Compositions and methods related to Ebola virus vaccines

Abstract

The present invention provides novel engineered Ebola virus GP proteins and polypeptides, as well as scaffolded vaccine compositions that display the engineered proteins. The present invention also provides methods of using such engineered Ebola virus GP proteins and vaccine compositions for various therapeutic applications, e.g., to prevent or treat Ebola virus infection.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 063,530, filed August 10, 2020, currently pending. The complete disclosure of the priority application is incorporated herein by reference in its entirety for all purposes.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under AI129698 and AI123861 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Filoviruses, such as Zaire Ebola virus (EBOV) and Marburg virus, can cause fatal hemorrhagic fever in humans and non-human primates (NHPs). The viral glycoprotein GP mediates cell entry by initiating attachment and membrane fusion. Structures of GP-binding neutralizing antibodies (NAbs) from human survivors demonstrated that GP harbors all NAb epitopes known to date and is the only target for vaccine design. Similar to the HIV-1 envelope glycoprotein (Env), filovirus GP is metastable and produces non-functional forms when expressed in various cell lines.

[0004] A sustained campaign is underway to identify and characterize NAbs against EBOV and other filoviruses such as Marburg virus (MARV). As a result, a large panel of NAbs has been isolated from human survivors, vaccinated humans, and immunized animals. Crystallography and electron microscopy (EM) have revealed multiple sites of vulnerability on the EBOV GP. A recent study of 171 monoclonal antibodies (mAbs) defined eight epitope classes, six of which can be recognized by broadly neutralizing antibodies (bNAbs). Recombinant virus-like particles (VLPs) can protect against EBOV challenge in animals, but production difficulties have hindered their further development as human vaccines. Summary of the Invention

[0005] Despite recent advances in the development of therapeutic regimens against filoviruses, there remains a great need in the art for more effective means of treating and preventing human infection and death caused by Ebola virus. The present invention is directed to this and other unmet needs in the art.

[0006] In one aspect, the present invention provides an engineered or redesigned Ebola virus glycoprotein (GP) sequence. Compared to the wild-type Ebola virus GP sequence, the engineered sequence contains (a) a substitution of residue W615 in heptad repeat 2 (HR2) with a smaller hydrophobic residue, and (b) one or more proline substitutions in the heptad repeat 1C segment (HR1C). The amino acid numbering is based on the Zaire Ebola virus GP sequence, which has an ectodomain sequence identified by UniProtID Q05320 (SEQ ID NO: 1). In various embodiments, the engineered Ebola virus GP sequence can be modified or derived from the wild-type GP sequence of Zaire Ebola virus (EBOV), the wild-type GP sequence of Sudan virus (SUDV), the wild-type GP sequence of Tai Forest virus (TAFV), the wild-type GP sequence of Bundibugyo virus (BDBV), or the wild-type GP sequence of Reston Ebola virus (RESTV).

[0007] In some embodiments, residue W615 in the engineered Ebola virus GP sequence can be replaced with residues L, A, V, I, or F. In some embodiments, the proline substitution in HR1C comprises T577P and / or L579P. In some embodiments, the engineered Ebola virus GP sequence further comprises a truncation at the C-terminus of the membrane proximal external region (MPER). In some of these embodiments, the engineered Ebola virus GP sequence may further comprise (a) a deletion of the mucin-like domain (MLD) deleted from the GP1 subunit, and / or (b) a deletion of the MPER from the GP2 subunit.

[0008] In another aspect, the invention provides an engineered Ebola virus GP sequence that contains (a) a truncated soluble Ebola virus GP with the MLD deleted from the GP1 subunit and the MPER deleted from the GP2 subunit, and (b) one or more modifications in the HR2 and HR1 regions. In various embodiments, the additional modifications in the HR2 and HR1 regions include (i) replacement of W615 in heptad repeat 2 (HR2) with a smaller hydrophobic residue, (ii) extension of HR2 at the C-terminus, (iii) a deletion of the heptad repeat 1C segment (HR1 C ), one or more proline substitutions in the GP domain, and (iv) one or more engineered inter-GP disulfide bonds. Again, amino acid numbering is based on the Zaire Ebola virus GP sequence having UniProt ID Q05320 (SEQ ID NO:1). In various embodiments, these engineered Ebola virus GP sequences may be derived from Zaire Ebola virus (EBOV), Sudan virus (SUDV), Tai Forest virus (TAFV), Bundibugyo virus (BDBV), or Reston Ebola virus (RESTV). Some engineered Ebola virus GP sequences are based on the truncated soluble GP sequence shown in SEQ ID NO:2 or SEQ ID NO:3, or conservatively modified or substantially identical variants thereof. In some embodiments, the engineered GP sequence may have an N-terminal leader sequence. By way of example, the N-terminal leader sequence may contain SEQ ID NO:41 or a conservatively modified variant.

[0009] Some engineered soluble Ebola virus GP sequences contain a substitution of residue W615. In some of these embodiments, residue W615 may be replaced with L, A, V, I, or F. Some of these engineered Ebola virus GP proteins contain the amino acid sequence shown in SEQ ID NO:4, conservatively modified variants thereof, or substantially identical sequences. In some embodiments, the engineered soluble Ebola virus GP protein may contain an extension of HR2 at the C-terminus. In some of these embodiments, the HR2 extension may be (a) an extension of the HR2 C-terminus with an N-terminal fragment of the adjacent MPER of the Ebola virus glycoprotein, or (b) a replacement of the HR2 C-terminal fragment with a longer leucine zipper motif. By way of illustration, an HR2 extension in an engineered soluble Ebola virus GP protein can include (a) an extension of the HR2 C-terminus in MPER from residue 632 to residue 637, i.e., the addition of residues KTLPD (SEQ ID NO: 32); (b) an extension of the HR2 C-terminus in MPER from residue 632 to residue 643, i.e., the addition of residues KTLPDQGDNDN (SEQ ID NO: 33); or (c) a replacement of residues 617-632 with the GCN4 leucine zipper sequence set forth in SEQ ID NO: 34.

[0010] Some engineered soluble Ebola virus GP sequences of the invention contain both the HR2 extension and a W615 substitution in HR2 as described above. In some of these embodiments, the W615 substitution can be a W615L or a P612G / W615F double mutation. In some embodiments, the engineered GP sequence contains (a) a W615L substitution and (b) an HR2 C-terminal extension from residue 632 to residue 637 in the MPER of the Ebola virus GP. In some other embodiments, the engineered GP sequence contains (a) a P612G / W615F double mutation in HR2 and (b) a substitution of residues 617-632 with the GCN4 leucine zipper sequence set forth in SEQ ID NO:34. Some particular engineered soluble Ebola virus GP sequences contain the amino acid sequence set forth in any one of SEQ ID NOs:5-8, a conservatively modified variant thereof, or a substantially identical sequence.

[0011] In addition to the HR2 extension and / or the W612 substitution, the engineered GP sequence may in some embodiments also include a C-terminal trimerization motif. In some of these embodiments, the C-terminal trimerization motif may contain SEQ ID NO:29 or SEQ ID NO:30, or a conservatively modified variant or substantially identical sequence thereof. In addition to the W612 substitution, the engineered GP sequence in some embodiments also includes an HR1 extension and / or a W612 substitution. C It may contain one or more proline substitutions in the segment. C Any residue in HR1 may be replaced with a proline residue. In some of these embodiments, the proline substitution includes T577P or L579P. C Some specific examples of engineered Ebola virus GP sequences having a proline substitution at 200 C to 250 C contain the amino acid sequence set forth in any one of SEQ ID NOs:9-16, a conservatively modified variant thereof, or a substantially identical sequence.

[0012] Some engineered GP sequences of the present invention include HR2 extension, HR1 C In some of these embodiments, the engineered Ebola virus GP sequence contains a proline substitution at residue 632 of MPER, and a substitution at residue W615. In some of these embodiments, the W615 residue can be replaced with L, A, V, I, or F. In some of the embodiments, the engineered Ebola virus GP sequence contains an extension of the HR2 C-terminus from residue 632 to residue 637 of MPER. Some specific examples of such engineered Ebola virus GP sequences contain the amino acid sequence set forth in any one of SEQ ID NOs: 17 and 20, conservatively modified variants thereof, or substantially identical sequences. In some of the embodiments, the engineered Ebola virus GP sequence can further include a C-terminal trimerization motif. For example, the engineered Ebola virus GP protein can include a C-terminal trimerization motif set forth in SEQ ID NO: 29 or SEQ ID NO: 30, or a conservatively modified variant or substantially identical sequence thereof.

[0013] Some engineered Ebola virus GP sequences contain (a) a W615L substitution, (b) a proline substitution T577P or L579P, and (c) an HR2 extension in MPER from residue 632 to residue 637. Some specific examples of these engineered Ebola virus GP sequences contain the amino acid sequence set forth in any one of SEQ ID NOs: 18, 19, 21 and 22, a conservatively modified variant thereof, or a substantially identical sequence.

[0014] Some engineered Ebola virus GP sequences contain one or more engineered disulfide bonds between two adjacent GP promoters. In various embodiments, the engineered disulfide bonds can be created by cysteine ​​substitutions at residue pairs G91 / A575 (SS2), F153 / Y534 (SS1), T520 / A575 (SS3), G157 / I532 (SS4), D522 / A575 (SS5), and / or K56 / G599 (SS6). Some specific examples of these engineered Ebola virus GP sequences have the amino acid sequence set forth in any one of SEQ ID NOs: 23-28, conservatively modified variants thereof, or substantially identical sequences.

[0015] In some related aspects, the invention provides nucleic acid or polynucleotide sequences encoding the engineered full-length or truncated Ebola virus GP protein sequences described herein. Some engineered Ebola virus GP sequences also contain a 5' leader peptide coding sequence. Pharmaceutical compositions containing the engineered Ebola virus GP protein or encoding polynucleotide sequences described herein and a pharma- ceutically acceptable carrier are also provided herein.

[0016] In another aspect, the present invention provides a vaccine composition containing an engineered Ebola virus GP protein displayed on the surface of a self-assembling nanoparticle. In some embodiments, the C-terminus of the engineered Ebola virus GP protein is fused to the N-terminus of a subunit of a self-assembling nanoparticle (NP). In some embodiments, the C-terminus of the engineered Ebola virus GP protein is fused to a subunit of a self-assembling nanoparticle via the linker sequence GGGGS (SEQ ID NO: 43) or (GGGGS)2 (SEQ ID NO: 42). In some embodiments, the engineered Ebola virus GP protein in the NP vaccine composition contains an amino acid sequence set forth in any one of SEQ ID NOs: 4-28, a conservatively modified variant thereof, or a substantially identical sequence. In some embodiments, the subunit of the self-assembling nanoparticle contains a polypeptide sequence set forth in SEQ ID NO: 36 (E2p), SEQ ID NO: 37 (I3-01v9), or SEQ ID NO: 38 (ferritin), a conservatively modified variant thereof, or a substantially identical sequence.

[0017] Some NP scaffold vaccine compositions of the invention may further comprise a locking domain (LD) fused to the C-terminus of the NP subunit sequence. In some of these embodiments, the LD used contains the sequence shown in SEQ ID NO: 39 or 40, a conservatively modified variant thereof, or a substantially identical sequence. Some of the scaffold vaccines may further comprise a T cell epitope fused to the C-terminus of the locking domain. In some of these embodiments, the T cell epitope used contains the sequence shown in SEQ ID NO: 31, a conservatively modified variant thereof, or a substantially identical sequence.

[0018] Some NP vaccine compositions of the invention contain (1) a polypeptide sequence that contains, from N-terminus to C-terminus, (a) an engineered Ebola virus GP protein, linker sequence G4S (SEQ ID NO: 43), nanoparticle sequence ferritin, (b) an engineered Ebola virus GP protein, linker sequence G4S (SEQ ID NO: 43), nanoparticle sequence E2p, or (c) an engineered Ebola virus GP protein, linker sequence (G4S)2 (SEQ ID NO: 42), nanoparticle sequence I3-01v9, or (2) a conservatively modified variant of the polypeptide sequence of (1). In some of these embodiments, the engineered Ebola virus GP sequence contains a W615L substitution, a proline substitution T577P, and an HR2 extension of MPER from residue 632 to residue 637. These engineered Ebola virus GP sequences can further contain a locking domain and / or a T cell epitope fused to the C-terminus of the nanoparticle subunit sequence. In some embodiments, the vaccine composition contains (1) a polypeptide sequence that contains, from N-terminus to C-terminus, (a) an engineered Ebola virus GP protein set forth in SEQ ID NO: 17, a nanoparticle subunit sequence set forth in SEQ ID NO: 36 (E2p), a locking domain set forth in SEQ ID NO: 39 (LD4), and a T cell epitope set forth in SEQ ID NO: 31 (PADRE); or (b) a polypeptide sequence that contains an engineered Ebola virus GP protein set forth in SEQ ID NO: 17, a nanoparticle sequence set forth in SEQ ID NO: 37 (I3-01v9), a locking domain set forth in SEQ ID NO: 40 (LD7); or (2) a conservatively modified variant of the polypeptide sequence.

[0019] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. [Brief description of the drawings]

[0020] [Figure 1]FIG. 1 shows (A) an overview of the Ebola virus glycoprotein (GP) structure and (B) a general redesign strategy for generating immunogenic proteins for the engineered Ebola virus GP vaccine of the present invention.

[0021] [Diagram 2] FIG. 2 shows a sequence alignment of the H1 (SEQ ID NOs: 44 to 48) and HR2 (SEQ ID NOs: 49 to 53) regions of the glycoprotein (GP) of randomly selected strains from all five Ebolaviruses, namely Bundibugyo Ebolavirus (AYI50316), Zaire Ebolavirus (AER59712), Reston Ebolavirus (ASU06443), Sudan Ebolavirus (ALH21228) and Tai Forest Ebolavirus (AWK96625).

