on stability, antigenicity, and expression. All combinations of substitutions resulted in variants with higher trimer content and higher trimer yield than any of the corresponding single substitutions based on SEC-MALS (Figure 3A).

All multiple substitution variants have higher melting temperatures than the ConC_SOSIP backbone and most single cumulative substitutions increase melting temperatures. The addition of both D589V (data not shown) and I573F resulted in melting curves that display two separate melting events, labeled Tm1 and Tm2 (Figures 3A and 3B). Because L556P increased the trimer percentage only slightly and because E647F hampered PGT151 binding, a variant was produced with just 7 stabilizing substitutions (termed ConC_base0), which was slightly more stable than the variant with 9 substitutions (Figure 3A, #10). ConC_base0 trimer showed greatly reduced CD4 induction as assessed by binding of the CD4-induced antibody 17b (Figure 3C), remained stable at 37 C for at least 4 weeks based on antigenic evaluation (Figure 3D), and was measured to be 320 kDa (Figure 3E). The removal of SOS from ConC_base0 hardly affected its trimer yield, suggesting its 7 mutations to stabilize the base region of the Env timer even in the absence of the SOS disulfide (Figure S1B). Back mutation of P559I in the a6-a7 hinge loop did, however, reduce trimer yield, and a proline at position 556 or 558 was able to compensate for the instability in refolding region 1 (Figure S1B). Crystal Structure of ConC_base0 Variant To provide a structural basis for the introduced mutations, we determined the crystal structure of the ConC_base0 variant (Figure 4). The structure determination of this variant was facilitated by use of antibodies PGT122 and 35O22, which we modified to enhance crystallization. Data from a single crystal of ConC_base0 in complex with 3H+109L (a PGT122 family member) and a 35O22 variant diffracted anisotropically (to 2.7 A˚–3.8 A˚ resolution), with a nominal resolution of 3.5 A˚ resolution (Table S2). The resulting electron density allowed for relatively precise positioning of side chains in most regions in the structure. The overall structure of ConC_base0 was highly similar to the clade A BG505 structure (Garces et al., 2015) (0.78 A˚ between 547 Ca atoms) (Figure S2A) despite 24.9% of its surface residues being different in sequence (Figure S2B) and was also similar to a recently published 3.9 A˚ structure of stabilized clade C 16055 Env (0.76 A˚ between 462 Ca atoms) (Guenaga et al., 2017). By inspecting the ConC_base0 crystal structure, we observed clear density for all 7 stabilizing mutations as well as the introduced glycan at V295N (Figure S3). The local interactions verified the stabilizing effect of these mutations. For example, F651 and I655 located in a9 strengthened the trimer interface by interacting with L537, I603, and L602 of a neighboring gp41 (Figure 4B). N535, located in a6 on the other side of the a6/a9 trimer interface, was exposed so a hydrophilic residue was likely more favorable. Although a7 could be considered as the static base helix on top of which the refolding region 1 will mount after the large conformational change to the pre-hairpin structure, the

(C) Comparison of expression level plotted against PGT145 binding for single point mutation variants of ConC_SOSIP Env using AlphaLISA in cell culture supernatant 3 days after transfection. Substitutions with highest increase in trimer yield and trimer fraction are labeled. Substitutions at the same positions have identical color and largest dot was selected for further evaluation. (D) SEC profiles using crude cell culture supernatants 3 days after transfection with the best single point mutation variants compared with backbone ConC_SOSIP (dotted gray line) (colored as in C). The trimer peak is indicated with an asterisk and gp140 monomer peak is indicated with a degree symbol. See also Table S1.

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Figure 3. Combined Effect of Cumulative Substitutions

A Tm1 Tm2

B

C Tm1= 75.7°C Tm2= 79.1°C

ConC_SOSIP

(A) SEC-MALS analysis (left) and temperature stability as measured by DSC (right) of Env variants with cumulative combination of amino acid substitutions in ConC_SOSIP. The trimer peak is indicated with an asterisk. For constructs with two melting event, a Tm1 (minor) and Tm2 (main) is indicated. Construct number 10 (ConC_base0) has a Glu instead of Gln at position 588. (B) DSC pattern of ConC_base0. (C) 17b binding in the presence (solid lines) and absence of CD4 (dotted lines). (D) Antibody binding levels measured with Octet after 0 and 4 weeks incubation at 37 C of ConC_base0. (E) SEC-MALS of purified ConC_base0 with SEC (purple) and molar mass trace. See also Figure S1B.

