Epitope-focused immunogen design based on the ebolavirus glycoprotein HR2-MPER region

Abstract

The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 -membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies. To study whether this epitope can serve as an immunogen for the elicitation of broadly-reactive antibody responses, protein design in Rosetta was employed to transplant the HR2-MPER epitope identified from a co-crystal structure with the known broadly-reactive monoclonal antibody (mAb) BDBV223 onto smaller scaffold proteins. From computational analysis, selected immunogen designs were produced as recombinant proteins and functionally validated, leading to the identification of a sterile alpha motif (SAM) domain displaying the BDBV-HR2-MPER epitope near its C terminus as a promising candidate. The immunogen was fused to one component of a self-assembling, two-component nanoparticle and tested for immunogenicity in rabbits. Robust titers of cross-reactive serum antibodies to BDBV and EBOV GPs and moderate titers to SUDV GP were induced following immunization. To confirm the structural composition of the immunogens, solution NMR studies were conducted and revealed structural flexibility in the C-terminal residues of the epitope. Overall, our study represents the first report on an epitope-focused immunogen design based on the structurally challenging BDBV-HR2-MPER epitope.

Fig 1. Computational design and initial experimental validation of the BDBV-MPER based immunogens.

(A) BDBV GP, as observed in PDB: 6DZM [32], contains the soluble parts of GP1 and GP2, but not the HR2-MPER region. (B) Monoclonal antibody BDBV223 bound to the BDBV-MPER peptide (PDB: 6N7J) [21]. (C) A close-up view of the interactions of BDBV-GP with the BDBV-MPER peptide via critical interaction residues. (D) Rosetta grafting protocols are employed over a scaffold library containing small, highly resolved proteins, to transfer the epitope through Rosetta’s grafting protocols sidechain and backbone grafting, and FoldFromLoops [16, 18, 33]. (E) Out of eleven selected designs, six designs expressed from E. coli were tested for binding to BDBV223. Design 4, a sidechain graft of the epitope residues on the crystal structure PDB 1B0X, strongly bound to BDBV223 in ELISA (n = 2, duplicates, exemplary experiment plotted with standard deviation (SD); reference EC₅₀ value for BDBV223 binding to the BDBV-MPER peptide has been reported as 85 ng/ml [14]; for further reference curves compare S1 Fig). (F) Binding of all three known BDBV-MPER targeting antibodies BDBV223, BDBV317 and BDBV340, to the BDBV-MPER carrying immunogen, while the control antibody, 2D22, a dengue E protein targeting antibody [34], does not bind to the immunogen (n = 2, duplicates, exemplary experiment plotted with SD). (G) Designs from sidechain grafting were the most prominent selected group tested. The selected design based on FoldFromLoops and one of the selected designs from backbone grafting were expressed but did not bind. The binding immunogen was one out of eleven designs.

Fig 2. Antibody binding of BDBV223, BDBV317 and BDBD340 to (A) HR2-MPER peptides (data taken from Flyak et al. [14]) or to (B) designed immunogens carrying HR2-MPER sequences of BDBV, SUDV and EBOV GP or mutants to characterize sequence activity relationships.

2D22, a dengue antibody [34], was used as control for non-specific binding, in ELISA. Peptides were coated at a concentration of 4 μg/mL, whereas immunogens were used at 1 μg/mL, n = 2, duplicates (S1 Fig).

Fig 3. Design and characterization of self-assembling nanoparticles displaying the BDBV-MPER immunogen.

(A) Model of the self-assembling nanoparticle displaying the BDBV-MPER immunogen on the trimeric component of the two-component I53-40 nanoparticle. (B) Negative stain electron microscopy 2D classes for I53-40 nanoparticles decorated with the BDBV-MPER immunogen. (C) Dynamic light scattering confirmed a homogeneous size distribution (n = 2, triplicates, exemplary experiment shown). (D) BDBV223 binding to the BDBV-MPER-bearing I53-40 nanoparticle observed by ELISA (n = 2, duplicates, SD). BDBV223 did not show any binding to empty I53-40 nanoparticles.