[0022] [Figure 3-1]Figure 3 shows the design, screening and antigen characterization of stalk and HR1C engineered EBOV GPΔmuc trimers. (A) Schematic of mucin-deleted GP (GPΔmuc) and three stalk designs (GPΔmuc-2WPZ, GPΔmuc-L and GPΔmuc-Ext). (B) SEC profiles of the four GPΔmuc constructs from a Superdex 200 10 / 300 column after transient expression in 250 ml HEK293F cells and purification with mAb114. Left: SEC curve of GPΔmuc shown as black line; Right: three stalk designs shown for GPΔmuc-2WPZ, -L and -Ext, respectively. (C) BN-PAGE of GPΔmuc proteins purified by mAb114 (left) and mAb110 (right) columns. Note that an additional GPΔmuc construct in which the 8-aa HR1C is replaced with a flexible (GS)4 loop is included for comparison. (D) ELISA curves of four mAb100 / SEC purified stalk modified EBOV GP / GPΔmuc-foldon trimers binding to ten antibodies. (E) Summary of EC50 values ​​(μg / ml) of EBOV GP / GPΔmuc-foldon trimers binding to ten antibodies. (F) ELISA curves of mAb100 / SEC purified stalk / HR1C modified EBOV GP / GPΔmuc-foldon trimers binding to ten antibodies. (G) Summary of EC50 values ​​(μG / ml) of stalk / HR1C-modified EBOV GP / GPΔmuc-foldon trimers binding to ten antibodies. [Figure 3-2] (H) Plot of EC50 values ​​(μg / ml) for binding of five GP / GPΔmuc-foldon trimers to ten antibodies. Four NAbs common to Ebola virus are shown. In (E) and (G), EC50 values ​​were calculated for all ELISA data in Prism version 8.4.3.

[0023] [Figure 4-1]Figure 4 shows the design and analysis of properties of multilayered EBOV GPΔmuc-displayed NPs. (A) Locking domains (LDs) identified from the Protein Data Bank (PDB) to stabilize the NP-forming interface. The PDB IDs of the identified LDs and their full sequences (SEQ ID NOs: 54 to 62) are shown in the figure. The residues deleted at the N-terminus and / or C-terminus of the original sequence to generate the actual LDs used in the studies herein are listed in the sequence. (B)-(C) Antigenicity evaluation by ELISA. The stable immobilization of the GPΔmuc-WL2P2 trimers displayed by binding to the FR and the re-artificially generated E2P (E2P-LD4-PADRE or E2P-L4P) and I3-01 (I3-01-LD7-PADRE, I3-01-L7P) NPs against 10 antibodies is shown by the ELISA curves shown in (B) and the EC50 values ​​are outlined in (C). All GP-NPs were transiently expressed in ExpiCHO cells and purified by mAb100 column and further purified by SEC on a Superose 6 10 / 300 GL column. [Figure 4-2] (D)-(G) Antigenicity assessment by BLI using Octet96. Binding kinetics of GPΔmuc-WL2P2-foldon (D) and GPΔmuc-WL2P2 conjugated to FR (E), E2P-L4P (F) and I3-01-L7P (G) NPs for ten different antibodies. BLI sensorgrams were obtained from an Octet RED96 instrument using six consecutive concentrations (400 to 12.5 nM for GPΔmuc, 25 to 0.78 nM and 10 to 0.31 nM for 2-fold dilutions for FR and E2p-L4p / I3-01-L7P, respectively) and a quantitative biosensor (see Materials and Methods). [Figure 4-3] (H) SEC profile of GPΔmuc-WL2P2-10GS-I3-01v9-LD7. I3-01v9 is a mutant form of I3-01 with a redesigned NP-forming interface based on the PDB structure (1VLW). I3-01v9 was reported in our previous study under the construct name "1VLW-V9".

[0024] [Figure 5-1] Figure 5 shows immunogenicity evaluation of EBOV GP / GPΔmuc trimers and GPΔmuc-displaying NPs in BALB / c mice. (A) ELISA binding curves of mouse sera from three trimer groups in which mice were immunized with WT GP-foldon, GPΔMuc-foldon and GPΔMuc-WL2P2-fold-trimer and three NP groups in which mice were immunized with the indicated FR, E2P-L4P and I3-01v9-L7P NPs displaying GPΔMuc-WL2P2 at w2, w5, w8 and w11 to GPΔMuc-WL2P2. (B) EC50 titers measured for the three trimer groups. [Figure 5-2] (C) EC50 titers were measured in three groups. EC50 titers were expressed as a scale of dilution. Of note, binding of sera at w2 did not reach a plateau (or saturation) to allow accurate determination of EC50 titers. However, EC50 values ​​calculated in Prism were used as a quantitative measure of binding antibody titers to facilitate comparison of the various vaccine groups at week 2. (D) Neutralization of Ebolavirus-pp (Makona C15) by 10 Ebolavirus-specific antibodies in TZM-bl cells. (E) Neutralization of MLV-pp by 10 Ebolavirus-specific antibodies in 293T cells. (F) Neutralization of MLV-pp by purified mouse serum IgG in 293T cells. Mouse IgG was collected at week 11 from all six vaccine groups.

[0025] [Figure 6-1]Figure 6 shows the immunogenicity evaluation of EBOV GP / GPΔmuc trimers and GPΔmuc-presenting NPs in rabbits. (A) ELISA binding curves of rabbit sera from two trimer groups immunized with GPΔMuc-foldon and GPΔMuc-WL2P2-fold trimers and three NP groups immunized with FR, E2P-L4P and I3-01v9-L7P NPs presenting GPΔMuc-WL2P2 to GPΔMuc-WL2P2 at w0, w2, w5, w8, w11 and w13. (B) EC50 titers measured for the two trimer groups and the three NP groups. EC50 titers are expressed as a scale of dilution. It should be noted that the binding of sera at w2 did not reach a plateau (or saturation), but the OD450 was at approximately the same concentration as that obtained at w5 (where it had completely plateaued). Therefore, the EC50 values ​​calculated in Prism were considered to be sufficiently accurate to facilitate comparison of the different vaccine groups at w2. [Figure 6-2] Neutralization of Ebola virus-pp (EBOV-Makona and BDBV) and MLV-pp by purified rabbit IgG from w2, w5 and w8 in 293T cells is shown in (C), (D), (E) and (F). Data points not fitting the sigmodal dose-response model are shown with dashed lines. ADE in the two GPΔmuc trimer groups, the FR group and the two multilayer 60-mer NP groups appears in w2, w5 and w8, respectively, and is highlighted by dashed rectangles. [Figure 6-3] (E) and (F)

[0026] [Figure 7-1]Figure 7 shows unbiased NGS repertoire analysis of bulk-sorted EbolaGP-specific mouse splenic B cells. (A) SEC profile of biotinylated Avi-labeled EbolaGPΔmuc trimer, designated GPΔmuc-WL2P2-1TD0-Avi-Biot, obtained from a HiLoad Superdex 200 16 / 600 column. 1TD0 is a trimer scaffold used to stabilize GPΔmuc in the trimer state and fold the two trimer groups by deselecting B cells. (B) Overview of EbolaGPΔmuc-specific bulk sorting of mouse splenic B cells from the five vaccine groups. (C) Antibodyomics analysis of NGS data obtained on EbolaGPΔmuc-specific mouse splenic B cells. Distribution of key B cell signatures for the three vaccine groups immunized with (D) GPΔmuc-foldon, (E) GPΔmuc-WL2P2-5GS-FR and (F) GPΔmuc-WL2P2-10GS-I3-01v9-L7P. [Figure 7-2] For each group, the distribution of germline VH / VK gene usage (top), germline VH / VK genetic divergence (bottom left), and CDRH / K3 loop length (bottom right) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] I. Overview Ebola virus can cause severe hemorrhagic fever. There are five virus species in the Ebolavirus genus: Ebola virus (also known as Zaire Ebola virus; EBOV), Sudan virus (SUDV), Tai Forest virus (TAFV), Bundibugyo virus (BDBV) and Reston Ebola virus (RESTV). Of these, EBOV, SUDV, TAFV and BDBV all have pandemic potential in humans. For example, EBOV was the single cause of the largest filovirus outbreak in history between 2013-2016, which spread to nine African countries and caused 28,600 cases and 11,325 deaths. The previous EBOV outbreak resulted in 2103 deaths and was declared an international emergency by the World Health Organization (WHO) on July 17, 2019. Significant progress has been made in recent years to combat this deadly virus. Neutralizing antibodies (NAbs) are now established as effective treatments for EBOV infection. Vaccines based on diverse delivery systems have been tested in humans. rVSV-ZEBOV, a replication-competent recombinant vesicular stomatitis virus (VSV) expressing the Zaire EBOV glycoprotein (GP), is the most advanced vaccine candidate deployed during the 2018–2019 pandemic. However, GP-specific antibody titers did not increase significantly 7 days after vaccination with rVSV-ZEBOV in humans, in contrast to previous findings in nonhuman primates (NHPs). Furthermore, a recent study reported neurotropism of rVSV-ZEBOV, which caused damage to the eye and brain in neonatal mice. Antibody-dependent enhancement (ADE) of infection was found for EBOV antibodies isolated from human survivors, suggesting that weak or non-NAbs induced by a suboptimal vaccine may cause adverse effects. Currently, no EBOV vaccine has been approved by the US Food and Drug Administration (FDA) for use in humans.

[0028] Ebola virus (EBOV) glycoproteins (GPs) can be recognized by neutralizing antibodies (NAbs) and are a prime target for vaccine design. The present invention stems in part from our work to rationally redesign Ebola virus glycoproteins and engineer single-component multi-layered nanoparticles as vaccine candidates. As detailed herein, we explored the cause of EBOV GP metastability and designed a multi-layered NP vaccine for in vivo evaluation. To facilitate GP purification, we developed an immunoaffinity column based on mAb100 specific for native trimeric GP. We first characterized two regions in GP2, namely the HR2 (so-called "stalk") region and the heptad repeat 1C (HR1 C We investigated the contribution of the HR1 stalk to GP metastability in a mucin-deleted Zaire EBOV GP construct (GPΔmuc). We extended the HR2 stalk to residue 637 and introduced the W615L mutation based on a comparison of the EBOV and MARV GPs. C We assessed whether proline mutations in HR1 could prevent the pre- and post-fusion conformational changes. C Both -proline mutations increased trimer yields, but the latter appeared to have complex effects on the thermal stability of GP. Disulfide bonds (SS) between GP molecules were also found to increase trimer stability. We determined the crystal structures of two redesigned GPΔmuc constructs and compared the stalk mutations and HR1 mutations. C We verified the -proline mutation at the atomic level. We then presented the redesigned GPΔmuc trimer to ferritin, E2p, and I3-01 NPs. Locking domains (LDs) and helper T cell epitopes were incorporated into the E2p and I3-01 60-mers to stabilize the NP shell from the inside, creating multilayered NP carriers.

[0029] In mice and rabbits, it was observed that GP trimers and NPs could induce different antibody responses. Next-generation sequencing (NGS) of GP-specific B cells showed different profiles for NPs presenting large trimer spikes and NPs presenting small antigens such as the Hepatitis C virus (HCV) E2 core. These studies demonstrate the key factors of EBOV GP metastability and enable two single-component multilayer self-assembling NPs for designing VLP-type vaccines, providing an EBOV vaccine candidate that warrants further evaluation in NHPs and humans.

[0030] In accordance with these studies, the present invention provides novel engineered Ebola virus GP sequences that contain various modifications disclosed herein. Some embodiments of the present invention are directed to immunogenic polypeptides and polynucleotide vaccines derived from the redesigned Ebola virus GP sequences described herein. Also provided in the present invention are Ebola virus vaccine compositions that contain a presentation platform comprising self-assembling nanoparticles that present one or more of the engineered Ebola virus GP immunogens. Therapeutic uses of engineered Ebola virus GP immunogenic polypeptides and related nanoparticle vaccine compositions, such as treating or preventing Ebola virus infection, are also provided in the present invention.

[0031] Unless otherwise specified herein, the vaccine immunogens, encoding polynucleotides, expression vectors and host cells of the present invention, as well as related therapeutic applications, can all be produced or performed according to the procedures exemplified herein or routinely practiced methods well known in the art, such as, for example, Methods in Enzymology, Volume 289:Solid-Phase Peptide Synthesis, JN Abelson, MI Simon, GB Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906); US Pat. Nos. 4,965,343 and 5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3 rdBrent et al., Current Protocols in Molecular Biology, John Wiley&Sons, Inc. (ringbou ed., 2003); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, SLBerger and ARKimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells:A Manual of Basic Technique by R. Ian Freshney,Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998). The following sections provide further guidance for carrying out the compositions and methods of the invention.

[0032] II. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1 st Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3 rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1 st Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4 th ed., 2000). Further clarification of some of these terms that apply specifically to the present invention is provided herein.

[0033] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, "Env-derived trimer" can refer to both a single or multiple Env-derived trimer molecules and can be considered equivalent to the phrase "at least one Env-derived trimer."

[0034] As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, capable of inducing an immune response in a subject. The terms also refer to a protein that is immunologically active in the sense that it is capable of eliciting a humoral and / or cellular type immune response against the protein when administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein). Unless otherwise indicated, the term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogenic polypeptide."

[0035] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that code for identical or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. With respect to a polypeptide sequence, "conservatively modified variants" refers to variants that have conservative amino acid substitutions, amino acid residues replaced with other amino acid residues having side chains with similar charges. Families of amino acid residues having side chains with similar charges have been defined in the art and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0036] Epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic, eliciting a specific immune response, for example, an epitope is the region of an antigen to which B and / or T cells respond. Epitopes can be formed both from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0037] An amount of a vaccine or other agent that is effective enough to produce a desired response, such as reducing or eliminating a sign or symptom of a condition or disease, such as a viral infection. For example, this can be the amount necessary to inhibit viral replication or measurably change the outward symptoms of a viral infection. Generally, this amount is sufficient to measurably inhibit the replication or infectivity of a virus (e.g., Ebola virus). A dosage that achieves a target concentration that, when administered to a subject, has been shown to be sufficient for in vitro inhibition of viral replication is generally used. In some embodiments, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or underlying causes of any of the disorders or diseases, e.g., treating Ebola virus infection. In some embodiments, an effective amount is a therapeutically effective amount. In some aspects, an effective amount is an amount that prevents the onset of one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with Ebola virus infection.