ConC_SOSIP

D

E

I573F mutation at the N-terminal top of a7, close to the trimeric axis, stabilized the trimer through increased hydrophobic interaction. In addition to the stabilization between three protomers, stabilization between gp120 and gp41 within one protomer was also observed. Residue K588, D589, R585, E490, and K492 formed a cluster of charged residues in the gp120-gp41 interface that was balanced in the metastable structure but could harbor potential repulsions as a result of small local movements. The positive charge at position 588 could be partly compensated by D589, but in most crystal structures, the charged D589 is partly buried in the hydrophobic interface of gp120-gp41 (Lee et al., 2016; Pancera et al., 2014; Stewart-Jones et al., 2016). The D589V mutation on gp41 has hydrophobic interactions with Y40, P493, and L494 on gp120 while the K588E has electrostatic interactions with K46 and K492 on gp120 as well as R585 on gp41. Interestingly, R585H was recently shown to stabilize the BG505 trimer (Kulp et al., 2017; Steichen et al., 2016). From the ConC_base0 crystal structure, we calculated the difference in energy for each of the stabilizing mutants (Figure S4A) as well as the difference in energy for the mutation in the CD4bound prefusion structure (Figure S4B). In the prefusion closed structure, the most stabilizing mutation appeared to be from

588 Cell Reports 23, 584–595, April 10, 2018

the hydrophobic interactions gained by N651F. In addition, A204I, I535N, I573F, and D589V also showed favorable DDG. From the DDG calculations of the same mutations using the CD4-bound prefusion Env structure (PDB: 5VN3), I535N, I573F, and K588E appeared to destabilize the CD4-bound prefusion conformation, which may explain why K588E had a beneficial effect on formation of a prefusion-closed Env trimer (Figure S4C). In general, the energetic analysis was consistent with the observed stabilization for all mutants except K655I. Overall, the crystal structure of ConC_base0 verified inter-protomer and intra-protomer stabilization to have synergistic stabilization of the combination variant (Figure 4C). General Application of the Repair-and-Stabilize Approach for HIV-1 Env To investigate the universality of our approach, the effect of the stabilizing substitutions was assessed with other Env sequences. Because wild-type sequences from viruses isolated from infected patients may have acquired destabilizing mutations that impede correct folding, wild-type Env sequences of several clade C strains and the mosaic sC4 were first repaired by substituting each rare mutation with the corresponding consensus amino acid according to the conceptual framework described in Figure 5A. Among the selected Envs were typical strains known to express and fold properly (DU422), Envs known for their low expression and quality like ZM233M and CN97001(Julien et al., 2015), and the low quality Env ADM30337.1 that was described in Figure 1A. Next, stabilizing substitutions described in Figure 3 were transferred to the repaired sequence (Table S3). Supernatants of cells transiently transfected with wild-type, repaired, and stabilized Env variants were tested for binding to

A

gp41 Cα movement >5Å gp120 Cα movement >5Å Residues not present in 5VN3

B

C

α7 E647

α7

K588E

K588E

D589V

D589V

E647

N651F

N651F β4-

I535N

K655I K658

β26

α6

β26

β27

β27

α9

β4-

I535N K655I

E647

N651F K658

K658

α9 K655I

α6 I535N

Figure 4. Crystal Structure of ConC_base0 (A) Mutations in ConC_base0 are shown in red ball-and-stick with molecular surface. gp120 and gp41 are shown in ribbon for one protomer with the color scheme shown in Figure 2B. The other two protomers are shown in surface representation, colored peach and pink. Residues on gp120 and gp41 potentially interacting with the stabilizing mutations are shown in ball-and-stick without molecular surface. (B) Detail of trimer base showing the functionally critical a-9 helix in dark green. Interaction of stabilizing residues 651F and 655I at the trimer interface (red). Residue E647 and K658 that when substituted to hydrophobic residues are also stabilizing (Figure 2) are indicated in orange. (C) Both protomers shown as ribbons showing the functionally critical a-9 helix in dark green of one protomer that interacts with a6 and intersubunit b sheet of the other protomer (colored as in A) Clustering of stabilizing residues indicated on both sides of the intersubunit b sheet. See also Figures S2–S4 and Table S2.

