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Epitope-focused immunogen design based on the ebolavirus glycoprotein HR2-MPER region [1]

['Clara T. Schoeder', 'Department Of Chemistry', 'Vanderbilt University', 'Nashville', 'Tennessee', 'United States Of America', 'Center For Structural Biology', 'Institute For Drug Discovery', 'University Leipzig Medical School', 'Leipzig']

Date: 2022-07

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.

Recent breakthroughs in structure-based and computational-guided vaccine design have made methods available for transplanting epitopes from native antigens onto smaller scaffold proteins. We employed these methods to design epitope-focused immunogens based on the ebolavirus HR2-MPER epitope, which is targeted by potently neutralizing and broadly-reactive antibodies. Our efforts resulted in an immunogen that bound with high affinity to monoclonal antibodies and elicited high titers of epitope-specific polyclonal antibodies following rabbit immunization but failed to induce neutralizing antibodies. Structural characterization of the grafted epitope confirmed the overall immunogen model but also highlighted the inherent flexibility and complexity of the HR2-MPER epitope. This study demonstrates the current capabilities and remaining challenges of computational epitope-focused vaccine design. It also represents the first study of epitope-focused immunogen design based on an ebolavirus.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: J.E.C. has served as a consultant for Takeda Vaccines, Sanofi-Aventis U.S., Pfizer, Novavax, Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from IDBiologics. N.P.K. is a co-founder, shareholder, paid consultant, and chair of the scientific advisory board of Icosavax, Inc. and has received an unrelated sponsored research agreement from Pfizer. All other authors declare no conflict of interest.

Funding: This work was supported by the NIH under awards R01AI141661 (A.B., J.E.C., J.M.), U19AI117905 (J.E.C., J.M.) and U01AI150739 (J.E.C., J.M.); the Defense Threat Reduction Agency under HDTRA1-18-1-0001 (N.P.K.); and the Bill & Melinda Gates Foundation (OPP1156262 to N.P.K.). Supported in part by grants for NMR instrumentation from the National Science Foundation (NSF 0922862), National Institutes of Health (NIH S10 RR025677) and Vanderbilt University matching funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Schoeder et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we explored epitope-focused immunogen design using epitope grafting protocols in Rosetta for the HR2-MPER epitope, as defined by the co-crystal structure of BDBV223 with the BDBV-HR2-MPER peptide (PDB: 6N7J) [ 21 ]. We tested designs experimentally for their ability to elicit antibody responses to the BDBV-HR2-MPER epitope and structurally validated the immunogen model through biomolecular nuclear magnetic resonance (NMR) spectroscopy.

The display of engineered antigens on self-assembling nanoparticles can enhance the induction of epitope-specific antibodies in immunization studies. Antigens including respiratory syncytial virus (RSV) prefusion-stabilized fusion (F) protein, human immunodeficiency virus (HIV) envelope glycoprotein, and the SARS-CoV-2 receptor binding domain have been displayed on computationally designed self-assembling protein nanoparticles, leading to significant increases in neutralizing potency and protective breadth [ 25 – 28 ]. Thus, a combination of epitope-focused immunogens and multivalent display on nanoparticle scaffolds ensures induction of high antibody titers [ 29 ].

An apo-structure of BDBV223 and a peptide-antibody complex containing the BDBV-HR2-MPER epitope has been determined at a resolution of 2.03Å and 3.68 Å, respectively, by King et al. [ 21 ] providing the information required for structure-based immunogen design. Antibody recognition of the HR2-MPER epitope nevertheless remains enigmatic since the observed binding mode of the HR2-MPER peptide and BDBV223 is inaccessible to the antibody at most times due to its spatial localization close to the viral membrane [ 21 ]. In addition, HR2-MPER-reactive antibodies are low in abundance in immune subjects [ 22 ]. Similar epitopes have been described in other class I fusion proteins, e.g., the HIV envelope glycoprotein or SARS-CoV-2, where antibody recognition patterns raise the same questions [ 23 , 24 ]. Nevertheless, designed proteins presenting solely the epitope could enhance the immune response to the HR2-MPER epitope.