[0038] As used herein, a fusion protein is a recombinant protein that contains amino acid sequences from at least two unrelated proteins linked together via peptide bonds to create a single protein. The unrelated amino acid sequences can be linked directly to each other or can be linked using a linker sequence. As used herein, proteins are unrelated if their amino acid sequences are not normally found linked together via peptide bonds in their natural environment (e.g., inside a cell). For example, the amino acid sequences of a bacterial enzyme, such as B. stearothermophilus dihydrolipoyl acyltransferase (E2p) and the amino acid sequence of Ebola virus GP are not normally found linked together via peptide bonds.

[0039] The term "Ebola virus" refers to members of the Filoviridae family and is associated with outbreaks of highly fatal hemorrhagic fever in humans and non-human primates. Human Ebola virus pathogens include Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), and Tai Forest virus (TAFV). Reston virus (RESTV) is a monkey pathogen and is not currently considered a human pathogen. There is no known natural reservoir for Ebola virus, and currently no available vaccine or effective therapeutic treatment for Ebola virus infection. The Ebola virus genome consists of a single strand of negative-sense RNA approximately 19 kb in length. This RNA contains seven consecutively arranged genes that produce eight mRNAs upon infection. The Ebola virion contains seven proteins: the surface glycoprotein (GP), the nucleoprotein (NP), four virion structural proteins (VP40, VP35, VP30, and VP24), and an RNA-dependent RNA polymerase (L) (Feldmann et al. (1992) Virus Res. 24, 1-19; Sanchez et al., (1993) Virus Res. 29, 215-240; reviewed in Peters et al. (1996) In Fields Virology, Third ed. pp. 1161-1176. Fields, BN, Knipe, DM, Howley, PM, et al. eds. Lippincott-Raven Publishers, Philadelphia).

[0040] The term "Ebola virus glycoprotein (GP)" refers to the only surface antigen of Ebola virus that is expressed as a trimer on the viral surface. It is unusual in that it is encoded in two open reading frames. Transcriptional editing is required to express the transmembrane form that is incorporated into virions (Sanchez et al. (1996) Proc. Natl. Acad. Sci. USA 93, 3602-3607; Volchkov et al, (1995) Virology 214, 421-430). The unedited form produces a nonstructural secreted glycoprotein (sGP) that is synthesized in large amounts early in the infection process. The GP of Ebola virus is a transmembrane glycoprotein (GP) that is involved in both receptor binding and membrane fusion. During virus assembly, the glycoprotein undergoes proteolytic cleavage by host proteases such as furin, resulting in two subunits, GP1 and GP2, linked by disulfide bonds. The GP1 subunit (amino acids 33–501) contains a glycoprotein core, a receptor-binding domain (RBD), a glycan cap, and a large mucin-like domain (MLD) that spans the RBD. The GP2 (amino acids 502–676) subunit contains an internal fusion loop (IFL), heptad repeats 1 and 2 (HR1 and HR2), a membrane-proximal external region (MPER), a transmembrane region (TM), and a cytoplasmic tail (CT). During transport of Ebola virus particles to late endosomes, low pH leads to proteolytic processing of GP by host cysteine ​​proteases such as cathepsins, and the exposed receptor-binding site of the proteolytically digested GP is thought to interact with the host receptor Niemann-Pick C1, followed by membrane fusion. Unless otherwise stated, the amino acid residue numbering of GP from Zaire Ebola virus strain Mayinga-76 is used herein as a reference, with the complete genomic sequence identified by the GenBank ID of AF272001, the GP ectodomain sequence of which (UniProt ID Q05320; GenBank ID AAG40168.1) is set forth herein in SEQ ID NO:1.

[0041] As used herein, corresponding residues refer to residues present at aligned loci. Related or variant polypeptides are aligned by any method known to those of skill in the art (such methods typically include methods that maximize matches and use manual alignment, as well as methods using the many alignment programs available (e.g., BLASTP) and other programs known to those of skill in the art). By aligning the sequences of the polypeptides, one of skill in the art can identify corresponding residues using conserved and identical amino acid residues as a guide. For example, by aligning the sequences of GP polypeptides from different Ebola virus species or different isolates of the same species, one of skill in the art can identify corresponding residues using conserved and identical amino acid residues as a guide. Corresponding positions can also be based on structural alignments, for example, by using computer-simulated alignments of protein structures.

[0042] Immunogen as used herein refers to a protein or portion thereof capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of the immunogen can result in protective and / or prophylactic immunity against the pathogen of interest.

[0043] An immune response refers to a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In some aspects, the response is specific for a particular antigen (an "antigen-specific response"). In some embodiments, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In some other embodiments, the response is a B cell response, resulting in the production of a specific antibody.

[0044] An immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against a virus expressing the immunogenic polypeptide or that induces a measurable B cell response (such as the production of antibodies) against the immunogenic polypeptide.

[0045] Sequence homology or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the homology or similarity between the sequences. Sequence homology can be measured in terms of the percentage of homology, with the higher the percentage, the more closely the sequences are identical. Two sequences are "substantially identical" when they have a specified percentage of amino acid residues or nucleotides that are considered to be identical (i.e., 60% homology over a specified region, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% homology, or if not specified, homology over the entire sequence), when compared and aligned for maximum correspondence over a window of comparison, or when compared and aligned over a designated region as determined using one of the sequence comparison algorithms described below, or when compared and aligned over a designated region as determined by manual alignment and visual inspection. Optionally, the region of homology spans a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably, a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0046] Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence homology / similarity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Also, Altschul et al., J. Mol. Biol. 215:403-10, 1990, provides a detailed discussion of sequence alignment methods and homology calculations.

[0047] The term "subject" refers to any animal classified as a mammal, e.g., humans, and non-human mammals. Examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Unless otherwise indicated, the terms "patient" and "subject" are used interchangeably herein. Preferably, the subject is a human.

[0048] The terms "treat" or "alleviate" include administration of a compound or agent to a subject to prevent or delay the onset of symptoms, complications, or biochemical signs of a disease (e.g., Ebola virus infection), alleviate the symptoms, or halt or inhibit further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder and those at risk of developing the disorder. Treatment can be prophylactic (to prevent or delay the onset of the disease or to prevent its clinical or subclinical manifestations) or for the therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0049] A vaccine refers to a pharmaceutical composition that induces a preventative or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response against a pathogen, e.g., an antigen of a viral pathogen, or a cellular component that correlates with a pathological condition. A vaccine may comprise a polynucleotide (e.g., a nucleic acid encoding an Ebola virus GP polypeptide disclosed herein), a peptide or polypeptide (e.g., an Ebola virus GP polypeptide antigen disclosed herein), a virus, a cell, or one or more cellular components. In some embodiments of the invention, a vaccine or vaccine immunogen or vaccine composition is expressed from a fusion construct and self-assembles into a nanoparticle that displays an immunogenic polypeptide or protein on its surface.

[0050] Virus-like particle (VLP) refers to a non-replicating viral shell derived from any of several viruses. VLPs are generally composed of one or more viral proteins, including but not limited to proteins called capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of proteins in an appropriate expression system. Methods for producing specific VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art (e.g., electron microscopy, biophysical characterization, etc.). See, e.g., Baker et al. (1991) Biophys. J. 60:1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and / or identified by characteristic density banding. Alternatively, cryo-electron microscopy can be performed on a vitrified aqueous sample of the VLP preparation in question and images recorded under appropriate exposure conditions.

[0051] Self-assembling nanoparticles refer to ball-shaped protein shells with diameters of tens of nanometers and well-defined surface shapes formed by identical copies of non-viral proteins that can automatically assemble into nanoparticles with a similar appearance to VLPs. Known examples include ferritin (FR), which is conserved across species and forms a 24-mer, as well as B. stearothermophilus dihydrolipoyl acyltransferase (E2p), Aquifex aeolicus lumazine synthase (LS), and Thermotoga maritima encapsulin, which all form 60-mers. Self-assembling nanoparticles can form spontaneously upon recombinant expression of proteins in an appropriate expression system. Methods for the production, detection, and characterization of nanoparticles can be performed using the same techniques developed for VLPs. III. Engineered Ebola virus GP sequences

[0052] The present invention provides engineered or redesigned Ebola virus glycoprotein (GP) sequences (polypeptide or polynucleotide sequences) for producing Ebola virus vaccines. Compared to the wild-type GP sequence, these engineered GP polypeptides may contain various modifications, particularly in the GP2 subunit. The GP2 subunit of Ebola virus GP contains an N-terminal peptide, an internal fusion loop, two consecutive heptad repeat regions (HR1 and HR2), a membrane proximal external region (MPER) and a C-terminal transmembrane domain (see FIG. 1A). HR1 in turn mediates the HR1 fusion loop, which is a fusion loop that mediates the GP2 fusion loop, and a C-terminal transmembrane domain. A、 HR1 B、 HR1 Cおよび HR1 D The first two segments, HR1, A and HR1 B (residues 554-575) forms an α-helix with a twist of about 40° at Thr565, which is related to HR1. A HR1 B Depict from HR1 C is HR1 B and HR1 D An extended coil linker is formed between the segments. HR1 D forms an amphipathic helix, and the hydrophobic faces of the three helices in the trimer come together to form the peplomer interface. C and HR1 D were initially defined to correspond to residues 576–582 and 583–598, respectively (Lee et al., Future Virol. 2009; 4: 621–635). Based on the higher resolution and more complete crystal structure of GP reported in Zhao et al. Nature 535, 169-172, 2016, HR1 was C is redefined herein to include aa 576-583, and HR1 Drefers to aa 584 to 598. HR2 is a mostly alpha-helical section of the protein, also called the "HR2 stalk", that connects the GP core to the viral membrane. In the EBOV sequence, the HR2 stalk encompasses amino acid residues 599 to 632.

[0053] As detailed herein, the engineered Ebola virus GP polypeptide is primarily involved in HR1. C and HR2 motifs (FIG. 1B). Unless otherwise stated, the organization and amino acid numbering for the various domains or regions of Ebolavirus GP are based on the GP sequence of Zaire Ebolavirus strain Mayinga-76, which has an ectodomain sequence described by GenBank ID AAG40168.1 (SEQ ID NO: 1). Due to substantial sequence conservation in structural motifs among different Ebolaviruses and different strains of the same Ebolavirus species, the corresponding amino acid residues flanking the various domains, regions and loops of Ebolavirus GP from any other Ebolavirus and strains can be readily determined (e.g., by sequence alignment) or are otherwise known in the art. As an illustration, a sequence alignment of the HR1 and HR2 regions of EBOV with other Ebolaviruses is shown in FIG. 2. The sequence of the entire HR1+HR2 region is about 84.8% identical across five different species: EBOV, SUDV, TAFV, BDBV and RESTV. As shown in the figure, the HR1 and HR2 regions of EBOV are ... CThe residues in the segment are highly conserved among different Ebolaviruses. The first few N-terminal residues in the MPER motif for HR2 extension described herein are also highly conserved among different Ebolavirus species. The W615 residue exemplified herein for substitution is conserved in all Ebolavirus species. Furthermore, the logo analysis of the sequence conservation of the HR2 stalk described in the following examples showed that W615 is 100% conserved across all strains of all six species of the Ebolavirus genus. Therefore, it is clear that the Ebolavirus GP sequence modifications exemplified in EBOV can be easily applied to other Ebolavirus species and different strains of the same virus species.

[0054] Compared to a full-length wild-type Ebola virus GP (see, e.g., FIG. 1A), an engineered Ebola virus GP sequence of the invention typically contains structural motifs corresponding to the GP ectodomain, excluding the N-terminal leader, MLD, and MPER, in addition to the mutations or modifications described herein (see, e.g., SEQ ID NO: 2). In various embodiments, an engineered Ebola virus GP sequence of the invention can include additional structural motifs or domains of the full-length GP beyond the structural components present in SEQ ID NO: 2. Thus, some engineered Ebola virus GP sequences of the invention can further contain one or more of: (1) a leader sequence, (2) part or all of the MLD, (3) part or all of the MPER, (4) part or all of the transmembrane domain, and (5) part or all of the cytoplasmic tail.

[0055] Some embodiments of the present invention are directed to engineered Ebola virus GP sequences that correspond to full-length Ebola virus GP with one or more stabilizing modifications or mutations in the HR2 and HR1 regions described herein. In some embodiments, the engineered Ebola virus GP sequence may contain a substitution at residue W615 in HR2 of the wild-type GP sequence. In some of these embodiments, the W615 residue may be replaced with a small hydrophobic residue, e.g., L, A, V, I, or F. Additionally or alternatively, the engineered Ebola virus GP sequence may contain a substitution at residue W615 in HR1. C As exemplified herein, HR1 may have one or more proline substitutions in the segment. C Any of the residues in the segment, T576, T577, E578, L579, R580, T581, F582 and S583, may be substituted with proline. In some of these embodiments, the engineered Ebola virus GP sequence is HR1. C The modified Ebola virus GP protein comprises a polynucleotide sequence having a cysteine ​​substitution at the 5' end of the polynucleotide sequence, e.g., SEQ ID NO: 41, which encodes a leader peptide. In some embodiments, the ...

[0056] In some embodiments, the engineered Ebola virus GP sequence of the invention contains only the ectodomain (i.e., MPER at the C-terminus) or otherwise soluble portion of the Ebola virus GP protein, along with the modifications of the HR1 and HR2 regions described herein. In some of these embodiments, the truncated or modified soluble Ebola virus GP sequence also lacks the MLD. In some embodiments, the truncated soluble GP sequence further lacks the MPER at the C-terminus of the GP ectodomain. In various embodiments, the expressed and assembled trimeric protein also does not contain the leader peptide sequence (SEQ ID NO:41). An example of such a truncated GP soluble sequence based on the Zaire Ebola virus (EBOV) GP ectodomain sequence (SEQ ID NO:1) is shown in SEQ ID NO:2, which has the leader, MLD and MPER sequences deleted from the wild-type ectodomain sequence. In some embodiments, the truncated soluble GP sequence further contains a T42A mutation in the GP1 base motif (GP1 muc; SEQ ID NO:3).