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Figure 5. Prefusion Closed HIV Env_SOSIP Trimers Through Sequence Repair and Mutational Stabilization (A) Universal concept for repairing HIV-1 Env sequence illustrated for strain C97ZA. Occurrence of highest consensus residue in the total HIV-1 database (top bars) and strain C97ZA residue (bottom bars) sorted from low to high occurrence percentage of C97ZA residue position. C97ZA sequence positions to be substituted to consensus were selected based on the following criteria: positions with a C97ZA residue that occurs <2% in consensus Env sequence (blue bars); positions with a C97ZA residue that occur between 2% and 7.5% in consensus Env and are buried or partly buried (magenta bars); positions that are exposed and hydrophobic in C97ZA and hydrophilic in consensus (yellow bars); and a position that is a PNGS in the consensus (green bar). (B) AlphaLISA signals in cell culture supernatant for all SOSIP variants normalized to the ConC_SOSIP for broadly neutralizing (left) and non-neutralizing or strainspecific (right) mAbs. For the non-bNAbs the intensities are further normalized for expression levels. (C) Analytical SEC profile of control Env_SOSIPs (red line) repaired according to the concept described in a (blue line) and additional stabilizing substitutions according to Table S3 (green line). Analysis was performed on crude cell culture supernatant instead of purified protein. The trimer peaks are indicated with asterisks. (D) Repaired and stabilized (REP & STAB) C97ZA_SOSIP compared with CZA97_SOSIP.4.2-M6.IT-2 (Ringe et al., 2017) according to the approach previously described. The upper panel shows the analytical SEC pattern on crude supernatant and the lower panel shows the AlphaLISA signals normalized to those of ConC_SOSIP. See also Figures S5 and S6 and Table S3–S5.

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several trimer-specific neutralizing antibodies directed to the apex as well as several non-neutralizing antibodies. The repair substitutions, and especially the stabilizing substitutions, had a dramatic impact on trimer content (Figures 5B and 5C). Moreover, the antigenicity of the trimers was improved as shown by a reduction of binding to non-neutralizing antibodies and melting temperatures increased (Figures 5B and S5; Table S4). Similarly, the single substitutions D589V, N651F, K655I, and I535N increased trimer yield and content in a consensus clade B SOSIP (Figure S6). The yields of repaired and stabilized C97ZA_SOSIP was more than 10 mg/L after transient transfection in Expi293F culture, which is higher compared with the optimized C97ZA Env previously described (Ringe et al., 2017). Because cell type, signal peptide, and the residue at position 152 are different for both proteins, we compared the expression levels of both optimized proteins with identical signal peptide based on ConC (Table S3) and Lys at position 152 (Table S5). Our repaired and stabilized C97ZA_SOSIP yielded approximately four times more native-like closed trimers (Figure 5D). DISCUSSION HIV-1 Env is highly variable in sequence and conformation, which are two major hurdles that may need to be addressed to obtain an efficacious vaccine that induces bNAbs against HIV. Human antibodies preferentially recognize immunogenic nonglycosylated Env regions, such as those exposed on open conformations; even minor imperfections in Env folding may hamper the induction of the desired glycan shield-penetrating antibodies. A prerequisite for an effective HIV vaccine that focuses on induction of broadly neutralizing antibodies may thus be high expression of multiple closed trimers with high-quality folding and stability. In this study, improved quality and stability of the native trimer were pursued by repairing folding through a consensus approach and stabilizing trimer in the prefusion-closed conformation through a structure-based screening approach that optimized regions critical to the folding process. In specific, we hypothesized that a consensus sequence might possess characteristics of founder virus Envs, which have higher fitness and more optimal folding than chronic virus Envs (Carlson et al., 2014; Tully et al., 2016). This study showed that a consensus clade C sequence and repair of selected accumulated mutations of wild-type to consensus, yields superior expression, trimer formation, and antigenicity compared with the closest wild-type Env sequences. Because the prefusion spike is a highly dynamic and metastable fusion machine, the repaired sequence was further stabilized by modifications that arrest conformational changes needed for the fusion process. We therefore systematically searched for conserved weak interactions between the protomer interface and subunit interface based on the known X-ray and electron microscopy (EM) structures (Lee et al., 2016; Pancera et al., 2014; Stewart-Jones et al., 2016; Wang et al., 2016). Although several regions were identified in this study that stabilized the closed prefusion conformation of the trimer, several of the most successful stabilizing substitutions were located at the