Rational, structure-based vaccine design has made enormous progress in the past decade. In particular, epitope-focused immunogen design transplants a specific epitope of interest onto a scaffold protein for display in a non-native environment. This allows for the targeting of a specific antibody or an antibody population with the goal of eliciting humoral responses focused on epitopes targeted by protective antibodies. Computational protein design methods are employed during this technique to ensure the epitope maintains the correct backbone and side chain conformations [ 15 , 16 ]. A number of methods and protocols in the protein design software Rosetta, such as focusing on the transfer of sidechain residues involved in the interaction between antibody and antigen [ 17 ], the transfer of the whole backbone of either a linear or discontinuous epitope [ 18 , 19 ] and the transfer of flexible regions such as loops [ 16 , 20 ], have resulted in a number of antigens successfully targeted by single antibody populations. To reliably design a new immunogen, a high-resolution co-crystal structure of the epitope-antibody interface is necessary.

One class of neutralizing antibodies identified from survivors of the 2007 BDBV outbreak in Uganda targets the conserved HR2-MPER epitope [ 7 ]. This epitope consists of a linear peptide sequence in the GP2 subunit of the viral GP, framed by a N-terminal glycosylation site and in close proximity to the C-terminal transmembrane region of the protein. The HR2-MPER targeting antibody, BDBV223, shows extraordinary binding breadth across all three human pathogenic viruses and neutralizes BDBV and EBOV. In immunization studies using the BDBV-HR2-MPER peptide conjugated to keyhole limpet hemocyanin (KLH), the antigen elicited GP-reactive and neutralizing antibodies in animal serum with varying levels of neutralizing activity against EBOV, BDBV, and SUDV [ 14 ].

The human antibody response against ebolavirus GPs has been characterized in depth, and major antigenic targets of the humoral response have been identified and characterized functionally and structurally [ 6 – 10 ]. However, only few of the identified epitopes evoke antibody responses that recognize all three human pathogenic ebolaviruses, although many serological studies have shown occurrence of cross-reactivity at least for EBOV, BDBV and SUDV.[ 11 , 12 ] These epitopes include at least two non-overlapping internal fusion loop epitopes, the GP1-core, the GP1-2 interface, and the HR2-MPER epitope. In contrast, many other epitopes are virus-specific, such as those in the glycan cap and mucin-like domain [ 6 , 12 , 13 ]. Epitopes targeted by cross-reactive antibodies are of major interest for rational vaccine design.

Ebolaviruses and marburgviruses belong to the Filovirus family, named after the filamentous appearance of the virus in electron micrographs, and contain similarly structured genomes and protein compositions. A surface GP of the class I fusion protein family mediates host cell attachment and fusion and is the major target of the protective humoral immune response [ 4 ]. The GP is a trimer in which each protomer has two subunits: a GP1 head that is heavily glycosylated and a GP2 subunit that comprises the fusion machinery [ 5 ].

Ebolavirus disease (EVD) is caused in humans by three viruses of the genus Ebolavirus: Ebola virus (EBOV), being the most common; Bundibugyo virus (BDBV), which was first described in 2007 [ 1 ]; and the less phylogenetically related Sudan virus (SUDV). Although a vaccine was approved for EBOV in 2019, its development was based on EBOV GP sequences and it is not indicated for protection against BDBV and SUDV [ 2 , 3 ].