[0057] In addition to truncations at the C-terminus, the engineered soluble Ebola virus GP sequences of the invention typically contain additional sequence modifications in the HR1 and HR2 regions of the soluble GP sequence (e.g., SEQ ID NO: 2 or 3). In various embodiments, the additional sequence modifications include substitution of residue W615 in HR2, (1) adding one or more flanking residues in the MPER, or (2) extending HR2 by replacing several C-terminal HR2 residues with a longer heterologous sequence, e.g., replacing residues W615 in HR2 ... C These additional sequence modifications include the substitution of one or more residues in the segment with proline and the introduction of one or more disulfide bonds. As described herein, these additional sequence modifications, alone or in any combination, can promote GP trimerization, reduce metastability, and stabilize the Ebola virus GP in a native-like trimeric conformation.

[0058] In some embodiments, the engineered Ebola virus GP sequence of the invention contains a truncated or shortened soluble GP sequence (e.g., SEQ ID NO:2 or SEQ ID NO:3, or conservatively modified or substantially identical variants thereof) with additional modifications at HR2. In the exemplified EBOV ectodomain GP sequence (SEQ ID NO:1), the HR2 motif encompasses residues 599-632. In some of these embodiments, the inwardly facing residue in the three-dimensional structure W615 is replaced with a smaller, more hydrophobic amino acid residue. In various embodiments, the W615 residue can be replaced with Leu, Phe, Ala, Val, or Ile to improve packing of the HR2 stalk. These substitutions are intended to stabilize the Ebola virus GP trimer. In some exemplary embodiments, the sequence substitution is W615L. In some other embodiments, HR2 can contain an additional substitution at residue 612 in addition to the W615 substitution. In some of these embodiments, the amino acid substitution is P612G / W615F. Some specific examples of engineered Ebola virus GP sequences containing the W615 substitution are shown in SEQ ID NOs:4 and 5.

[0059] In addition to substitutions in HR2, the engineered soluble Ebola virus GP sequence can alternatively or additionally contain an extension of the HR2 motif. In some of these embodiments, the extension of the HR2 motif (ending at residue 632) comprises the addition of one or more adjacent residues in the MPER motif (starting at residue 633) naturally occurring in the Ebola virus GP sequence. In various embodiments, the HR2 extension can be the addition of its C-terminal adjacent residues in MPER up to or beyond residues 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646. In some exemplary embodiments, the HR2 extension comprises an extension to the TACE cleavage site in MPER (residue 637), i.e., with the MPER N-terminal residues KTLPD (SEQ ID NO: 32). The TACE cleavage site is involved in GP shedding from the virion surface following host TACE cleavage. In some other embodiments, the HR2 extension can include six additional flanking residues in the MPER motif, i.e., with the N-terminal residues KTLPDQGDNDN (SEQ ID NO: 33) of MPER. In some embodiments, the extension of the HR2 motif includes replacing some of the C-terminal residues of HR2 with a longer heterologous sequence. In some of these embodiments, some of the C-terminal residues of HR2 can be replaced with a longer GCN4 leucine zipper sequence. For example, HR2 can be extended by replacing residues 617-632 in HR2 with a 31 aa sequence from the GCN4 leucine zipper, having PDB ID 2WPZ (SEQ ID NO: 34), as exemplified herein. Some specific embodiments of engineered Ebola virus GP proteins containing HR2 extensions are shown in SEQ ID NOs: 5-7. Specific embodiments of engineered Ebola virus GP proteins containing both an HR2 extension and a residue W615 substitution are shown in SEQ ID NOs: 6 and 8.

[0060] In addition to or instead of the above modifications in the HR2 region, the engineered Ebola virus GP protein of the present invention may further comprise a modification of the C segment of the HR1 region (HR1 C ) can also contain a mutation in HR1.C The mutation in contains one or more proline substitutions that can stabilize the GP trimer and reduce metastability. In various embodiments, the proline substitutions are C As exemplified herein with the EBOV GP sequence (SEQ ID NO:1), a proline substitution can be introduced at each of residues 576 to 583 (TTELRTFS; SEQ ID NO:35). Thus, in various embodiments, an engineered Ebola virus GP protein of the invention can contain one or more of the following HR1C mutations: T576P, T577P, E578P, L579P, R580P, T581P, F582P, and S583P. In some exemplary embodiments, an engineered Ebola virus GP protein of the invention has an HR1C mutation of T577P (P 2) Or L579P(P 4) Specific embodiments of engineered Ebola virus GP sequences containing one or more proline substitutions in the HR1C segment are shown in SEQ ID NOs:9-16.

[0061] In some embodiments, the engineered Ebola virus GP sequence of the invention comprises the HR1 C It contains both a proline substitution at HR1 and a modification at HR2. For example, an engineered GP protein may contain a proline substitution as well as a W615 substitution and / or a further HR2 extension to the MPER. The proline substitution is C The W615 substitution can be any residue in the MPER (e.g., residues 577 or 579). The W615 substitution can be either W615L, W615F, W615A, W615V, or W615I. These HR2 extensions in these engineered GP proteins can be, for example, extensions to residue 637 in MPER (i.e., the TACE cleavage site). HR1 C Specific embodiments of engineered Ebola virus GP sequences containing both a proline substitution at and also an HR2 modification are shown in SEQ ID NOs: 17-22.

[0062] Other than the modifications in the heptad region described above, some engineered Ebola virus GP sequences of the present invention may alternatively or additionally contain one or more engineered cysteine ​​residues for forming inter-GP disulfide bonds. By forming disulfide bonds between adjacent promoters of the GP trimer, these Cys substitutions and the resulting engineered disulfide bonds may similarly function to promote trimer formation and stabilize the GP in a native-like trimer conformation. Depending on the particular GP sequence used, suitable positions for introducing one or more inter-GP disulfide bonds can be easily determined. This can be done, for example, through analysis of potential amino acid pairs in the three-dimensional structure of the GP, followed by biochemical and immunological characterization as described herein. As exemplified in the EBOV GP sequence (SEQ ID NO:1) herein, an engineered GP trimer protein may contain one or more engineered interprotomer SS bonds between G91 / A575 (SS2), F153 / Y534 (SS1), T520 / A575 (SS3), G157 / I532 (SS4), D522 / A575 (SS5) and K56 / G599 (SS6). In some embodiments, an engineered GP trimer protein of the invention contains an engineered disulfide bond at G91 / A575. Specific embodiments of engineered Ebola virus GP proteins containing such engineered disulfide bonds are shown in SEQ ID NOs:23-28.

[0063] In some embodiments, the engineered Ebola virus GP sequence of the present invention can contain a C-terminal trimerization motif. This motif functions to further stabilize the trimer and also to increase the trimer ratio within the total protein yield. Trimerization motifs suitable for the present invention include, for example, the T4 fibritin foldon (PDB ID: 4NCV) and the viral capsid protein SHP (PDB: 1TD0). T4 fibritin (foldon) is well known in the art and constitutes the C-terminal 30 amino acid residues of the trimeric protein fibritin from bacteriophage T4 and functions to promote fibritin folding and trimerization. See, for example, Papanikolopoulou et al., J. Biol. Chem. 279: 8991-8998, 2004; and Guthe et al., J. Mol. Biol.337:905-915,2004. Similarly, SHP proteins and their use as functional trimerization motifs are also well known in the art. See, e.g., Dreier et al., Proc Natl Acad Sci USA 110:E869-E877, 2013; and Hanzelmann et al., Structure 24: 140-147, 2016. In some exemplary embodiments, the trimerization motif in the engineered GP protein comprises the Foldon sequence shown in SEQ ID NO:29 or the 1TD0 protein sequence shown in SEQ ID NO:30. In some other embodiments, the trimerization motif used may contain a conservatively modified variant of the exemplified sequences or a sequence that is substantially identical (e.g., at least 90%, 95% or 99% identical) sequence. In some embodiments, the trimerization motif may be inserted with a short GS linker. In various embodiments, the linker may contain 1-6 tandem repeats of GS. In some embodiments, a His6 tag can be added to the C-terminus of the trimerization motif to facilitate protein purification, for example, by using a nickel column.

[0064] In addition to the specific examples of GP polypeptides set forth in SEQ ID NOs: 4-28, engineered Ebolavirus GP sequences of the invention also encompass sequences having an amino acid sequence substantially identical to one of these sequences, including conservatively modified variant sequences. In various embodiments, an engineered Ebolavirus GP protein of the invention can have an amino acid sequence that is identical to any of SEQ ID NOs: 4-28, except for one or more amino acid residue substitutions of non-conserved residues in HR1 and HR2 between different Ebolavirus species or strains, or substitutions in non-conserved regions or motifs of the GP sequences of different Ebolavirus species or strains.

[0065] As exemplified herein with EBOV strain Mayinga-76 (SEQ ID NO:1), GP sequences from different Ebola virus species and strains can all be easily used to generate engineered Ebola virus GP sequences according to the strategies described herein. Ebola viruses suitable for the present invention include any of EBOV, SUDV, TAFV, BDBV and RESTV, as well as any strain of a given Ebola virus species. In some embodiments of the present invention, the engineered Ebola virus GP proteins are chimeric. These chimeric Ebola virus GP proteins or immunogenic polypeptides can contain chimeric GP sequences with different subunits or domains derived from multiple Ebola virus species or multiple strains of the same Ebola virus species. For example, the GP1 and GP2 subunit sequences in a chimeric engineered Ebola virus GP sequence can be derived from two different Ebola virus species (e.g., GP2 from EBOV and GP1 from SUDV or TAFV) or two different strains of the same Ebola virus species (e.g., two different EBOV strains).

[0066] Many GP sequences of the five different Ebolavirus species and many different strains of a given Ebolavirus species are known in the art, and such sequence information can be readily obtained from, for example, the Ebola database maintained by Los Alamos National Laboratory (LANL) and other Ebolavirus-related databases, such as the Virus Pathogen Resource (ViPR). See, for example, Kuiken et al., Nucleic Acids Res. 40: D587-92, 2012; Pickett et al., Nucleic Acids Res. 40: D593-98, 2012; and Swetha et al., Adv Bioinformatics. 2016: 1673284. Clearly, there is a significant degree of variability between the GP sequences of different Ebolavirus species and different strains of the same Ebolavirus species. However, as noted above, there are a number of conserved residues and motifs in HR1 and HR2 of all Ebola virus species, and these are positions at which mutations or modifications may be introduced into the engineered GP proteins of the present invention. In engineering the Ebola virus GP proteins of the present invention, suitable residues for modification can be readily determined through sequence alignment, also taking into account the conserved Ebola virus GP motifs and residues known in the art. For example, Leroy et al., J. General Virol. 83: 67?73, 2002; Arslan et al., Bioinformatics. 32: 151-154, 2017; Wec et al., Cell 169: 878?890, 2017; Jun et al., FEMS Microbiol Rev. 39: 764?778, 2015; Pappalardo et al., Sci. Rep. 6: 23743, 2016; and Ruedas et al., J. Virol. 91:e00392-17, 2017.Engineered Ebola virus GP sequences based on any of the other Ebola virus species and strains, or combinations of different strains or species, can all be generated using the same strategy as exemplified herein for the EBOV GP sequence (SEQ ID NO:1).

[0067] As detailed below, the engineered Ebola virus GP protein can be conjugated to a display platform (e.g., a nanoparticle or VLP) via various means. Preferably, conjugation is achieved via covalent linkage, e.g., protein fusion or insertion. In some preferred embodiments, the protein sequence is fused to the display platform sequence via a linker sequence. In various embodiments, other modifications can also be made to the engineered Ebola virus GP protein or binding partner to improve stability or antigenicity.

[0068] The various engineered Ebola virus GP molecules of the present invention can be obtained or produced according to the protocols exemplified herein or methods known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3 rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). Upon recombinant expression (e.g., in HEK293 F cells as detailed herein), the protein can be purified by any of the routine procedures. See, for example, Guide to Protein Purification, Ed. Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982. Substantial purification means purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Once purified, the antigenicity and other properties of the engineered Ebola virus GP protein can also be readily examined using standard methods, such as antigen profiling using known bNAbs and non-NAbs, differential scanning calorimetry (DSC), electron microscopy, binding analysis by ELISA, biolayer interferometry (BLI), surface plasmon resonance (SPR) and co-crystallography as exemplified herein. IV. Scaffolded Ebola GP Vaccine Compositions

[0069] The present invention provides Ebola virus GP-based vaccine compositions that contain a heterologous scaffold that displays or incorporates the engineered Ebola virus GP protein described herein. Any heterologous scaffold can be used to display the engineered Ebola virus GP protein in the construction of the vaccine of the present invention. These include nanoparticles, virus-like particles, protein carriers (e.g., immunoglobulin chains or domains such as Fc, KLH, BSA, tetanus toxoid, and diphtheria toxoid), and various chemical scaffolds. In some embodiments, bacteriophage Qβ Virus-like particles (VLPs), such as VLPs and nanoparticles, can be used. In some preferred embodiments, the heterologous scaffold for presenting or displaying the engineered Ebola virus GP protein is a nanoparticle. A variety of nanoparticle platforms can be used in the generation of the vaccine composition of the present invention. In general, the nanoparticles used in the present invention should be formed by multiple copies of a single subunit. The nanoparticles are typically ball-like in shape and / or have rotational symmetry (e.g., with three-fold and five-fold axes), e.g., icosahedral structures as exemplified herein. Additionally or alternatively, the amino termini of the particle subunits must be exposed and close to the three-fold axis, and the spacing of the three amino termini must closely match the spacing of the carboxyl termini of the Ebola virus GP protein. In some preferred embodiments, the immunogen comprises a self-assembled nanoparticle with a diameter of about 20 nm or less (usually constructed from 12, 24, or 60 subunits) and a three-fold axis on the particle surface.

