Design and characterization of HIV-1 vaccine candidates to elicit antibodies targeting multiple epitopes

Abstract

A primary goal in the development of an AIDS vaccine is the elicitation of broadly neutralizing antibodies (bNAbs) that protect against diverse HIV-1 strains. To this aim, germline-targeting immunogens have been developed to activate bNAb precursors and initiate the induction of bNAbs. While most preclinical germline-targeting HIV-1 vaccine candidates only include a single bNAb precursor epitope, an effective HIV-1 vaccine will likely require bNAbs that target multiple epitopes on Env. Here, we report a newly designed germline-targeting Env SOSIP trimer, named 3nv.2, that presents three bNAb epitopes on Env: the CD4bs, V3, and V2 epitopes. 3nv.2 forms a stable trimeric Env and binds to bNAb precursors from each of the desired epitopes. Immunization experiments in rhesus macaques and mice demonstrate 3nv.2 elicits the combined effects of its parent immunogens. Our results provide proof of concept for using a germline-targeting immunogen presenting three or more bNAb epitopes and a framework to develop improved next-generation HIV-1 vaccine candidates.

Figure 1.

Schematic illustrating how a combined triple immunogen could elicit a lower proportion of off-target Abs than a combination of three single immunogens, each presenting only one epitope.

Figure 2.

Design and biochemical characterization of 3nv.2 SOSIP. (A) Design of 426c-based triple immunogen to present CD4bs (blue), V3 (green), and V2 (purple) epitopes. (B) Schematics of single (IGT2), double (IGT2–RC1), and triple (3nv.2) immunogen constructs used in this study. (C and D) 3nv.2 and 3nv.1 triple immunogen characterization by (C) SEC and (D) SDS-PAGE. (E) Top: 2D class averages demonstrating 3nv.2 is predominantly trimeric. Bottom: 6.6-Å single-particle 3nv.2 cryo-EM density map. (F) Schematic for the generation of SOSIP–mi3 nanoparticles using the SpyCatcher–SpyTag system. (G) Characterization of purified SOSIP–mi3 nanoparticles by SDS-PAGE. R, reduced; NR, non-reduced; SC, SpyCatcher. (H) Negative-stain EM of SOSIP–mi3 nanoparticles. Scale bar = 100 nm.

Figure S1.

Schematics of constructs used in this study.

Figure 3.

3nv.2 binds to iGLs targeting three bNAb epitopes. (A) SPR sensorgrams of SOSIP–mi3 nanoparticles injected over bNAb iGLs at a concentration of ∼0.5 mg/ml. First row: The CD4bs-specific single immunogen IGT2 only binds to CD4bs bNAb precursors (IOMA iGL, left), and not V3 (10-1074/PGT121 iGL, middle) or V2 (PG9 or PG16 iGL, right) bNAb precursors. Second row: Incorporating V3-targeting mutations into IGT2 creates an immunogen that binds CD4bs and V3 bNAb precursors, but not V2 bNAb precursors. Third row: Incorporating V3- and V2-targeting residues into the CD4bs-targeting IGT2 SOSIP creates an immunogen (3nv.2) that binds to the CD4bs (IOMA iGL, left), V3 (10-1074/PGT121 iGL, middle), and V2 (PG9 or PG16 iGL, right) bNAb precursors. Fourth row: The parental 426c Env does not bind to any of the bNAb precursors. (B) 3nv.2 SOSIP injected in a dilution series over bNAb iGLs starting at top concentrations of 10 µM or 5 µM as indicated. 3nv.2 SOSIP binds to multiple CD4bs precursors, including IOMA iGL (left), BG24 iGL (middle), and VRC01 iGL (right). (C) 3nv.2 SOSIP injected in a dilution series over bNAb UCAs and an iGL starting at a top concentration of 10 µM. 3nv.2 SOSIP binds to multiple V3 precursors, including BG18 iGL (left), DH270 UCA (middle), and BF520 UCA (right). Representative sensorgrams are from at least two independent experiments. UCA, unmutated common ancestor; RU, resonance unit.

Figure 4.

3nv.2 triple immunogen elicits combined responses of IGT2 and RC1 immunogens in RMs. (A) Schematic and timeline of immunization regimens for RMs (n = 5 per group). (B–E) Serum neutralization ID50s against (B) IGT2, (C) IGT1, (D) RC1, and (E) 11MUTB pseudoviruses. The dotted line at y = 10² indicates the lowest dilution evaluated. Significance was demonstrated using an unpaired t test (P ≤ 0.05). (F) Serum ELISA binding to IGT1 and IGT1 CD4bs KO SOSIPs for RMs immunized with IGT2-mi3 or 3nV.2-mi3. All samples are from week 8 after prime immunizations. Significance was demonstrated using a paired t test (P ≤ 0.05). (G) Mouse serum neutralization ID50s against IGT2, IGT1, RC1, and 11MUTB pseudoviruses (n = 8). The dotted line at y = 10² indicates the lowest dilution evaluated. * denotes 0.01 < P ≤ 0.05, ** denotes 0.001 < P ≤ 0.01, and *** denotes 0.0001 < P ≤ 0.001.

Figure S2.

Data processing of the 3nv.2 SOSIP dataset. * denotes the dataset that is deposited in the EMDB.