Results

Rosetta epitope grafting protocols were used to design small protein immunogens carrying the HR2-MPER epitope The Rosetta software suite contains different methods for epitope-focused immunogen design, including sidechain and backbone grafting, and a protocol called FoldFromLoops, which is now succeeded by FunFolDes [16, 18, 20, 29, 30]. In the work presented here, these grafting protocols were used to transplant the BDBV-HR2-MPER epitope, as observed in the crystal structure PDB: 6N7J, onto smaller scaffold proteins [21]. In this co-crystal structure, BDBV223 was crystallized in complex with a synthetic peptide containing the linear BDBV-HR2-MPER epitope to a resolution of 3.68 Å. The BDBV-HR2-MPER peptide forms an α helix that interacts especially via residues D621, D624, H628, I631, and K633 with BDBV223 (Fig 1A, 1B and 1C). It is noteworthy that the C-terminal residues diverge from α-helical angles and the helical fold unravels. A glycan borders the N-terminal end of the HR2-MPER epitope in the full-length ebolavirus GP, which is not included in this structure. The Protein Database (PDB) was screened for small proteins with a resolution < 2.5 Å and reported to be expressible from E. coli for transplantation of the BDBV-HR2-MPER epitope from the co-crystal structure (PDB: 6N7J). Extensive filtering and screening steps were included, such as measures of protein stability, interaction with the BDBV223 crystal structure, and forward folding to ensure high quality of the designs (Fig 1D). In total, eleven designs were chosen for experimental validation. Designs were derived from different approaches, consisted of diverse sequences, and contained a total of three different scaffold proteins. In the case of the SAM domain (PDB: 1B0X) [31], additional refinement steps were undertaken to remove the dimer interface of the original crystal structure and help stabilize the monomeric protein. In total, one designed protein was chosen from a FoldFromLoops protocol run, two from backbone grafting, and eight from sidechain grafting (compare Fig 1F and S1 and S2 Tables for detailed protocol descriptions and designed sequences). PPT PowerPoint slide

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TIFF original image Download: 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 EC 50 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. https://doi.org/10.1371/journal.ppat.1010518.g001 The designed proteins were expressed in E. coli, purified from the soluble fraction of lysed cells, and tested for binding by BDBV223. Out of the eleven selected designed proteins, six designs were purified. However, only one design bound to BDBV223 in ELISA (Fig 1E). The binding was specific for BDBV-HR2-MPER antibodies BDBV223, BDBV317 and BDBV340, but did not bind to 2D22, a dengue virus-specific antibody [34] (Fig 1F). This designed immunogen, designated BDBV-MPER, was derived from a Mus muculus SAM domain, which was observed in its native crystal structure as dimer [31] and was modified in order to remove interactions from the dimer interface for stabilization of the monomeric protein.

