The Chimpanzee SIV Envelope Trimer: Structure and Deployment as an HIV Vaccine Template

Abstract

Epitope-targeted HIV vaccine design seeks to focus antibody responses to broadly neutralizing antibody (bnAb) sites by sequential immunization. A chimpanzee simian immunodeficiency virus (SIV) envelope (Env) shares a single bnAb site, the variable loop 2 (V2)-apex, with HIV, suggesting its possible utility in an HIV immunization strategy. Here, we generate a chimpanzee SIV Env trimer, MT145K, which displays selective binding to HIV V2-apex bnAbs and precursor versions, but no binding to other HIV specificities. We determine the structure of the MT145K trimer by cryo-EM and show that its architecture is remarkably similar to HIV Env. Immunization of an HIV V2-apex bnAb precursor Ab-expressing knockin mouse with the chimpanzee MT145K trimer induces HIV V2-specific neutralizing responses. Subsequent boosting with an HIV trimer cocktail induces responses that exhibit some virus cross-neutralization. Overall, the chimpanzee MT145K trimer behaves as expected from design both in vitro and in vivo and is an attractive potential component of a sequential immunization regimen to induce V2-apex bnAbs.

Keywords: HIV envelope trimer; HIV vaccine; V2-apex bnAb site; broadly neutralizing antibodies; chimpanzee SIV envelope trimer; glycan shield; human immunodeficiency virus; immunization strategies; simian immunodeficiency virus.

Graphical abstract

Figure 1

Design of a Chimpanzee Env-Stabilized Trimer and Binding to V2-Apex bnAb iGL Abs (A) Structural arrangement of the V2-apex bnAb core epitope region on the BG505.664 soluble Env trimer (modified from Garces et al., 2015; PDB: 5CEZ). The ribbon representation of V1V2 loop strands that form the trimer apex show a cluster of positively charged lysine-rich peptide regions (HXB2-R166-K171: R or K residues shown as blue spheres) and the two glycans N156 and N160 (depicted in green spheres with lines). The side chains of the positively charged residues intersperse with the side chains of residues from adjacent protomers to form a continuous positively charged surface at the tip of the trimer to provide a minimal V2-apex bnAb epitope. (B) Amino-acid sequence alignment of strand B and C V2 of HIV CRF250, CAP256.SU, chimpanzee SIV MT145 WT, and its V2-modified variant (Q171K), MT145K. Glutamine (Q) at position 171 (shown in red) was substituted with lysine (K) in MT145 Env to gain binding to V2-apex bnAb inferred germline (iGL) Abs. (C) ELISA binding of mature V2-apex bnAbs, PG9, CAP256.09, and CH01 and their iGL versions to WT MT145 (red) and MT145K SOSIP trimers. (D) Octet binding curves (association, 120 s [180–300]; dissociation, 240 s [300–540]) of CAP256 UCA and CH01 iGL Abs and their respective mature Ab versions (CAP256.09 and CH01) to MT145K trimer, its glycan knockout (N160K) variant, K-rich core epitope substituted variants, and the corresponding monomeric gp120. The Abs were immobilized on human IgG Fc capture biosensors and 1uM trimer or gp120 proteins used as analytes. The binding response is shown in nanometers (nm).

Figure 2

Cryo-EM Structure of the MT145K Trimer (A) Schematic showing the MT145K SOSIP soluble trimer design from its full-length gp160 Env sequence. The gp120 constant (C1-C5) and variable (V1-V5) regions and the gp41 regions (fusion peptide, FP; heptad repeat, HR1 and HR2; membrane proximal external region, MPER; transmembrane, TM; and cytoplasmic tail, CT) are indicated. The N-linked glycan positions for each NXT or NXS residue are labeled according to the HIV HXB2 numbering scheme. The SOSIP trimer stabilizing modifications include (i) disulfide bond, A501C-T605C; (ii) R6 cleavage site; (iii) I559P; and (iv) 664-residue truncation in gp41 MPER. The substitution to incorporate a K residue at position 171 (Q171K) to gain binding for V2-apex iGL Abs is indicated in blue. (B and C) Side (B) and top (C) views of the unliganded MT145K trimer model based on the cryo-EM density map at ∼4.1-Å resolution. Ribbon representations of the MT145K trimer spike, in which the subunits gp120 (cornflower blue) and gp41 (orange) are depicted on one protomer. The gp120 variable loops (V1-V5) positioned to the trimer periphery are depicted in different colors (V1, khaki; V2, red; V3, magenta; V4, yellow; and V5, chartreuse). The fusion peptide region of gp41 is shown in cyan. Glycan sugar residues modeled based on density are represented in forest green stick form. (D) Superimposition of variable loops (V1-V5) and fusion peptide region for MT145K and unliganded HIV clade A BG505 (PDB: 4ZMJ) SOSIP trimers. The dotted lines indicate regions in the V loops or FP for which the observed electron density was absent or unclear. (E) Structural comparison of gp41 regions of the MT145K (orange) and BG505 (gray) trimers. The gp41 structural elements overall show a similar arrangement except for the fusion peptide region (colored cyan on MT145K and pink on BG505), which is exposed on the BG505 trimer but remains hidden in a pocket inside the MT145K trimer.