[0070] In some preferred embodiments, the engineered Ebola virus GP protein is displayed on a self-assembling nanoparticle, such as the self-assembling nanoparticles derived from E2p, I3-01v9, and ferritin (FR) exemplified herein. E2p is a redesigned variant of dihydrolipoyl acyltransferase from Bacillus stearothermophilus that has been shown to self-assemble into thermostable 60-mer nanoparticles. See, e.g., He et al., Nat. et al., Commun. 7:12041, 2016. Similarly, I3-01 is an engineered protein that can self-assemble into ultrastable nanoparticles. See, e.g., Hsia et al., Nature 535, 136-139, 2016. Here, I3-01v9, a modified version of I3-01, is used as an example. Ferritin is a globular protein found in all animals, bacteria, and plants. The globular form of ferritin is composed of monomeric subunit proteins (also referred to as monomeric ferritin subunits), which are polypeptides having a molecular weight of about 17-20 kDa. The amino acid sequences of E2p, I3-01v9 and ferritin nanoparticle subunits exemplified herein are shown in SEQ ID NOs:36-38, respectively. Compared to the original sequence, the E2p sequence shown in SEQ ID NO:36 contains an Ala substitution at residue 92, as underlined in the sequence below. The sequences of several other suitable nanoparticle sequences are also known in the art (see, e.g., WO2017 / 192434, WO2019 / 089817 and WO19 / 241483). In various embodiments, the Ebola virus nanoparticle vaccine of the present invention can use any of these known nanoparticles, as well as conservatively modified variants thereof or variants having substantially identical (e.g., at least 90%, 95% or 99% identical) sequences.

[0071] E2p subunit sequence (SEQ ID NO:36) AAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTAIDDETEEIIQKHYYNIGIAADTDRGLLVP VIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM

[0072] I3-01v9 subunit sequence (SEQ ID NO:37) MKMEELFKKHKIVAVLRANSVEEAKMKALAVFVGGVHLIEITFTVPDADTVIKELSFLKELGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKE KGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTIAEVAAKAAAFVEKIRGCTE

[0073] Ferritin sequence (SEQ ID NO:38) DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK

[0074] In addition to these exemplary nanoparticle sequences, many other nanoparticles or VLPs known in the art can be used in the practice of the invention, including, for example, Aquifex aeolicus lumazine synthase, Thermotoga Maritima encapsulin, Myxococcus xanthus encapsulin, bacteriophage Q beta virus particles, Flock House virus (FHV) particles, ORSAY virus particles, and infectious bursal disease virus (IBDV) particles. Other molecules that can be used as presentation platforms for the nanoparticle vaccines of the invention include, for example, molecules with the following PDB IDs: 1JIG (12-mer Dlp-2 from Bacillus anthracis), 1UVH (12-mer DPS from Mycobacterium smegmatis), 2YGD (24-mer ocular lens chaperone alpha B-crystallin), 3CS0 (24-mer DegP24), 3MH6 and 3MH7 (24-mer HtrA protease), 3PV2 (12-mer HtrA homolog DegQ WT), 4A8C (12-mer DegQ from E. coli), 4A9G (E. coli), 4EVE (12-mer HP-NAP from Helicobacter pylori strain YS29), and 4GQU (24-mer HisB from Mycobacterium tuberculosis).

[0075] In various embodiments, the Ebola virus GP protein displayed on the nanoparticle platform may optionally contain a trimerization motif as described above, e.g., Foldon or SHP. Some Ebola virus nanoparticle vaccine compositions may further contain other structural components that function to further enhance the stability and antigenicity of the displayed immunogen. In some embodiments, a locking protein domain may be inserted into the nanoparticle construct, e.g., by covalent fusion to the C-terminus of the nanoparticle subunit. The locking domain may be any dimeric protein that can form an interface through specific interactions such as hydrophobic (van der Waals) contacts, hydrogen bonds, and / or salt bridges. General guidelines and specific examples for the selection of locking domains are described in the art, e.g., PCT2019 / 036917. In some exemplary embodiments, the locking domain used in the Ebola virus nanoparticle vaccine of the present invention may contain the locking domains LD4 or LD7 exemplified herein.

[0076] In some embodiments, the Ebola virus nanoparticle vaccine of the present invention can also contain a T cell epitope to promote a robust T cell response and direct B cell development to bNAb. The T cell epitope can be located at any position relative to other structural components, as long as it does not affect the presentation of the immunogenic polypeptide on the nanoparticle surface. Any T cell epitope sequence or peptide known in the art can be used in the practice of the present invention. They include any polypeptide sequence that contains an MHC class II epitope and can effectively activate CD4+ and CD8+ T cells upon immunization, for example, a T helper epitope that activates CD4+ T helper cells. See, e.g., Alexander et al., Immunity 1, 751-761,1994;Ahlers et al., J. Clin. Invest. 108:1677-1685, 2001;Fraser et al., Vaccine 32, 2896-2903, 2014;De Groot et al., Immunol. Cell Biol. 8:255-269, 2002;and Gene Ther. 21: 225-232, 2014. In some embodiments, the T cell epitope inserted into the Ebola virus nanoparticle vaccine construct is a universal pan-DR epitope peptide (PADRE). See, for example, Hung et al., Mole. Ther. 15: 1211-19, 2007;Wu et al., J. Biomed. Sci. 17: 88, 2010;and Bissati et al., npj Vaccines 2: 24, 2017. Other examples of suitable T cell epitopes, such as D and TpD epitopes, have also been described in the art (Fraser et al., Vaccine 32, 2896-2903, 2014). In some embodiments, the PADRE peptide used contains the sequence AKFVAAWTLKAAA (SEQ ID NO: 31), a conservatively modified variant thereof, or a substantially identical (e.g., at least 90%, 95% or 99% identical) sequence.In some of these embodiments, the PADRE epitope is inserted into the C-terminus of a locking domain, such as LD4 or LD7 exemplified herein. In some of these embodiments, a GS restriction site can be added between the LD and the PADRE epitope.

[0077] The scaffolded Ebola virus vaccine compositions of the invention can be made according to standard recombinant techniques and methods described herein (see, e.g., the Examples herein) and / or other methods described in the art, e.g., He et al., Nat. Comm. 7, 12041, 2016; Kong et al., Nat. Comm. 7, 12040, 2016; He et al., Sci Adv. 4(11):eaau6769, 2018; and PCT publications WO2017 / 192434, WO2019 / 089817, and WO19 / 241483. In various embodiments, nanoparticles displaying any of the engineered Ebola virus GP proteins can be constructed by fusing an Ebola virus GP polypeptide to a nanoparticle subunit (e.g., an E2p subunit). Preferably, the C-terminus of the Ebola virus GP polypeptide is fused to the N-terminus of the nanoparticle subunit. In some embodiments, a short peptide spacer can be used to connect the Ebola virus GP polypeptide to the nanoparticle. For example, the spacer may have a GS restriction site and / or a longer G4S (SEQ ID NO: 43) or (G4S)2 (SEQ ID NO: 42) linker, as exemplified herein. Some embodiments of the invention utilize engineered Ebola virus GP protein GP-muc-WL with various combinations of nanoparticle subunit sequences, locking domain sequences and / or T cell epitopes. 2 P 2 The present invention relates to nanoparticles presenting the protein (SEQ ID NO: 17). Some specific examples of such nanoparticle vaccine compositions include GPΔmuc-WL2P2-AS-G4S-ferritin, GPΔmuc-WL2P2-AS-E2p-LD4-PADRE, and GPΔmuc-WL2P2-AS-(G4S)2-I3-01v9-LD7-PADRE.

[0078] Once constructed, the antigenicity and structural integrity of the nanoparticle-displaying Ebola virus GP polypeptide can be readily analyzed by standard assays, such as antibody binding assays, biolayer interferometry, and negative staining electron microscopy (EM). As exemplified herein, a variety of fusion molecules can all self-assemble into nanoparticles that display immunogenic epitopes of the Ebola virus GP protein. By eliciting a strong neutralizing antibody response, these nanoparticles are useful for vaccinating individuals against a wide range of Ebola virus infections. V. Polynucleotides and Expression Constructs

[0079] The engineered Ebola virus GP proteins and nanoparticle vaccine compositions of the invention are typically produced by first generating an expression construct (i.e., an expression vector) that contains operably linked coding sequences for the various structural components described herein. Thus, in some related aspects, the invention provides polynucleotides (DNA or RNA) that encode engineered Ebola virus GP proteins or polypeptides and that encode subunits of nanoparticles that display engineered Ebola virus GP polypeptides as described herein, as well as expression vectors carrying such polynucleotides, and host cells (e.g., HEK293 F cells and ExpiCHO cells exemplified herein) for producing Ebola virus GP immunogenic polypeptides and vaccine compositions. Fusion polypeptides encoded by polynucleotides or expressed from vectors are also included in the invention.

[0080] Polynucleotides and associated vectors can be readily prepared using standard molecular biology techniques or protocols exemplified herein. For example, general protocols for cloning, transfection, i.e., for transient gene expression or stable transfected cell lines, can be found in the art, e.g., in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, (3 rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou edition, 2003. Introduction of mutations into polynucleotide sequences by PCR is described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0081] The selection of a particular vector depends on the intended use of the fusion polypeptide. For example, the selected vector must be capable of driving the expression of the fusion polypeptide in the desired cell type, whether that cell type is prokaryotic or eukaryotic. Many vectors contain sequences that allow both prokaryotic vector replication and eukaryotic expression of the operably linked gene sequence. The vectors useful in the present invention can replicate autonomously, i.e., the vector exists extrachromosomally and its replication is not necessarily directly related to the replication of the genome of the host cell. Alternatively, the replication of the vector can be linked to the replication of the host chromosomal DNA, for example, the vector can be integrated into the chromosome of the host cell, as is achieved by retroviral vectors and in stably transfected cell lines. Both viral-based and non-viral expression vectors can be used to produce immunogens in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors that typically carry expression cassettes for expressing proteins or RNA, and human artificial chromosomes (see, for example, Harrington et al., Nat. Genet. 15:345, 1997). Useful viral vectors include lentivirus or other retrovirus-based vectors, adenovirus, adeno-associated virus, cytomegalovirus, herpes virus, SV40-based vectors, papillomavirus, HBP Epstein-Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, e.g., Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0082] Depending on the particular vector used to express the fusion polypeptide, various known cells or cell lines can be used in the practice of the invention. The host cell can be any cell into which a recombinant vector carrying the fusion of the invention can be introduced, the vector being capable of driving the expression of the fusion polypeptide useful in the invention. It can be prokaryotic, such as any of a number of bacterial strains, or eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including, for example, rodent, monkey or human cells. The cell expressing the fusion polypeptide of the invention can be a primary cell or an established cell line. Thus, in addition to the cell lines exemplified herein (e.g., CHO cells), many other host cell lines that may be well known in the art can also be used in the practice of the invention. These include, for example, various Cos cell lines, HeLa cells, HEK293, AtT20, BV2 and N18 cells, myeloma cell lines, transformed B cells and hybridomas.

[0083] The use of mammalian tissue cell cultures to express polypeptides is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Fusion polypeptide expression vectors can be introduced into selected host cells by any of a number of suitable methods known to those skilled in the art. The method used to introduce a vector encoding a fusion polypeptide into a mammalian cell depends on the form of the vector. In the case of a plasmid vector, the DNA encoding the fusion polypeptide sequence can be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection ("lipofection"), DEAE-dextran mediated transfection, electroporation, or calcium phosphate precipitation. These methods are detailed, for example, in Brent et al., supra. Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs into eukaryotic cells, particularly mammalian cells, in culture. For example, LipofectAMINE™ (Life Technologies) or LipoTaxi™ (Stratagene) kits are available. Other companies that provide reagents and methods for lipofection include Bio-Rad Laboratories, Clontech, Glen Research, Life Technologies, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

[0084] For long-term, high-yield production of recombinant fusion polypeptides, stable expression is preferred. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with the fusion polypeptide coding sequence controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selection marker. The selection marker in the recombinant vector confers resistance to selection and allows the cell to stably integrate the vector into its chromosome. Commonly used selection markers include neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J.Mol.Biol., 150:1, 1981); and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30:147, 1984). With appropriate selection, the transfected cells can contain an integrated copy of the fusion polypeptide coding sequence. VI. Pharmaceutical Compositions and Therapeutic Uses

[0085] The present invention provides pharmaceutical or immunogenic compositions and related methods using nanoparticles presenting engineered Ebola virus GP sequences and GP proteins as described herein for preventing and treating Ebola virus infection. In some embodiments, nanoparticles presenting engineered Ebola virus GP sequences (polypeptide or polynucleotide sequences) or engineered proteins are included in a pharmaceutical composition. The pharmaceutical composition can be either a therapeutic or prophylactic formulation. Typically, the composition further comprises one or more pharmaceutically acceptable vehicles, and optionally other therapeutic components (e.g., antibiotics or antivirals). Various pharmaceutically acceptable additives can also be used in the composition.

[0086] Some of the pharmaceutical compositions of the present invention are vaccines. For vaccine compositions, suitable adjuvants can be further included. Examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL™ and IL-12. In some embodiments, the engineered Ebola virus GP sequences and related vaccines disclosed herein can be formulated as controlled release or sustained release formulations. This can be achieved in compositions containing slow release polymers, or via microencapsulated delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 19.sup.th Ed., Mack Publishing Company, Easton, Pa., 1995; Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978); U.S. Pat. Nos. 4,652,441 and 4,917,893; U.S. Pat. Nos. 4,677,191 and 4,728,721; and U.S. Pat. No. 4,675,189.