The BDBV-MPER-based immunogen binds strongly to three HR2-MPER epitope-specific antibodies BDBV223, BDBV317, and BDBV340 The designed immunogen incorporated residues I623 to D632 of the BDBV-HR2-MPER epitope. As observed in the crystal structure, residues K633 and P634 contact BDBV223 [21]. Therefore, we ensured that the designed immunogen recapitulated the known sequence-activity relationships that were reported by Flyak et al. [14] through introduction of the same mutations in our immunogens (Fig 2A). Using site-directed mutagenesis, the three C-terminal residues MHG were replaced with the last three residues of the BDBV-HR2-MPER epitope, specifically with the residues KPL (designated BDBV-MPER-KPL). Additionally, immunogens carrying the sequence of the SUDV- and EBOV-HR2-MPER were studied. Through ELISA binding, we observed strong dose-response binding for the three HR2-MPER antibodies (BDBV223, BDBV317 and BDBV340) to the BDBV-MPER and BDBV-MPER-KPL immunogens. In comparison to the native BDBV-HR2-MPER epitope sequence, both BDBV-MPER and BDBV-MPER-KPL immunogens contain a different starting amino acid in the third position (glutamate instead of a threonine and a serine instead of a lysine), which in the crystal structure PBD 6N7J failed to form any interactions with BDBV223 [21], and should not impact the binding properties of the immunogen. The last three amino acids in the BDBV-MPER immunogen, however, showed interactions with the antibody in the crystal structure. As the impact of these changes was unclear, we assessed the binding properties of both the BDBV-MPER and BDBV-MPER-KPL immunogens. Binding was comparable, with half maximal effective concentration (EC 50 ) values of <1 ng/mL, suggesting the last three amino acids minimally contribute to the binding interaction (Fig 2). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Antibody binding of BDBV223, BDBV317 and BDBD340 to (A) HR2-MPER peptides (data taken from Flyak et al. [ 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). https://doi.org/10.1371/journal.ppat.1010518.g002 For peptides derived from the BDBV-HR2-MPER sequence, a binding pattern was recently reported by Flyak et al. [14] for the three antibodies BDBV223, BDBV317 and BDBV340. All three antibodies revealed strongest binding to the homologous BDBV-HR2-MPER peptide, bound less strongly to the EBOV-HR2-MPER peptide, and showed reduced or no binding to the SUDV-HR2-MPER peptide [14]. In order to investigate this loss of binding, we tested a BDBV-HR2-MPER-D624N immunogen, which contains one of the two amino acid changes of the SUDV-HR2-MPER peptide to test the hypothesis that BDBV223 binds differentially compared to BDBV317 and BDBV340 [14]. Immunogens carrying the sequences for SUDV-, EBOV-HR2-MPER, and respective mutations were expressed, purified, and assessed by ELISA. BDBV223, BDBV317 and BDBV340 bound strongly with EC 50 values of < 10 ng/mL to BDBV-MPER, BDBV-MPER-KPL, or EBOV-MPER immunogens, showing no preference for the BDBV-HR2-MPER epitope sequences compared to the native EBOV-HR2-MPER sequence. BDBV317 exhibited weaker binding to the SUDV-MPER immunogen, but still displayed an EC 50 value < 100 ng/mL. When introducing mutations to the immunogen similar to the mutations studied for HR2-MPER peptides [14], only the BDBV-MPER-I631V mutant diminished binding to BDBV317. For antibody BDBV223 and BDBV340, the binding potency for all immunogens stayed the same (S1 Fig).

Multivalent display on self-assembling nanoparticle platform for enhanced immune recognition In an initial experiment, two New Zealand White (NZW) rabbits were injected with KLH-bound BDBV-MPER immunogen, and serum was collected after a prime immunization and three boosts to assess for antigen binding. Serum collected on day 90 after immunogen inoculation was tested for binding to ebolavirus GPs, and although serum binding to BDBV-GP was observed, neutralization activity against EBOV was not detected (S2 Fig). In order to achieve higher antibody titers, multivalent display of the immunogen on a self-assembling two-component nanoparticle platform was chosen [25, 27, 35]. Briefly, trimeric and pentameric scaffold proteins are mixed together to form regular icosahedral complexes comprising 60 copies of each subunit in the final complex [36]. Two-component nanoparticles have been used to multivalently display several class I fusion proteins through genetic fusion to the trimeric scaffold protein (e.g., the prefusion-stabilized RSV F protein [DS-Cav1] [25], the influenza hemagglutinin [28] or the HIV-envelope protein [26]). The BDBV-MPER immunogen is a small monomeric unit that we fused genetically to the trimeric components of three different nanoparticle scaffolds via a flexible linker (Fig 3). Trimeric proteins based on the reported designs of I53-40, I53_dn5, and I53-50 were tested for successful expression, assembly, and antibody binding. Additionally, two designs based on the assembly of a trimer and a dimer (termed I32-28 [36]) were tested. However, these did not express in the soluble fraction and were not considered for further studies. For all three successfully expressed nanoparticle component-immunogen fusions, surface exposure of the epitope was confirmed by ELISA binding to HR2-MPER antibodies (BDBV223, BDBV317, and BDBV340) (S3 Fig). For all three designs, the assembled nanoparticle was generated by mixing the trimeric component with the pentameric component and purified through size exclusion chromatography (SEC). Epitope exposure was confirmed by ELISA binding to BDBV223, BDBV317, and BDBV340 and off-targeting binding was probed using nanoparticles without surface decorated immunogen (S3 Fig). The I53-50 nanoparticle without immunogen displayed weak binding to BDBV223 in ELISA studies and was therefore excluded from further studies. Both I53_dn5 and I53-40 nanoparticles did not show any off-target antibody binding. I53-40-based nanoparticles were chosen for further studies based on a similar antibody binding profile to the BDBV-MPER immunogen (Figs 3D and S3). Structural integrity was verified using negative stain electron microscopy and dynamic light scattering. Both methods confirmed a homogenous composition of the BDBV-MPER immunogen-decorated nanoparticles (Fig 3B and 3C). However, representative negative stain EM 2D classes and a 3D reconstruction of the particle showed only the scaffold proteins since the immunogen could not be resolved, presumably due to flexibility in the linker between the displayed antigen and the nanoparticle scaffold (S4 Fig). PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1010518.g003