Figure 3

Site-Specific Glycoform Composition of MT145K Trimer (A) Site-specific glycoform quantification of the MT145K SOSIP soluble trimer. MT145K trimers from transiently transfected HEK293F cell expressed supernatants were affinity purified by the quaternary trimer-specific antibody, PGT145. The purified MT145K trimers were treated separately with three proteases—trypsin, chymotrypsin, and elastase—and the digests were enriched for glycopeptides and analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS). The individual glycan compositions of the N-linked glycan sites (n = 26) are represented by bar graphs that indicate the relative abundance of each glycoform species and are derived from the mean of two analytical replicates. The pie charts summarize the proportion of glycoforms for each site and this information is color coded: oligomannose type in green and complex and/or hybrid glycans in pink. The glycoforms at N262 and N268 positions (indicated by “^∗”) could not be separately determined by enzymatic digestion and the bars represent the average glycan compositions across both sites. (B) Hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) profiles of the total N-linked glycans released from the MT145K trimers. The proportions of oligomannose plus hybrid glycan contents and complex-type glycans are represented in green and pink colors, respectively. (C) Modeled glycan shields for the MT145K and BG505 SOSIP trimers. Man9GlcNAc2 oligomannose-type glycans were docked and rigid-body fitted at each of the corresponding Env glycan positions using the MT145K structure (determined in this study [PDB: 6OHY]) and the unliganded BG505 SOSIP.664 trimer structure (Kwon et al., 2015; PDB: 4ZMJ). Top and side views of the trimers are shown and the individual glycan sites are labeled and color coded based on the content of oligomannose: green, 100%–80%; orange, 79%–20%; and pink, 19%–0%.

Figure 4

Antigenic Profile of the MT145K Trimer (A) HIV Env-specific mAbs were used to characterize the antigenicity of the MT145K Env trimer. MAbs targeting neutralizing and non-neutralizing epitope specificities, including V2-apex, V3-N332, linear V3, CD4bs, CD4i, and the gp120-41 interface were tested with MT145K and BG505 Env-encoding pseudoviruses in a TZM-bl cell-based reporter assay. The reciprocal IC₅₀ neutralization titers for each virus are indicated as dot plots; plots for individual epitope specificities are depicted separately. The neutralization sensitivity comparison of BG505 and MT145K viruses against the mAb panel shows a selectively potent neutralization of MT145K by V2-apex bnAbs but no other bnAbs, except a single gp120-gp41 interface bnAb, 35022. The BG505 virus was neutralized by bnAbs targeting diverse Env sites. (B) The above mAb panel was further tested with PGT145 Ab-purified MT145K trimer and Galanthus nivalis lectin (GNL)-purified MT145K gp120 monomer by ELISA. The binding, represented as EC₅₀ binding titers, shows selective binding of MT145K by V2-apex bnAbs. Two of the gp120-gp41 interface bnAbs and a CD4i mAb also showed significant binding to the MT145K trimer. Four of the non-neutralizing mAbs specific to a linear V3 epitope exhibited binding to MT145K gp120, but not to the trimer.

Figure 5

A Close-Up View of Regions on the MT145K Trimer That Correspond to Those Recognized by HIV bnAbs on HIV Trimers (A) V2-apex bnAb binding region: cryo-EM model of PGT145 bnAb (HC, transparent sandy brown; LC, transparent orchid) in complex with the BG505 SOSIP trimer depicting V1V2 loops in ribbon representation (Lee et al., 2017; PDB: 5V8L). The strand C K-rich region (¹⁶⁶RDKKQK¹⁷¹; red spheres) and the glycan N160 (forest green sticks) that form the epitope for PGT145 bnAb are indicated. The elements in the core epitope interact with the CDRL1 loop and the long CDRH3 loop that penetrates through glycans to reach the positively charged surface underneath. Both glycan N160 and the positively charged protein residues are conserved between BG505 HIV and MT145K SIV Env trimers. (B) V3-glycan bnAb binding region: cryo-EM model of PGT128 bnAb (HC, transparent sandy brown; LC, transparent orchid) in complex with the BG505 SOSIP trimer (Lee et al., 2015; PDB: 5ACO). The V3 loop protein backbone residues (³²⁴GDIR³²⁷; depicted in purple spheres) and the glycans N301 and N332 form the bnAb epitope and are shown to interact with the antibody CDR loops. The MT145K trimer has a glycan at N334 rather than N332 and the glycan points away from the expected location of the PGT128 Ab paratope. In addition, MT145K Env has glycans at two positions, N412 (positioned differently on HIV Env) and N442 (absent on HIV Envs), and particularly the latter will clash with PGT128 CDRH2 and prevent it from interacting with the protein part of the epitope. (C) The gp120-gp41 interface bnAb binding region: cryo-EM model of PGT151 bnAb bound to a membrane-extracted clade B JRFL Env trimer. The structure depicts PGT151 bnAb CDRs interacting with gp120 and the gp41 interface regions (Lee et al., 2016; PDB: 5FUU). PGT151 CDRH3 interacts with the epitope formed by the protein backbone (in both gp120 and gp41), including the fusion peptide (depicted in pink) and the gp120 (N88, N448) and gp41 (N611 and N637) glycans (not shown). PGT151 Ab CDR loops interact with the FP region on the BG505 trimer. The MT145K trimer FP region (cyan) remains hidden inside the trimer. (D) Cryo-EM model of two-domain human sCD4 with the B41 SOSIP trimer (Ozorowski et al., 2017; PDB: 5VN3). The structure shows how the Phe43 residue on sCD4 stacks into the Env cavity lining Trp427. This Trp427 cavity is conserved between HIV and MT145K Envs to accommodate CD4 binding. (E and F) The CD4bs bnAb binding region: crystal structure of VRC01 bnAb in complex with 93TH057 gp120 (Zhou et al., 2010; PDB: 3NGB). The structure depicts VRC01 CDRH3, CDRL3, and CDRL1 loops interacting with the protein residues in loop D (HXB2: 278-282) and the glycan at N276 (E and F, left panels). The MT145K trimer lacks the N276 glycan and bears glycan N236 (unique to SIV Env) in place of N234 that would clash with the VRC01 CDRL1 loop (E and F, right panels). Additionally, the MT145K Env trimer has a longer gp120-V5 loop due to a six-amino-acid insertion at HIV HXB2-456 residue that would shift the loop such that it clashes with the CDRH2 the VRC01 Ab.