membrane-proximal base of the trimer, in the lower part of the fusion subunit gp41 where all the essential contacts between gp120 termini and gp41 refolding region 1 and 2 are located. Several stabilizing substitutions (i.e., E647F, N651F, K655I, and D658V) in a9 improved the interaction at the trimer interface with a6 and b27, locking refolding region 1 and 2 and preventing the dissociation of a9 with a6 and b27 (Figures 4B and 4C). Stabilization of the closed conformation was supported by the observation that CD4 induction leading to 17b binding was reduced in the stabilized ConC_base0 (Figure 3C). b27 is part of refolding region 2 and is the only structural element that is part of a secondary structure that also involves gp120. This intersubunit gp120/gp41 b sheet (b4, b26, b27) connects gp41 with the N and C terminus of gp120 and stabilizes the prefusion conformation of refolding region 2, preventing it from unfolding. This intersubunit b sheet has a pivotal role in Env stability, and we found stabilizing substitutions on both sides of the b sheet that keep b27 in place: the aforementioned stabilizing substitutions in a9 on one side and stabilizing substitutions in the highly charged region on the other side. Substitutions 588E and 589V improved the interaction of gp41 with the gp120 termini, stabilizing Y40 ensuring the intersubunit b4b26b27 sheet to not be disrupted (Figures 4A and 4C). The stabilizing substitutions we identified in the base are in line with the structure of the CD4bound prefusion state of Env (Ozorowski et al., 2017; Wang et al., 2016), which shows that both the apex and the base are able to breathe and that, when the gp120 termini and the a6-b27 loop are pulled upward, the interaction between a9 and b27 and interactions in the charged network are lost. Therefore, substitutions may prevent breathing of the base that may also reduce breathing of the apex. The strong effect on increased prefusion trimer yields by substitutions at the base indicate an important step in the refolding process to be regulated at the trimer base. We envision extreme motions of gp120 to provide freedom for the loop between a6 and a7 to hinge: when b27 contacts are lost, the gp120 N and C termini would be pulled from the gp41 clamp, and refolding regions 1 and 2 would be liberated. Because the hinge loop preceding the base helix is a common element of refolding region 1 in all type I fusion proteins, it had previously been suggested that stabilizing the hinge loop by introduction of prolines may be a general solution to stabilize this metastable element (Krarup et al., 2015), which also has been successfully applied to other type I fusion proteins (Hastie et al., 2017; Pallesen et al., 2017). The current study thus draws attention to a conserved structural element essential for the stability of refolding region 2, which comprises the intersubunit b sheet composed of b strands of the head and the fusion domain. Detachment or ‘‘splaying’’ of the head of the type I fusion proteins is expected ultimately to disrupt the intersubunit sheet and to destabilize contacts with refolding region 2 (Figure 6). Therefore, stabilizing the intersubunit sheet may be a general strategy for conformationally fixing the prefusion state of other metastable type I fusion proteins, a potentially important step in their use as vaccine immunogens. In summary, we describe a ‘‘repair-and-stabilize’’ approach— that could be generally applicable to all HIV-1 clades—to obtain high levels of stable prefusion-closed HIV Env trimers. Our approach utilized optimization of prefusion-closed folding based