Rabbit immunization with surface-displayed BDBV-MPER immunogen nanoparticles results in high antibody titers for Ebola GPs Four antigens were selected for immunization studies: 1.) the BDBV-MPER immunogen, 2.) the BDBV-MPER-KPL immunogen and as control antigens: 3.) an immunogen void of any HR2-MPER epitope, but the native sequence of the SAM domain, and 4.) the HR2-BDBV-MPER peptide (called BDBV-MPER peptide), each of which were displayed on the surface of the nanoparticle. Each group consisted of four NZW rabbits with prime and boost immunizations on days 1, 14, 42 and 56. Antigens were administered in Complete Freund’s Adjuvant for prime immunization and Incomplete Freund’s Adjuvant for boost immunizations. Blood was drawn on days 0, 28, 56 and 70. To assess immunogenicity, serum binding to the respective antigen was evaluated first. Strong serum binding was observed to the respective antigen for days 28, 56 and 70, while day 0 serum showed minimal or no binding (Figs 4B and S6). Next, serum binding to BDBV, EBOV, or SUDV GP was tested, and higher antibody titers were observed for rabbit sera immunized with nanoparticle-displayed immunogens, similar to the KLH-linked BDBV-MPER immunogen (S2, S7, S8 and S9 Figs). For the control group carrying the unmodified SAM domain scaffold protein, serum antibody binding was not detected at any time point to the three tested ebolavirus GPs. However, serum binding to the reverted scaffold antigen was strong. This finding confirms that serum antibody binding activity is mediated by the displayed epitope. In all three epitope-carrying groups, serum binding to BDBV GP was observed, except for rabbit #2 and #3 in the BDBV-MPER immunogen group, which showed very weak or no serum binding, despite having high antibody titers against the antigen (Figs 4B, S6, S7, S8 and S9). All other sera from the BDBV-MPER and BDBV-MPER-KPL groups contained antibodies that bound to BDBV and EBOV GP with up to a 1:100,000 dilution. Interestingly, serum binding was stronger for the BDBV-MPER peptide control group, with 3 out of 4 rabbit sera from day 70 binding to BDBV and EBOV GP with up to a 1:10,000,000 dilution. Cross-reactive binding to SUDV GP was observed for all three epitope carrying groups, with the BDBV-MPER peptide immunized group showing the highest antibody titers. Serum antibodies of rabbit #3 in the BDBV-MPER-KPL group did not bind to SUDV GP at all, despite having similar antibody titers for BDBV and EBOV GP as the other three animals in the group. Overall, antibody titers for SUDV GP seemed to be lower and delayed in response compared to BDBV or EBOV GP (Figs 4B, S6, S7, S8 and S9). PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1010518.g004