[0087] The therapeutic methods of the invention include administering a pharmaceutical composition having an engineered Ebola virus GP sequence or polypeptide of the invention to a subject having or at risk of developing an Ebola virus infection (e.g., EBOV infection). In some embodiments, the pharmaceutical composition of the invention is used in therapeutic or prophylactic applications to treat an Ebola virus infection in a subject or to induce a protective immune response against an Ebola virus species or strain. For example, the composition can be administered to a subject to induce an immune response against an Ebola virus species, e.g., to induce the production of broadly neutralizing antibodies against an Ebola virus species. A subject at risk of developing an Ebola virus infection can be administered a vaccine composition of the invention to provide prophylactic protection against the viral infection. Depending on the particular subject and condition, the pharmaceutical composition of the invention can be administered to a subject by various modes of administration known to those skilled in the art, for example, intramuscular, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, or parenteral routes. In general, the pharmaceutical composition is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate a selected disease or condition or one or more symptoms thereof. Symptoms of Ebola virus exposure or infection include, for example, liver inflammation, loss of appetite, fatigue, abdominal pain, jaundice, flu-like symptoms, itching, muscle and joint pain, intermittent low-grade fever, sleep disorders, nausea, indigestion, cognitive changes, depressive headaches, and mood changes.

[0088] Typically, the immunogenic compositions of the invention are administered in an amount sufficient to induce an immune response against the Ebola virus. For therapeutic applications, the composition should contain a therapeutically effective amount of the engineered Ebola virus GP sequence or nanoparticle vaccine composition described herein. For prophylactic applications, the composition should contain a prophylactically effective amount of the nanoparticles presenting the engineered Ebola virus GP sequence or GP protein. The appropriate amount of the polypeptide immunogen or nanoparticle composition can be determined based on the particular disease or condition being treated or prevented, the severity of the subject, the age, and other personal attributes of the particular subject (e.g., the general state of the subject's health and the robustness of the subject's immune system). The determination of an effective dosage is further guided by an administration protocol that significantly reduces the occurrence or severity of the target disease symptom or condition in the subject, guided by animal model studies followed by human clinical trials.

[0089] For prophylactic applications, the immunogenic composition is provided prior to any symptoms, e.g., prior to infection. Prophylactic administration of the immunogenic composition serves to prevent or ameliorate any subsequent infection. Thus, in some embodiments, the subject being treated is a subject who has or is at risk of developing an Ebola virus infection, e.g., due to exposure or potential exposure to the Ebola virus. After administration of a therapeutically effective amount of the disclosed therapeutic composition, the subject can be monitored for Ebola virus infection, symptoms associated with Ebola virus infection, or both.

[0090] For therapeutic applications, the immunogenic compositions are provided at or after the onset of symptoms of disease or infection, e.g., after the onset of symptoms of Ebola virus infection or after diagnosis of Ebola virus infection. Thus, the immunogenic compositions can be provided prior to expected exposure to Ebola virus, after exposure or suspected exposure to the virus, or after the actual onset of infection to reduce the expected severity, duration, or extent of infection and / or associated disease symptoms.

[0091] The pharmaceutical compositions of the present invention can be combined with other agents known in the art for treating or preventing Ebola virus infection. Administration of the pharmaceutical composition and the known antiviral agent can be either simultaneous or sequential.

[0092] A pharmaceutical composition containing an engineered Ebola virus GP protein or nanoparticle vaccine of the invention can be provided as a component of a kit. Optionally, such a kit will include additional components including packaging, instructions and various other reagents (e.g., buffers, substrates, antibodies or ligands (e.g., control antibodies or ligands), and detection reagents). Optional instructions can further be provided in the kit. VII. Sequences of some engineered Ebola virus GP polypeptides or structural motifs EBOV GP ectodomain sequence (GenBank AAG40168.1) (SEQ ID NO:1): MGVTGILQLP RDRFKRTSFFLWVIILFQRTFSI-PLGVIHN STLQVSDVDK LVCRDKLSST NQLRSVGLNL EGNGVATDVP SATKRWGFRS GVPPKVVNYE AGEWAENCYN LEIKKPDGSE CLPAAPDGIR GFPRCRYVHK VSGTGPCAGD FAFHKEGAFF LYDRLASTVI YRGTTFAEGV VAFLILPQAK KDFSSHPLR EPVNATEDPS SGYYSTTIRY QATGFGTNET EYLFEVDNLT YVQLESRFTP QFLLQLNETI YTSGKRSNTT GKLIWKVNPE IDTTIGEWAF WETKKNLTRK IRSEELSFTV VSNGAKNISG QSPARTSSDP GTNTTTEDHK IMASENSSAM VQVHSQGREA AVSHLTTLAT ISTSPQSLTT KPGPDNSTHN TPVYKLDISE ATQVHHRR TDNDSTASDT PSATTAAGPP KAENTNTSKS TDFLDPATTT SPQNHSETAG NNNTHHQDTG EESASSGKLG LITNTIAGVA GLITGGRRTR REAIVNAQPK CNPNLHYWTT QDEGAAIGLA WIPYFGPAAE GIYIEGLMHN QDGLICGLRQ LANETTQALQ LFLRATTELR TFSILNRKAI DFLLQRWGGT CHILGPDCCI EPHDWTKNIT DKIDQIIHDFVD - KTLPDQGD NDNWWTGWRQ WIPAGIGVTG VIIAVIALFC ICKFVF Shortened EBOV GP ECTO Sequence (Leader / MLD / MPER deletion) (SEQ ID NO:2): PLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVD Leaderless GPΔmuc (leader / MLD / MPER deletion + T42A GP ECTO )(SEQ ID NO: 3) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVD Leaderless GPΔmuc-W615L (SEQ ID NO: 4) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTG PCAGDFAFHKEGAFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTT QDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEEPHDLTKNITDKIDQIIHDFVD Leaderless GPΔmuc-2WPZ (612 / 615 double mutation + 2WPZ extension) (SEQ ID NO:5) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEGHDFTKQLEDKVEENLSKVYHNENEVARLKKLVGER Leaderless GPΔmuc-L (C-terminal extension to residue 637) (SEQ ID NO: 6) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPD Leaderless GPΔmuc-Ext (C-terminal extension to residue 643) (SEQ ID NO: 7) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPC AGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYT SGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIG LAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDN Leaderless GPΔmuc-W615L-L- (also known as GPΔmuc-WL 2 -foldon) (SEQ ID NO: 8) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAF HKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLI WKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIE GLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNITDKIDQIIHDFVDKTLPDASGYIPEAPRDGQAYVRKDGEWVLLSTFL Leaderless GPΔmuc-W615L-P1(T576P) (SEQ ID NO: 9) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWYPIFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRAPTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNIDTKIDQIIHDFVD リーダーレスGPΔmuc-W615L-P2(T577P) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATPELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNIDTKIDQIIHDFVD Leaderless GPΔmuc-W615L-P3(E578P) (SEQ ID NO: 11) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTG PCAGDFAFHKEGAFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTT QDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTPLRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEEPHDLTKNITDKIDQIIHDFVD Leaderless GPΔmuc-W615L-P4 (L579P) (SEQ ID NO: 12) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWYPIFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTEPRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNIDTKIDQIIHDFVD リーダーレスGPΔmuc-W615L-P5(R580P) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWYPFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELPTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNIDTKIDQIIHDFVD PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWYPFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRPFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNIDTKIDQIIHDFVD リーダーレスGPΔmuc-W615L-P7(F582P) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWYPIFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTPSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNIDTKIDQIIHDFVD Leaderless GPΔmuc-W615L-P8(S583P) (SEQ ID NO: 16) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTG PCAGDFAFHKEGAFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTT QDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFPILNRKAIDFLLQRWGGTCHILGPDCCIEEPHDLTKNITDKIDQIIHDFVD Leaderless GPΔmuc-W615L-L-P2 (SEQ ID NO: 17) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATPELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNITDKIDQIIHDFVDKTLPD Leaderless GPΔmuc-W615L-L-P2 + restriction site “AS” and Foldon (SEQ ID NO: 18) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATPELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNITDKIDQIIHDFVD KTLPDASGYIPEAPRDGQAYVRKDGEWVLLSTFL Leaderless GPΔmuc-W615L-L-P2 + restriction site "AS" + G4S linker + 1TD0 (SEQ ID NO: 19) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRG TTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTH HQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATPELRTFSILNRKAIDFLLQRWGGTCHILG PDCCIEPHDLTKNITDKIDQIIHDFVDKTLPDASGGGGSEVRIFAGNDPAHTATGSSGISSPTPALTPLMLDEATGKLVVWDGQKAGSAVGILVLPLEGTETALTYYKSGTFATEAIHWPESVDEHKKANAFAGSALSHAA Leaderless GPΔmuc-W615L-L-P4 (also known as GPΔmuc-WL 2 P 4 ) (SEQ ID NO: 20) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGP CAGDFAFHKEGAFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNET IYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDE GAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTEPRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEEPHDLTKNITDKIDQIIHDFVDKTLPD Leaderless GPΔmuc-W615L-L-P4 + restriction site "AS" and foldon (SEQ ID NO:21) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTEPRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNITDKIDQIIHDFVDKTLPDASGYIPEAPRDGQAYVRKDGEWVLLSTFL Leaderless GPΔmuc-W615L-L-P4 + restriction site "AS" + G4S linker + 1TD0 (SEQ ID NO: 22) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRG TTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTH HQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTEPRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDLTKNITDKIDQIIHDFVDKTLPDASGGGSEVRIFAGNDPAHTATGSSGISSPTPALTPLMLDEATGKLVVWDGQKAGSAVGILVLEGTETALTYYKSGTFATEAIHWPESVDEHKKANAFAGSALSHAA リーダーレスGPΔmuc-SS1(Y534C / F153C,rC β -C β =3.819A) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFACHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPCFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVD リーダーレスGPΔmuc-SS2(A538C / G91C,rC β -C α =3.994A) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSCVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLWIPYFGPCAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVD リーダーレスGPΔmuc-SS3(T520C / A575C,rC β -C β =4.008A) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTCQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRCTTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVD リーダーレスGPΔmuc-SS4(I532C / G157C,rC β -C α =4.202A) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKECAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWCPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVD リーダーレスGPΔmuc-SS5(D522C / A575C,rC β -C β =4.410A) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQCEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRCTTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVD リーダーレスGPΔmuc-SS6(K56C / G599C,rC β -C α =4.597A) PLGVIHNSALQVSDVDCLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTG PCAGDFAFHKEGAFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLN ETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTT QDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGCTCHILGPDCCIEEPHDWTKNITDKIDQIIHDFVD Foldon (SEQ ID NO: 29) GYIPEAPRDGQAYVRKDGEWVLLSTFL: 1TD0 (SEQ ID NO: 30) EVRIFAGNDPAHTAGSSGISSPTPALTPLMLDEATGKLVVWDGQKAGSAVGILVLPLEGTETALTYYKSGTFATEAIHWPESVDEHKKANAFAGSALSHAA: GCN leucine zipper motif (SEQ ID NO:34) KQLED KVEEN LSKVY HNENE VARLK KLVGE R Locking domain LD4 (SEQ ID NO: 39): FSEEQKKALDLAFYFDRRLTPEWRRYLSQRLGLNEEQIERWFRRKEQQIGWSHPQFEK Locking domain LD7 (SEQ ID NO: 40): SPAVDIGDRLDELEKALEALSAEDGHDDVGQRLESLLRRWNSRRAD Leader sequence of EBOV GP (SEQ ID NO:41) MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSI EXAMPLES

[0093] The following examples are offered to illustrate, but not to limit, the present invention. Example 1. Label-free immunoaffinity purification of EBOV GP trimers

[0094] EBOV GP has a highly glycosylated mucin-like domain (MLD) that shields the glycan cap and neutralizing epitopes in GP1 and GP2. A soluble mucin-deleted form of Zaire EBOV GP (GPΔmuc), which generates a high-resolution crystal structure (Zhao et al., Nature 535: 169-172, 2016), was used as a base construct to investigate the metastability of GP. In HIV-1 vaccine research, immunoaffinity chromatography (IAC) columns based on bNAb 2G12 and PGT145 (67, 68) have been widely used for the purification of native envelope (Env) trimers. 2G12 targets a single glycan patch on gp120, while PGT145 binds to the trimer apex and can separate the closed trimer from the partially open and misfolded Env. These two bNAb columns have also been used to purify NPs presenting gp140 of HIV-1. Such GP-specific antibody columns have not been reported in EBOV vaccine studies. Recently, Corti et al. identified two potent NAbs, mAb114 and mAb100, from human survivors (Science 351, 1339-1342, 2016). Misasi et al. elucidated the structural basis of neutralization by mAb114, which targets the receptor binding site (RBS), and mAb100, which interacts with the GP1 / GP2 interface and internal fusion loop (IFL) of the two GP subunits (Science 351, 1343-1346, 2016).

[0095] Here, we investigated the utility of mAb114 and mAb100 as IAC columns. GPΔmuc constructs with and without the C-terminal trimerization motif foldon were transiently expressed in 250 ml HEK293F cells and purified on an antibody column followed by size-exclusion chromatography using a Superdex 200 10 / 300 GL column and blue native polyacrylamide gel electrophoresis (BN-PAGE). With mAb114, both GPΔmuc samples showed aggregate (from 9 ml), dimer (from 12 ml) and monomer (from 13.5-14 ml) peaks in the SEC profile, but only GPΔmuc foldon showed a visible trimer peak (from 10.5-11 ml) on SEC and a faint band slightly smaller than 440 kD on the BN gel. After mAb100 purification, the yield of GPΔmuc was generally low, but GPΔmuc-foldon showed high trimer purity and had no dimer or monomer peaks. Consistently, GPΔmuc-foldon left a single trimer band on BN gels. In summary, both mAb114 and mAb100 columns can be used for GP purification, but mAb100 recognizes the quaternary structure of the GP epitope, making it an effective IAC method for purifying GP trimers closer to the native form. The significant difference in trimer yield between the two soluble GPΔmuc constructs suggests a strong tendency for trimer dissociation. Example 2 Effect of HR2 stalk on EBOV GP metastability

[0096] EBOV GP has a long and extended HR2 stalk. Even in the high-resolution GPΔmuc structure, the HR2 stalk still has less helical structure than most coiled coils in the database, from 15 aa pairs to 30 aa, and unwinds toward the C-terminus, suggesting inherent instability in HR2. Recently, King et al. solved the structure of the MARV GPΔmuc trimer in complex with a therapeutic human mAb, MR191 (Cell Host Microbe 23, 101-109.e104, 2018), at 3.17 Å resolution. Unexpectedly, the coiled coil is well formed in the MARV HR2 stalk, with canonical side chain packing along the three folding axes. To identify the source of this difference, we obtained EBOV and MARV GP sequences from the Viral Pathogens Database and Analysis Resource (ViPR). A total of 274 EBOV GPs and 87 MARV GPs were used for sequence conservation analysis, spanning the CX6CC motif and the HR2 stalk, from aa 601 to 632 in EBOV and aa 602 to 633 in MARV, respectively. Most of the inward-facing amino acids were conserved, except for W615 in EBOV or L616 in MARV. Indeed, structural analysis revealed a crucial difference at this position, with the three W615s in EBOV GP (PDB: 5JQ3) forming a wide-angle triangle at the neck of the coiled coil, with a Cα distance of 11.1 Å and a Cβ distance of 9.0 Å. In contrast, the smaller and more hydrophobic L616 was observed to have a Cα distance of 10.5 Å and a Cβ distance of 8.3 Å in MARV GP (PDB: 6BP2).