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Figure 6. Intersubunit Sheets of Head Domain and Fusion Domain in Type I Fusion Proteins (A–D) Prefusion trimer structures are shown with 2 protomers as light and dark gray surface representations and one protomer in ribbon with helices represented as cylinders for Lassa GP (A) (Hastie et al., 2017), MERS S (B) (Pallesen et al., 2017), Influenza HA (C), and HIV Env (6CK9) (D). Color coding of structural elements in the fusion protein is based on refolding region 1 (blue), base helix (green), refolding region 2 (purple), and head domain (red) according to (E), with inserts depicting the intersubunit sheet composed of part of the head region (red) and refolding region 2 or both refolding regions. Detachment or splaying of the head is expected to destabilize the hybrid sheet and to effect refolding. (E) Color scheme.

stains, resulting in soluble gp140 proteins with the SOSIP.R6.664.V295N mutations (Table S5 for the sequences). The DS_sC4_SOSIP was, however, truncated after 655 and contained the 201C-433C substitution.

on the Env fitness expected for a consensus sequence and stabilization based on the arrest of refolding events at the base of the trimer critical to the fusion process.

Repair of Wild-Type Clade C Sequences To search for non-optimal mutations in wild-type sequences an alignment of all HIV-1 Env sequences in the UniProt database and the Los Alamos database HIV database (90,000 sequences) was made, and the amino acid distribution was calculated for each amino acids. In general, a number of relatively rarely occurring amino acids in Env sequences were substituted into more prevalent amino acids (Table S3). Furthermore, two additional substitutions, Y353F and Q171K at the apex of C97ZA_SOSIP, were introduced to possibly improve the binding of apex targeting antibodies, and extra glycan sites were introduced by the substitution of D411N, K236T, and V295N because these PNGS were conserved >50%.

EXPERIMENTAL PROCEDURES Design of HIV Envelope Antigen Sequences C4 is a mosaic sequence we previously created during research toward synthetic Env antigens suitable for expression in adenoviral vectors (Langedijk, 2017). For the consensus C (based on clade C) an alignment was downloaded (HIV Sequence Alignments. 3,434 sequences in total 7DEC14) from the Los Alamos database (https://www.hiv.lanl.gov/content/index). From this alignment, we selected C-clade ENVs (1,252 sequences), and these were used in the consensus maker in the Los Alamos HIV database website. Because the consensus contains a few question marks, the consensus sequence was used to Blast the closest wild-type homologs, which was then also used to substitute the amino acids that have a question mark in the consensus sequence. The resulting sequence was designated ConC (Langedijk, 2017). Furthermore, two wild-type sequences with the highest homology to C4 and two wild-type sequences with the highest homology to ConC were identified with BLAST. The two sequences with the highest homology to C4 are the sequences with GenBank: AAS98045.1 and AAW57669.1. They are 93% and 84% identical to C4, respectively. The two sequences with the highest homology to ConC are the sequences with GenBank: ADM30337.1 and ADM30340.1, both having 90% sequence identity to ConC. The sequences were further modified by addition of the so-called SOSIP substitutions, optimization of the furin site by replacing the furin site (positions 508–511) by 6 Arg residues (R6), a C-terminal truncation of gp160 at residue 664, and a V295N mutation to introduce a PNGS that is present in more than 50% of all HIV-1

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Expression of HIV Env Constructs The constructs were synthesized and codon-optimized at GenScript (Piscataway, NJ 08854) and cloned into pCDNA2004 or generated by standard methods involving site-directed mutagenesis and PCR. HEK-Expi293 cells were transiently transfected with 90% of pCDNA2004 plasmid with the Env insert and 10% of Furin-pCDNA2004, according to the manufacturer’s instructions and cultured for 5 days at 37 C and 10% CO2. Culture supernatants were spun for 10 min at 1,250 3 g. For expressions in 96-well format, the cells were cultured for 3 days at 37 C and 10% CO2. 4 mL of Opti-MEM was mixed with 4 mL 100 ng/mL DNA, and 8 mL Expi293F mix (54 mL/mL Opti-MEM) as added and incubated for 20 min. Subsequently, 200 mL/well Expi293F cells were added at 2.5 3 10E6 cells/mL. We use special culture plates and incubation system for this. The culture supernatant was harvested and spun for 10 min at 200 3 g to remove cells and cellular debris. The spun supernatant was subsequently sterile-filtered using a 0.22 mm vacuum filter and stored at 4 C until use. For crystallization, the protein was produced in HEK293 GnTI / cells via transient transfection with 10% of Furin-pCDNA2004. Purification of HIV gp140 Trimer Using Lectin and SEC The recombinant HIV Envs were purified by a 2-step purification protocol applying a Galantus nivalis-lectin column (Vectorlabs, AL-1243, lot Z0325) for the initial purification and subsequently a Superdex200 Increase column (GE) for the polishing step to remove residual contaminants. For