Figure 6

Immunogenicity of MT145-WT Compared to MT145K Trimers in CH01 UCA HC-Only Knockin Mice (A) Schematic showing immunization schedule of CH01 UCA HC-only KI mice with MT145-WT and engineered MT145K trimers. The CH01 UCA HC-only KI mice were immunized twice with 25 μg of the soluble trimer with glucopyranosyl lipid adjuvant stable emulsion (GLA-SE) as adjuvant. Time points for immunization and bleeds are indicated. (B) ELISA binding of the MT145-WT and MT145K group trimer-immunized CH01 UCA HC-only KI mice serum samples (pre-bleed, Pre; 2 weeks post prime, Bleed #1; and 2 weeks post boost-1, Bleed #2) with soluble MT145K SOSIP and its glycan knockout variant (MT145K N160K) trimers. (C) Neutralization titrations of the MT145-WT and MT145K group trimer immunized CH01 UCA HC-only KI mice sera (pre-bleed, Pre; post prime, Bleed #1; and post boost-1, Bleed #2) with MT145K virus and a CH01-sensitive virus (Q23_17). The 3-fold diluted sera were tested against the viruses in a TZM-bl reporter cell assay. (D) The ID₅₀ neutralization titers of the MT145-WT and MT145K group trimer-immunized CH01 UCA HC-only KI mice sera (pre- and post-immunization bleed time points). Neutralization was assessed against the priming immunogen-matched autologous viruses in each group (MT145-WT group, MT145-WT virus; MT145K group, MT145K virus), the N160 glycan knockout variant of the MT145K virus (MT145K N160A), and a highly CH01-sensitive virus, Q23_17. The numerical values shown in the table represent the ID₅₀ neutralization titers of the immune serum samples and were calculated by non-linear regression method from the percent neutralizations of serum titrations with virus.

Figure 7

Boosting Chimpanzee SIV MT145K Trimer-Induced B Cell Responses with an HIV Trimer Cocktail in CH01 UCA HC-Only Knockin Mice (A) Schematic showing immunization schedule of CH01 UCA HC-only KI mice with a chimpanzee SIV MT145K trimer followed by an HIV Env-derived three-trimer cocktail. A group of five animals was immunized with two doses (prime, week 0; boost-1, week 4) of the MT145K trimer (as also shown in Figure 6). The animals were further boosted (boost-2 at week 8) with an HIV Env-derived three-trimer cocktail (C108, WITO, and ZM197-ZM233V1V2). The V1V2 loops on trimer cartoons are depicted in red to highlight that the region is shared between HIV and SIV Env trimers. The CH01 UCA HC-only KI mice were immunized with 25 μg of the soluble MT145K trimer or HIV three-trimer cocktail (25 μg total) with GLA-SE as adjuvant. Time points for the immunizations and the bleeds are indicated. (B) The ID50 virus neutralization titers of the Bleed #4 serum samples collected post boost-2 immunization with an HIV three-trimer cocktail. The neutralization of the post-immune sera was assessed against the priming immunogen-matched autologous virus, MT145K, the boosting immunogen-matched, CH01 sensitive viruses, and global panel HIV Env-encoding viruses. Each filled circle in the plot represents virus ID50 neutralization values for the individual animals; an asterisk (^∗) indicates that 50% neutralization was not reached at a 1:100 serum dilution.