[0097] Based on this finding, we hypothesized that the W615L mutation might stabilize the EBOV GP trimer. To further explore the effect of the stalk, we generated three GPΔmuc constructs, but replacing aa 617-632 with a GCN4 leucine zipper (PDB:2WPZ,aa 3-33) and extending the C-terminus to 637 and 643, containing a newly identified bNAb epitope (King et al., Nat Commun 10, 1788, 2019) spanning HR2 and the membrane proximal external region (MPER), termed "L" and "Ext", respectively (Figure 3A). The double mutation P612G / W615F was introduced into the GPΔmuc-2WPZ construct between the CX6CC motif and the coiled-coil to reduce any structural distortion at this site. These constructs were characterized by SEC and BN-PAGE after transient expression in 250 ml 293F cells and mAb114 purification. Indeed, all three designs increased trimer yields, with GPΔmuc-2WPZ showing the most visible trimer peak in SEC (Figure 3B). Consistently, trimer bands were observed for all three constructs on BN gels, although they were less intense for GPΔmuc-L and -Ext (Figure 3C, left). Upon mAb100 purification, all three GPΔmuc mutants exhibited a more intense trimer band than wild-type GPΔmuc (Figure 3C, right), supporting the idea that the HR2 stalk is important for GP trimer stability.

[0098] Next, we identified GPΔmuc-W615L-L-foldon or simply GPΔmuc-WL 2 The W615L mutation and the "L" extension were combined in a single construct designated GPΔmuc-foldon. This construct, together with GPΔmuc-foldon, was transiently expressed in 1 liter of 293F cells and purified using a mAb100 column followed by purification on a HiLoad Superdex 200 16 / 600 GL column. In three production runs, GPΔmuc-WL 2The -foldon consistently outperformed the wild-type construct, showing a 2-fold higher trimer peak in the SEC profile and approximately 2.6-fold higher trimer yield after SEC (1.3 mg vs. 0.5 mg). Thermal stability was assessed by differential scanning calorimetry (DSC) for the two purified GP trimers. The thermal denaturation midpoint (Tm) of the stalk-stabilized trimer was 3°C higher than that of the wild-type trimer (67°C vs. 64°C). Consistently, stalk stabilization also increased the onset (Ton) from 52.4°C to 62.5°C, resulting in a narrower half-width peak (ΔT 1 / 2 ) were observed. Antigenicity of the four mAb100 / SEC-purified EBOV GP trimers was assessed in an enzyme-linked immunosorbent assay (ELISA) (Figure 3D-E). A panel of 10 antibodies was used, including three NAbs targeting the base (KZ52, c2G4, and c4G7), two human NAbs - mAb100 (IFL) and mAb114 (RBS), a non-NAb targeting the glycan cap (c13C6), and four pan-Ebola virus bNAbs targeting the HR2-MPER epitope (BDBV223) and IFL (ADI-15878, ADI-15946, and CA45). GPΔmuc trimers were GP ECTO The antibody binding was significantly improved compared to the trimer, and the half-maximal effective concentration (EC 50 ) showed a maximum difference of 7.6-fold, indicating that MLD can effectively shield the GP from antibody recognition. Two HR2 stalk modifications further enhanced the recognition of the RBS by mAb114, CA45, and IFL, respectively, but did not enhance the recognition of the EC 50 The values ​​showed only moderate changes. EC 50 Approximately 40% of the GP-muc-WL in BDBV223 was reduced. 2 -foldon binding. Collectively, the two HR2 stalk mutations can effectively improve the trimer yield, thermostability, and antigenicity of EBOV GP. Example 3. Effect of HR1 on EBOV GP metastability C Bend Effects

[0099] HR1 CWe hypothesized that HR1 of wild-type EBOV GP is essential for the metastability of EBOV GP. C The truncated HR1 in the prefusion-optimized HIV-1 Env N Since the length of the EBOV GP is equal to that of the HR1 gene (8 aa), the metastability of the EBOV GP is C It is likely that a different solution will be required rather than being affected by the length of HR1. C Proline mutation (P 1-8 ) is HR1 C We hypothesized that this would fix the bend in the EBOV GP trimer and improve the stability of the EBOV GP trimer.

[0100] To investigate this possibility, each C Eight GPΔmuc-W615L mutants, which carry a proline mutation at HR1 but lack the C-terminal L extension and foldon, were experimentally validated. All constructs were transiently expressed in 250 ml 293F cells and purified using a mAb114 column, which captures all GP species. C The proline mutations at most positions in the SEC profile were T577P (P 2 ) and L579P(P 4 In a separate experiment, all eight constructs were transiently expressed in 250 ml of 293F cells and purified using a mAb100 column. 2 and P 4 Only P 4 A significantly high SEC peak was observed at 100 nm, which corresponds to a well-formed trimer. 2 and P 4 Overall, the T577P mutant P 2 can substantially increase the yield of trimers, while the L579P mutant P 4 showed a less pronounced effect.

[0101] Next, the T577P mutation (P 2) to GPΔmuc-WL 2 -foldon construct, GPΔmuc-WL 2 P 2 The construct, designated GPΔmuc-WL-foldon, was obtained. This construct was transiently expressed in 1 liter of 293F cells and purified on a HiLoad Superdex 200 16 / 600 GL column using a mAb100 column for SEC characterization. In three actual runs, GPΔmuc-WL 2 P 2 -Foldon is GPΔmuc-WL 2 GPΔmuc-WL and GPΔmuc-WL showed two-fold and four-fold more trimer peaks than wild-type GPΔmuc-WL, respectively, with an average yield of 2.6 mg after SEC. Protein collected in the SEC range of 55.5 to 62.0 ml was analyzed by BN-PAGE, showing a trimer band across all fractions with no trace of impurities. 2 P 2 The thermal stability of the -foldon was measured by DSC after mAb100 and SEC purification.

[0102] Unexpectedly, two transition peaks were observed in the thermogram, one recorded at a lower Tm of 61.6 °C and the other at a higher Tm of 68.2 °C. To this end, GPΔmuc-WL 2 P 4 -The L579P mutation, called foldon (P 4 A second construct, bearing the proline-rigidified HR1, was also evaluated by DSC. Only one peak was observed in the thermogram with a Tm of 67.0 °C, but the slight broadening at the onset of the peak suggested similar unfolding behavior upon heating. Thus, DSC demonstrated that the proline-rigidified HR1 C This revealed the complexity of the bend in GP, ​​but may increase the yield of trimers at the cost of decreasing the thermal stability of GP. 2 P 2 The antigenicity of the -foldon was assessed using the same 10 antibodies by ELISA (Figure 3F-G) and biolayer interferometry (BLI).2 ) is GPΔmuc-WL 2 -foldon (Figure 3G) appears to improve GP binding for most antibodies, with the bNAb BDBV223 targeting HR2-MPER showing an EC 50 A 40% decrease in GFP-foldon activity was observed. Although the BLI profiles were nearly indistinguishable between wild-type and redesigned GPΔmuc-foldon trimers, both had fast on-rates and flat dissociation curves, with a 2-fold higher signal at the lowest concentration (12.5 nM) than GPΔmuc-WL. 2 P 2 -foldon binding to bNAb BDBV223, consistent with the ELISA data.

[0103] From the above, HR1 C Metastability of EBOV GP is important and unexpectedly HR1 for proline mutations C Recently, Rutten et al. C Proline mutations in HR1, along with the K588F mutation, were tested to stabilize filovirus GP trimers (Cell Rep. 30, 4540–50, 2020). A similar pattern of increased trimer yield was observed with the T577P mutant, but the reported thermostability data did not seem to match our DSC measurements. C Further studies are needed to fully understand the role of and its impact on GP stability. Example 4 GP stabilization by disulfide bond mutation

[0104] Because the EBOV GP already contains an endogenous disulfide bond (C53-C609) linking GP1 and GP2, we investigated whether disulfide bonds between the GPs could be used to promote trimer formation and lock the GP in a “closed” trimer. Based on the high-resolution EBOV GPΔmuc (PDB: 5JQ3), we found that C α -C αAmino acid pairs between GPs were identified that were within a cutoff of 4.72 Å spacing. A total of nine pairs were identified. After visual inspection, three pairs were removed due to potential interference with existing SS bonds or hydrophobic clusters. The remaining six pairs were divided into three groups: IFL-head group (SS1 / 2 / 4), IFL-NHR group (SS5), and HR2 group (SS6). Six GPΔmuc-SS constructs were designed and then characterized by SEC after transient expression in 250 ml 293F cells and mAb114 purification. Diverse SEC profiles were observed, with SS2 showing a substantial trimer peak, identified by a band slightly below 440 kD on BN gels. mAb100 purification was also analyzed by BN-PAGE, and trimer bands were observed for SS2, SS3, and SS5. Antigenicity was assessed for the three SS variants in ELISA using six antibodies. All three SS mutants outperformed wild-type GPΔmuc, but SS2 showed higher affinity for NAbs targeting the base and IFL. Taken together, SS bonds between appropriately positioned GPs can effectively stabilize the EBOV GP in a native-like trimeric conformation. Example 5. Crystallographic analysis of the redesigned EBOV GPΔΔmuc trimer.

[0105] Stoke and HR1 C To understand how the mutations affect the EBOV GP, we solved the crystal structure of the uncoordinated GPΔmuc foldon trimer, but WL 2 and W.L. 2 P 2 are 2.3 Å and 3.2 Å, respectively. Both proteins exhibit a three-lobed chalice-like structure, with Cα root-mean-square deviations (rmsd) of 0.92 to 1.14 Å relative to the high-resolution structure (PDB: 5JQ3) at the single subunit level. WL 2 P 2 In the glycan cap (R302-V310) and HR2 stalk (.I627-D637), WL 2 A more complete structure was obtained than with WL.2 P 2 In the structure, the glycan cap covers the RBS with glycan moieties found at N238 / N257 / N268 in GP1 and N563 in GP2, while WL 2 In the structure, the positions were N238 / N257 in GP1 and N563 / N618 in GP2. GP1 is composed mainly of β-strands and forms a wide semicircular groove that clamps the α3 helix and the β19-β20 strand in GP2. The T577P mutation is located at HR1. C This is expected to have minimal effect on the conformation of the bend, as the Cα rmsd for the 8-aa segment has been shown to be 0.19 Å.

[0106] W.L. 2 P 2 In the structure, the main chain carbonyl (CO) groups of R574, A575, and T576 of one subunit formed moderate to strong hydrogen bonds with the head group of the R164 side chain of the neighboring subunit, whereas only one CO-NH distance was within the 3.5 Å cutoff in wild-type GPΔmuc. The W615L mutation had a visible structural effect on the HR2 stalk. We speculated that the bulky inward-facing W615 at the top of the coiled coil destabilizes the HR2 stalk and that the W615L mutation improves its packing. Indeed, the Cα-Cα / Cβ-Cβ spacing between the W615 of two neighboring subunits in wild-type GPΔmuc is 11.1 / 9.0 Å, whereas the W615L spacing between the W615 of two neighboring subunits in wild-type GPΔmuc is 11.1 / 9.0 Å. 2 and W.L. 2 P 2 The EBOV HR2 stalk was reduced to 10.1 / 8.0 Å and 10.6 / 8.2 Å, respectively. As a result, the coiled-coil region of the EBOV HR2 stalk contains an additional helical turn, making it more similar to the MARV HR2 stalk. 2 P 2The "L" extension of the structure could be fully modeled for D637 as a well-ordered loop anchored to the C-terminal foldon motif, rendering the complete HR2 stalk and partial MPER. Superposition of the HR2 stalk yielded Cα rmsd values ​​of 1.46 Å, 2.05 Å and 1.85 Å for EBOV-Mayinga (PDB: 5JQ3), SUDV (PDB: 3S88) and BDBV (PDB: 6EA5) GPs, respectively, suggesting some structural variability in this region.