the lectin step, the culture supernatant was diluted with 40 mM Tris, 500 mM NaCl pH7.5, and passed over a 4 mL CV Tricorn 10-50 lectin agarose column at 300 cm/hr. Subsequently, the column was washed with 4 column volumes (CV) of 40 mM Tris, 500 mM NaCl pH7.5, and eluted with 4 CV of 40 mM Tris, 500 mM NaCl, 1 M mannopyronoside pH 7.5 with an upflow of 120 cm/hr. The eluate was concentrated using a spin concentrator (50 K, Amicon Ultra, Millipore) and the protein was further purified using a Superdex200 Increase 10/300 column using 20 mM citrate, 75 mM NaCl, 5% sucrose, and 0.03% Tween 80 pH 6.0 as running buffer. The second peak (Figure 1) contained the HIV gp140 trimer. The fractions containing this peak were pooled, and the protein concentration was determined using OD280 and stored a 4 C until use. Purified protein samples were analyzed on 4%–12% (w/v) Bis-Tris NuPAGE gels, 13 MOPS (Life Technologies) under reducing or non-reducing conditions. All procedures were performed according to manufacturer’s instructions. The gels were scanned on an Odyssey instrument (Li-Cor) and images were analyzed using Image Studio 3.0 (Li-Cor). Size Exclusion Chromatography and Multi-angle Light Scattering Analysis Purity and mass was further confirmed by size exclusion chromatography (SEC) and multi-angle light scattering (MALS) using a high-performance liquid chromatography system (Agilent Technologies) and miniDAWN TREOS (Wyatt) instrument coupled to a Optilab T-rEX Refractive Index Detector (Wyatt). In total, 40 mg of purified protein was applied to a TSK-Gel G3000SWxl column (Tosoh Bioscience) equilibrated in running buffer (150 mM sodium phosphate, 50 mM sodium chloride, pH 7.0) at 1 mL/min. In other experiments, analysis was performed on supernatants instead of purified Env. The data were analyzed by the Astra 6 software package, and molecular weight calculations were derived from the refractive index signal. AlphaLISA AlphaLISA (Perkin-Elmer) is a bead-based proximity assay in which singlet oxygen molecules, generated by high energy irradiation of Donor beads, transfers to Acceptor beads, which are within a distance of 200 nm. It is a sensitive high throughput screening assay that does not require washing steps. A cascading series of chemical reactions results in a chemiluminescent signal (Eglen et al., 2008). For the AlphaLISA assay, the constructs were equipped with a Flag-His tag (AAALPETGGGSDYKDDDDKP(GGGGS)7H6). The HIV constructs were expressed in HEK-Expi293F cells, which were cultured for 3 days in 96-well plates (200 mL/well). Crude supernatants were diluted 120 times in AlphaLISA buffer (PBS + 0.05% Tween-20 + 0.5 mg/mL BSA). For 17b-based assays, supernatants were diluted 12 times. Subsequently, 10 mL of these dilutions were transferred to a half-area 96-well plate and mixed with 40 mL acceptor beads, mixed with donor beads and mAb. The beads were mixed well before use. After 2 hr of incubation at room temperature (RT), nonshaking, the signal was measured with Neo (BioTek) The donor beads were conjugated to ProtA (catalog #AS102M, Perkin Elmer), which could bind to the mAb. The acceptor beads were conjugated to an anti-His antibody (catalog #AL112R, Perkin Elmer) to detect the His-tag of the protein. For the quantification of the protein level, a combination of Nickel-conjugated donor beads (catalog #AS101M, Perkin Elmer) together with acceptor beads carrying antiFlag antibody (catalog #AL112R, Perkin Elmer) were used. For 17b in combination with sCD4-His, a combination of ProtA donor beads and anti-Flag acceptor beads were used. The average signal of mock transfections (no Env) was subtracted from the AlphaLISA counts measured for the different Env proteins. As a reference, the ConC_SOSIP Env plasmid was used. For normalization, the data were divided by the ConC_SOSIP signal. Trimer percentages (referred to as ‘‘trimer content’’ throughout the paper) were obtained by dividing these normalized signals through the normalized signal obtained for the quantification. Bio-Layer Interferometry (Octet) Antibodies were immobilized on anti-hIgG (AHC) sensors (Forte´Bio catalog #18-5060) at a concentration of 1 mg/mL in 103 kinetics buffer (Forte´Bio catalog #18-1092) in 96-half well black flat bottom polypylene microplates (Forte´Bio catalog #3694). The experiment was performed on an Octet RED384