Fig 4. Serum binding from rabbits immunized with BDBV-MPER immunogens.

(A) Immunization study set-up. Prime immunization, and three boosts were administered with 0.5 mg and 0.25 mg antigen, respectively. For prime immunization Complete Freund’s Adjuvant was used, while Incomplete Freund’s Adjuvant was used for boost immunizations. Blood was drawn on days 0, 28, 56, 70. (B) Serum binding titers to antigens used for immunization shows high titers for the nanoparticle formulation. (C) Serum binding titers for BDBV, EBOV, or SUDV GPs as determined by ELISA binding.

Fig 5. Serum from a MPER-KPL immunized rabbit shows cross-reactivity to MARV GP.

(A) Screening of rabbit sera by ELISA at a dilution of 1:30. (B) Sequence alignment of HR2-MPER regions for the viruses BDBV, EBOV, SUDV, and MARV GPs. (C) Serum of BDBV-MPER-KPL immunized rabbit #3 bound to MARV GP in ELISA at days 56 and 70. (D) Serum of BDBV-MPER-KPL immunized rabbit #3 strongly bound to the MARV-MPER peptide (GIEDLSRNISEQIDQIKKDEQKEG) in ELISA. (E) As a control, an unrelated antigen, the monomeric hemagglutinin head domain for H1 (A/California/07/2009), was tested for serum binding to exclude off-target binding. (ELISA, n = 2, duplicates, exemplary experiment shown).

Fig 6. Neutralization studies from immunized rabbit sera.

(A) Sera collected from individual immunized animals were tested for neutralization activity against BDBV, with no observable difference between the epitope carrying and non-carrying groups. (B) EBOV-515, a base-targeting antibody [8], neutralized chimeric infectious VSV/BDBV GP virus. Polyclonal antibodies from rabbits immunized on day 70 were not active, except for one sample from the BDBV-peptide immunization group.

Fig 7. Structural validation of the BDBV-MPER immunogen design using solution NMR.

(A) ¹H-¹⁵N TROSY spectra of BDBV-MPER, BDBV-MPER-KPL, and SUDV-MPER immunogens. Backbone resonance assignments are indicated by one-letter amino acid code and sequence number. Resonances that are unassigned correspond to the purification tags. (B) Overlay of ¹H-¹⁵N-TROSY spectra of BDBV-MPER and BDBV-MPER-KPL immunogens. The only change in the protein sequence is ⁷¹MHG⁷³ to ⁷¹KPL⁷³ meaning that there are no corresponding peaks for residues 71–73. Red arrows indicate shifted peaks. (C) Close-up view of the BDBV-MPER, BDBV-MPER-KPL and SUDV-MPER immunogen ¹H-¹⁵N-TROSY spectra with the BDBV-MPER backbone assignment. (D) The NMR-derived model of BDBV-MPER in green is overlaid with the Rosetta-designed BDBV-MPER immunogen in grey. The backbone RMSD between the experimental and predicted structure was 2.4 Å. (E) CD spectrum of the BDBV-MPER immunogen.

Fig 8

(A) Close-up of the interactions of BDBV-GP with the BDBV-MPER peptide via critical residues, PDB: 6N7J,—the only bound form of the EBOV MPER epitope [21]. (B) NMR solution structure of EBOV TM (PDB: 5T42), 20 lowest energy structures from DPC micelles at pH 5.5 [48]. (C) Post-fusion conformation of the EBOV-GP2 subunit (PDB: 2EBO). [52] (D) Close-up of a BDBV-GP structure (PDB: 6DZM) [32] with indicated glycosylation sites above the HR2-MPER epitope. (E) HIV envelope glycoprotein in a nanodisc in complex with 10E8, a HIV-MPER antibody, reconstructed from EM densities at a resolution of 5Å (PDB: 6VPX) [23].