Binding to MARV GP by one rabbit serum in the MPER-KPL group suggests cross-reactive immunization To test for cross-reactivity, rabbit sera were screened for binding to MARV GP at a dilution of 1:30 (Fig 5A). Initial binding was observed for serum from rabbit #3 of the BDBV-MPER-KPL immunized group. A possible MARV-HR2-MPER epitope has not been described in the literature. However, sequence alignment of all human pathogenic filoviruses displayed a hypothetical MARV-HR2-MPER epitope. This hypothetical epitope contains a number of different amino acids with some key residues sharing identity or amino acid characteristic similarities. For example, the following amino acids appear to be conserved: I623, D624, Q625, I626, D629, while other amino acids are exchanged for amino acids with similar properties, such as the exchange of D621 to E621 and H628 to K628. Interestingly, the MARV-HR2-MPER sequence is also preceded by a NxS sequence, which in all ebolaviruses is a NxT and carries a glycan as upper boundary of the epitope. It can therefore be rationalized that MARV-HR2-MPER might represent a possible epitope with similar properties to BDBV-HR2-MPER (Fig 5B). PPT PowerPoint slide

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TIFF original image Download: 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). https://doi.org/10.1371/journal.ppat.1010518.g005 Serum from rabbit #3 of the BDBV-MPER-KPL group was further analyzed for binding. At day 28, binding is barely observable at all, however, for day 56 and 70, binding is detectable for multiple dilutions, which suggests an induction of an antibody response. To test whether the observed binding is specific to the hypothetical MARV-HR2-MPER region, a peptide was used that contained the sequence GIEDLSRNISEQIDQIKKDEQKEG. Serum from days 56 and 70 strongly bound this peptide, whereas day 28 weakly bound. This suggests that cross-reactive antibodies to the hypothetical MARV-HR2-MPER region evolved gradually over time through somatic hypermutation. To confirm MARV-HR2-MPER region specific binding, serum was tested against an off-target antigen, hemagglutinin H1 head domain of A/California/07/2009, and failed to bind with specificity for any of the days tested. Interestingly, serum from rabbit #3 of the BDBV-MPER-KPL immunogen group bound to BDBV and EBOV GP only. These results suggest that immunization with an ebolavirus HR2-MPER epitope can elicit broadly-reactive polyclonal antibody responses, including MARV GP.

Serum antibodies do not reveal neutralizing activity against BDBV or VSV/BDBV GP viruses HR2-MPER antibodies, such as BDBV223, BDBV317 and BDBV340, potently neutralize authentic BDBV. However, these mAbs exhibit less neutralization activity and efficacy against EBOV and no observed activity against SUDV or Reston virus (RESTV) [14]. BDBV223 and BDBV317 protected mice inoculated with EBOV post-exposure but were far less effective in a guinea-pig challenge model [14]. When peptides derived from the HR2-MPER epitope were used to immunize rabbits, the serum antibodies displayed binding to BDBV, EBOV, and SUDV GP with varying levels of neutralization [14]. Serum derived from rabbits immunized with BDBV-MPER immunogen, reverted immunogen, BDBV-MPER-KPL, or BDBV-MPER peptide were tested for neutralization activity. Pooled serum alone did not show a difference in neutralization activity for epitope-carrying immunogens versus reverted immunogen for BDBV (Fig 6A). There seems to be some unspecific reaction to BDBV, which can be observed by comparing the reverted scaffold group with the immunogen groups (Fig 6A). Subsequently, polyclonal antibodies (pAbs) were purified from rabbit serum using affinity chromatography, and pAb reactivity was verified by ELISA (S11 Fig). However, when testing pAbs neutralizing activity using recombinant chimeric vesicular stomatitis virus (VSV) expressing BDBV GP (VSV/BDBV GP), only one serum from the BDBV-MPER peptide group and the positive-control broadly-neutralizing GP base-specific antibody, EBOV-515 [8, 37] neutralized the virus (Fig 6B). PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1010518.g006

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