instrument (Pall-Forte´Bio) at 30 C, shaking speed 1,000 rpm. Activation was 300 s, immobilization of antibodies 600 s, washing 300 s, and binding the Envs 1,200 s, followed by a dissociation of 1,200 s, all shaking at 1,000 rpm. The data analysis was performed using the Forte´Bio Data Analysis 8.1 software (Forte´Bio). Negative Stain Electron Microscopy HIV-1 Env DS_ConC_SOSIP trimer that contained a disulfide 201C- 433C (DS) was diluted to between 0.0005–0.05 mg/mL in Tris-buffered saline (TBS), pH 7.4, and adhered onto a carbon-coated 200 Cu mesh grid (EMS CF200-Cu) that had been glow discharged in air 2 3 10 1 mbar in air, 25 mA, 30 s, just before use. Subsequently, a 3 mL drop was applied for 1 min followed by blotting with filter paper (Whatman no. 1 or 4). After drying for 1 min, grids were then stained with 3 mL of 2.3% uranyl acetate (UAc) in filtered milliQ (filtered with Millipore filter 0.22 mm) for 60 s, followed by blotting with filter paper (Whatman no. 1 or 4). Data were collected using an FEI Tecnai F20 electron microscope operating at 120 keV, with a magnification of 25,0003 that resulted in a pixel size of 4.68 A˚ at the specimen plane. Images were acquired with a Gatan BM ultrascan. Differential Scanning Calorimetry Melting temperatures for HIV Env were determined using MicroCal capillary differential scanning calorimetry (DSC) system. 400 mL of a 0.5 mg/mL protein sample were used per measurement. The measurement was performed with a start temperature of 20 C and a final temperature of 110 C. The scan rate was 100 C/hr, and the feedback mode was low (= signal amplification). The data were analyzed using the Origin J software (MicroCal VP analysis tool). Purification of HIV gp140 Trimer Using CAP456-VRC26 Affinity Chromatography and SEC For crystallization, the ConC_base0 supernatant expressed in Expi 293 cells supplemented with kifunensin was passed over the CAP256-VRC26.09 IgG coupled to NHS-activated Sepharose 4 fast Flow (catalog #17-0906-01), washed with 20 mM Tris, 150 mM NaCl, pH 7.4, and eluted with the elution buffer containing 20 mM Tris, 3 M MgCl2, pH 7.2. The eluate was concentrated using a spin concentrator (50 K, Amicon Ultra, Millipore), and the protein was further purified using a Superdex200 Increase 10/300 column using 50 mM Tris, 150 mM NaCl, pH 7.4 as running buffer. HIV-1 Env Trimer-Fab Complex Preparation and Crystallization Purified trimers were mixed with a variant of 3H+109L (Garces et al., 2015) Fab and a variant of 35O22 (Pancera et al., 2014) scFv in a 1:3:3 molar ratio (gp140 protomer:Fab:scFv) and incubated overnight at room temperature. The 3H+109L variant contained two methionine substitutions in the light chain and the 35O22 variant three contained threonine and two serine mutations in the heavy chain. These alterations were designed to enhance the crystallization lattice (Y.-T.L., unpublished data). Complexes were further purified by SEC and concentrated to 10–15 mg/mL for crystallization. Crystals of the ConC_base0 in complex with 3H+109L Fab and 35O22 scFv were grown in 75 mM HEPES, pH 7.5, 2% PEG 8,000 and 10% PEG 400 with a protein-toreservoir ratio of 0.5 mL to 0.5 mL. X-Ray Data Collection, Structure Solution, and Model Building Crystals were harvested and incubated in crystallization condition supplemented with 20% ethylene glycol as cryo-protectant for 10 s before freezing in liquid nitrogen. Diffraction data were collected at SER-CAT beamline of the Advanced Photo Source at the Argonne National Laboratory. HKL-2000 was used to process the diffraction data, followed by anisotropy data truncation by the UCLA anisotropy server (https://services.mbi.ucla.edu/ anisoscale/). A structure of BG505.SOSIP in complex with PGT122 and 35O22 (4TVP.pdb) was modified by using Sculptor to change the BG505 sequence in the model to the corresponding ConC_base0 sequence and PGT122 Fab to 3H+109L Fab. Molecular replacement was carried out by using Phaser, utilizing the modified 4TVP.pdb as the search model. Refinement and structure evaluation was carried out in Phenix.

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Ca Deviation between Prefusion-Closed and CD4-Bound Conformation The Ca deviation between prefusion-closed trimer (PDB: 5FYL or the ConC_base0 structure) and CD4-bound prefusion trimer (PDB: 5VN3) were calculated by aligning gp120 using outer-domain residues (residue numbers 252–482) and aligning gp41 using a7 helix (residue numbers 572–595). Mutational Free Energy Change Calculations Mutational free energy change (DDG) were calculated using FoldX (Schymkowitz et al., 2005) based on the ConC_base0 structure and a Env trimer structure of strain B41 in the CD4-bound prefusion conformation (PDB: 5VN3), respectively. The input trimer structure was first repaired using FoldX’s RepairPDB command in order to optimize the structure by fixing steric clashes and lowering the global energy. Mutagenesis was performed using FoldX’s BuildModel command. Number of runs was set to five to insure convergence based on optimal rotamer positions. The average DDG over the five runs were reported for each mutation. DATA AND SOFTWARE AVAILABILITY The accession number for the coordinates and structure factors for the crystal structure of ConC-SOSIP_base Env trimer in complex with variants of antibodies PGT122 and 35O22 optimized for crystallization reported in this paper is PDB: 6CK9. SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and five tables and can be found with this article online at https://doi.org/10.1016/j.celrep.2018.03.061. ACKNOWLEDGMENTS We thank D. Peng for assistance with expression of antibody variants used in crystallization and members of Structural Biology and Structural Bioinformatics Core Sections, Vaccine Research Center for discussions and comments on the manuscript. We thank J.E. Robinson for providing antibody 14e, D.R. Burton and M. Feinberg for template versions of antibody PGT122, and M. Connors for antibody 35O22 that were modified for use in structural analysis. Funding was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases National Institutes of Health (ZIA AI005024-14), by the IAVI Neutralizing Antibody Consortium, and by Janssen Pharmaceuticals. Use of sector 22 (Southeast Region Collaborative Access team) at the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science (W-31-109-Eng-38). AUTHOR CONTRIBUTIONS L.R., Y.-T.L., D.T., D.v.M., H.S., P.D.K., and J.P.M.L. designed research and analyzed the data. L.R., Y.-T.L., S.B., N.M.S., D.T., A.K., I.J.M.B., G.-Y.C., and K.F. performed research/experiments. L.R., D.T., and J.P.M.L. performed the structure-based design of stabilizing substitution. Y.-T.L. and P.D.K. determined the Env trimer crystal structure, with K.F and G.Y.C. providing energetic calculations. L.R., Y.-T.L., D.T., P.D.K., and J.P.M.L. wrote the paper, with all authors providing comments or revisions. DECLARATION OF INTERESTS These studies were funded by Janssen Vaccines and Prevention. L.R., D.T., D.v.M., S.B., N.M.S., A.K., I.J.M.B., H.S., and J.P.M.L. are employees at Janssen. L.R., D.T., N.M.S., A.K., and J.P.M.L. are inventors on an international patent application describing trimer stabilizing HIV envelope protein mutations. The remaining authors declare no competing interests. Received: December 5, 2017 Revised: March 5, 2018 Accepted: March 13, 2018 Published: April 10, 2018

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