[0107] Next, W.L. 2 P 2 The structure was compared to the recently reported Makona GPΔmuc structure (PDB: 6VKM) containing the T577P / K588F mutations. 2 P 2 The structure matches 353 of 398 residues in the double mutant, with a Cα rmsd of 0.89 Å. A more complete cathepsin cleavage loop than in previous structures is found in the WL 2 P 2 (aa 197-210 vs. aa 193-213), suggesting that this loop bridges on IFL and interacts with NAbs directed against IFL, such as mAb100 (42). Furthermore, the electron density of the glycan cap (aa 294-310) and the β18 loop in the HR2 stalk was higher than in the double mutant. C For bends of WL 2 P 2 showed a more favorable hydrogen bonding pattern, with a Ca rmsd of 0.26 Å. A Ca rmsd of 1.7 Å was obtained for the IFL region between the two structures. Finally, WL 2 P 2 The structures were docked to a panel of known GP / antibody complexes. 2 P 2preserves all the important GP-antibody contacts. The mAb100 / GP complex is of most interest since mAb100 has been used for GP purification with substantial purity. Cryo-EM revealed additional electron density near the mAn100 light chain, possibly corresponding to a portion of the β13-β14 loop (aa 190-210) (Misari et al., Science 351: 1343-46, 2016). However, this density was not observed in the 6.7 Å resolution crystal structure of the same complex (PDB: 5FHC). WL 2 P 2 The structure shows density up to H197, which corresponds to the docked WL 2 P 2 The WL / mAb100 model was considered to be in the vicinity of the mAb100 light chain. Taken together, our structure validates the rationally designed mutations and provides an atomically detailed view of regions not available in previous structures. 2 P 2 The structure also potentially explains the increased trimer yield, although the origin of the two-peak thermogram remains unclear. Example 6 Display of EBOV GPΔΔmuc trimers on multilayer ultrastable nanoparticles

[0108] Self-assembling NPs provide an alternative to recombinant VLPs for vaccine development. We tested several protein NPs as "multivalent carriers" to present the redesigned GPΔmuc trimer for in vivo evaluation. Specifically, we modeled the GPΔmuc trimer structure on ferritin (FR), E2p, and I3-01, resulting in NPs presenting GP of 34.5 nm, 45.9 nm, and 49.2 nm, respectively. Superposition of the C-terminus of GPΔmuc onto the N-terminus of FR and E2p yielded Cα rmsds of 7.0 Å and 5.5 Å, respectively, suggesting that GPΔmuc could be fused to FR with a short G4S linker and to E2p without a linker. However, due to the large spacing between the N-terminus of I3-01 subunits (approximately 50.5 Å), it must be linked to the C-terminus of the GPΔmuc trimer by a long linker, resulting in the formation of a long, narrow stalk. Based on computational modeling, a 10-aa (G4S)2 linker was found to be sufficient, yielding a Cα rmsd of 0.8 Å. Here, we first characterize two GPΔmuc trimers, wild-type and WL 2 P 2 were presented on FR, E2P, and I3-01 with a 5-aa linker, no linker, and a 10-aa linker, respectively. All six GP-NP constructs were transiently expressed in 100 ml ExpiCHO cells, followed by mAb100 purification and SEC on a Superose 6 10 / 300 GL column. Overall, WL 2 P 2 had better NP yield and purity than wild-type GPΔmuc. Based on the molecular weight (mw), the SEC peak centered at 15 ml may represent a yet unorganized GP-NP species, suggesting inherent instability in wild-type E2p and I3-01. GPΔmuc-WL purified with mAb100 2 P 2 NP samples displaying were further analyzed by negative stain EM, which revealed that the NPs were contaminated with impurities.

[0109] Previously, we demonstrated the use of pan-reactive T cell epitopes both as linkers and as incorporated T cells in the HIV-1 NP construct based on I3-01 (55), suggesting that further structural and functional components can be incorporated into such a large 60-mer. Here, we sought to stabilize the E2p and I3-01 NPs via engineering. To this end, we fused a dimeric locking domain (LD) to the C-terminus of the NP subunit and then fused a T cell epitope to the C-terminus of the LD. We hypothesized that the LDs could stabilize the non-covalent NP formation interface and that the T cell epitopes could cluster at the NP core to induce a strong T cell response upon vaccination. To test this hypothesis, we selected nine LDs from 815 homodimers in the protein database (PDB) (Figure 4A). Based on structural modeling, LDs 1 to 7 were tested for E2P, and five LDs (4 to 5 and 7 to 9) were tested for I3-01, all of which were GPΔmuc-WL. 2 P 2 After transient expression in 100 ml of ExpiCHO cells and purification with mAb100, the NP sample containing 12 LDs was characterized by SEC. In particular, LD4 and LD7 were characterized by the peaks of NP (UV 280 Increasing the NP yield and purity (p<0.05) by 5-fold and 2.5-fold for E2p and I3-01, respectively, substantially improved the purity of the NPs. Further incorporation of the T cell epitope, PADRE, did not change or only slightly improved the NP yield and purity.

[0110] A total of seven GP-NP samples with three variants for each 60-mer were further analyzed by BN-PAGE. FR and the two E2p variants showed a single high molecular weight (mw) band corresponding to well-formed NP, whereas wild-type E2p and all three I3-01 samples showed a lower molecular weight (mw) band at 232-440 kD on the gel, which was considered to be a disorganized GP-NP species. mAb100 / SEC-purified GPΔmuc-WL 2 P 2 Samples of FR, E2P-LD4-PADRE (E2P-L4P) and I3-01-LD7-PADRE (I3-01-L7P) presenting WL were analyzed by negative stain EM. In addition to highly pure NPs for all three samples, well-formed arrays of GPΔmuc spikes were readily seen on the surface of FR and E2p-L4P NPs, consistent with SEC and BN-PAGE. Antigenicity was assessed for these three purified NP samples by ELISA against the same antibody panel (Figure 4B to C). Individual WL 2 P 2Compared to the trimer, the three NPs showed epitope-specific binding patterns. Overall, NP presentation seemed to improve antibody binding to the RBS and glycan cap in GP1 and reduce antibody binding to bNAbs targeting the base and IFL at the GP1 / GP2 interface and GP2 stalk. This finding raised the concern that these conserved bNAb epitopes at the base and IFL of GP on NP-presented trimers may not be as accessible as they are on the soluble trimer. To this end, BLI was performed to further probe antibody recognition of the various GP epitopes (Figure 4D to G). Comparing the molar concentrations of GP, the three NPs showed significantly higher binding signals than the soluble trimer, suggesting that NPs presenting GPs may be more effective in promoting BCR clustering via an avidity effect. However, three NPs based on FR, E2p-L4P and I3-01v9-L7P (Figure 4H), where I3-01v9 is a previously reported variant of I3-01, were selected for evaluation and comparison of trimers in vivo in mice and rabbits. Example 7 Immunogenicity of EBOV GP trimer and NP in BALB / c mice

[0111] BALB / c mice were immunized four times at 3-week intervals to obtain an initial readout of immunogenicity against the three EBOV GP / GPΔmuc trimers and the three NPs. ECTO The trimer, GP-foldon, was included to represent the wild-type GP (with MLD) used by EBOV vaccines in clinical trials. For the I3-01v9-L7P group, mice were immunized with 20 μg of mAb100-purified protein instead of 50 μg of mAb100 / SEC-purified protein due to the lower yield of this NP. We first evaluated GP-specific antibody responses in mouse serum, but not with GPΔmuc-WL. 2 P 2-1TD0 was used as a probe, where 1TD0 (PDB: 1TD0) is a trimerization motif (Figure 5A-C). Both GPΔmuc groups showed a significant increase in GPΔmuc expression by immunization (P < 0.0064). ECTO The MLD-treated GPΔmuc group was significantly superior to the WL group, suggesting that MLD can protect GP from antibody recognition. In contrast, little difference was observed between the two GPΔmuc groups, and the WL 2 P 2 EC is slightly higher at w2 and w5. 50 The mean titers are shown, which were reversed at later time points. 2 P 2 Compared with the trimer group, all NP groups had significantly lower EC at w2, except for the E2P-L4P NP group. 50 The titers were low, hence the P value was 0.0381.

[0112] This finding was somewhat unexpected, as a recent HCV vaccine study (He et al., Sci. Adv. 6: eaaz6225, 2020) found significant differences between the E2 core and NP groups at both w2 and w5, suggesting that antibody titers induced by antigen-presenting NPs may be significantly affected by antigen size, structure, and epitope distribution. NP display may block antibody access to base and stalk epitopes, which are targets of many bNAbs. This result may also be due to other factors, such as the dose. Since the NP carrier accounts for 21 to 33% of the total mass of the NP vaccine and the same dose (50 μg protein) was used for all vaccine groups except the I3-01v9-L7P NP group, mice in the NP group will receive significantly less GP antigen than mice in the trimer group.

[0113] We next validated the Ebolavirus pseudoparticle (Ebolavirus-pp) neutralization assay with a panel of 10 antibodies against EBOV-Makona and BDBV strains in 293T cells. As expected, the early EBOV NAbs KZ52, c2G4, and c4G7 neutralized EBOV but not BDBV. The NAbs mAb100 and mAb114 neutralized both Ebolavirus species with different potencies. Four bNAbs (three directed against IFL and one targeting HR2-MPER) cross-neutralized EBOV and BDBV and bound to the labeled lycan cap, while the non-NAb c13C6, part of the therapeutic antibody cocktail, seemed to enhance virus infection, suggesting a potential ADE effect. The Ebolavirus-pp assay was also performed in TZM-bl cells, which were used to screen small molecule inhibitors (Figure 5D). All NAbs / bNAbs tested blocked pseudovirus infection, but c13C6 showed a similar ADE pattern. When tested against pseudoparticles bearing murine leukemia virus (MLV) Env and MLV-pps, c13C6 showed enhanced infection, whereas NAbs and bNAbs remained non-reactive (Fig. 5E).

[0114] We next evaluated immunized mouse samples against EBOV and BDBV in an Ebola virus-pp assay. Immunoglobulin G (IgG) was purified from mouse serum at the last time point (w11) to remove nonspecific antiviral activity. Different patterns of antibody responses were observed, suggesting the induction of NAbs and non-NAbs causing ADE similar to c13C6. Among the three trimer groups, GP ECTO showed moderate NAb responses with ADE observed in 2 to 4 mice, whereas a substantial increase in both NAb and c13C6-like responses was observed in GPΔmuc, suggesting that removal of the MLD equally exposes NAb epitopes and glycan caps, which are the primary targets of antibodies causing ADE in natural infection. WL 2 P 2The mutations appeared to largely reverse the adversary effect of the loss of MLD. Among the three NP groups, E2p-L4P performed best, showing mostly NAb responses with few signs of ADE. However, c13C6-like antibody responses were observed in 2 to 3 mice in the FR group and in all mice in the I3-01v9-L7P group. Since c13C6 reacted with MLV-pps but not with NAb / bNAb (Figure 5E), we attempted to "gauge" the c13C6-like antibody responses induced by the different vaccines using the MLV-pp assay (Figure 5F). Indeed, we observed an enhancement of MLV-pp infection that was in close agreement with the ADE effect observed in the Ebola virus-pp assay. The MLV-pp analysis also demonstrated that E2p-L4P NPs were able to inhibit the GP with MLD shielding, which is a promising candidate for the ADE effect. ECTO The high levels of ADE effects observed for the GPΔmuc and I3-01v9-L7P groups appeared to be associated with the presence of open trimers and disorganized GP-NP species, respectively.

[0115] Mouse data provided important insights into the effect of different GP morphologies and NP carriers on vaccine-induced antibody responses. Briefly, multi-layered E2p NPs with 20 closed GPΔmuc trimers on their surface provide promising vaccine candidates. The benefits of NP presentation may not be fully assessed by binding antibody titers alone, but should be judged by the type of antibodies elicited. Antibodies similar to c13C6 that target the glycan cap and cross-react with small secreted GPs (ssGPs) may cause adverse effects in vaccination. Example 8 Immunogenicity of EBOV GP trimer and NP in rabbits

[0116] Two GPΔmuc trimers, wild type and WL 2 P 2 And WL 2 P 2The three trimer-presenting NPs were also evaluated in rabbits, with four injections at 3-week intervals. Rabbit sera collected at six time points during the immunization period were analyzed by ELISA using the same trimer probe (Figure 6A-B). Notably, immunization of rabbits with 20 μg of mAb100 / SEC-purified I3-01v9-L7P NPs reduced the c13C6-like response. Among the GPΔmuc groups, WL 2 P 2 The group had higher EC at all six time points except w11. 50 The EC titers were shown, and the P value obtained for w5 (2 weeks after the second injection) was 0.0229, indicating significance. A consistent pattern was observed among the three NP groups, with the I3-01v9 group showing the highest EC titers in this immunization. 50 The FR group showed the lowest score. Significant differences were observed between the I3-01v9-L7P and E2P-L4P groups at w8, w11, and w13 (4 weeks after the last injection), with P values ​​ranging from 0.0021 to 0.0053.

[0117] GPΔmuc-WL 2 P 2 Compared to the trimer group, the I3-01v9-L7P NP group had higher EC 50 The EC values ​​for w8, w11, and w13 were significant. In contrast, the FR and E2P-L4P groups had lower EC values ​​than the trimer groups at w2 and w5. 50 However, this trend was reversed at w8 and w11, with P values ​​indicating significance at w8 (0.0421 and 0.0492 for FR and E2P, respectively). However, the dominance of these two NP groups decreased at w11, with a lower EC than the trimer group at the last time point, w13. 50The titers of IgG purified from the pre, w2, w5, w8 and w11 time points were analyzed in Ebolavirus-pp and MLV-pp assays (Fig. 6C to F). At the last time point, all vaccine groups demonstrated NAb responses to both Ebolavirus species, with no signs of ADE, suggesting a different pattern compared to the mixed NAb / c13C6-like response in mice. Notably, the I3-01v9-L7P NP group had higher mean IC s for EBOV and BDBV than the other groups, at 211.3 μg / ml and 11.72 μg / ml, respectively. 50 (50% inhibitory concentration) titers, confirming that disorganized GP-NP species, and not NP itself, are responsible for ADE upon mouse immunization.

[0118] Consistently, all vaccine groups at w11 did not show enhanced MLV-pp infection, in contrast to the mouse data (Fig. 5F). Thus, c13C6-like antibodies appeared to be absent in the serum towards the end of rabbit immunization. However, rabbit IgG obtained at early time points showed a decline in c13C6-like antibody responses, along with an increase in Nab responses (Fig. 6C-F). In Ebolavirus-pp neutralization assays against EBOV-Makona, ADE was first observed at w2, w5, and w8 for the two trimer groups, the FR group, and the two multi-layered NP groups, but then disappeared at w5, w8, and w11, respectively. Thus, our analysis revealed a unique pattern of vaccine-induced c13C6-like antibodies in rabbits, which may change epitope specificity upon repeated antigenic challenge via a mechanism of "gene conversion." Example 9. B cell response profiles associated with EBOV GP trimers and NP

[0119] Previously, we combined antigen-specific B cell sorting and NGS to quantitatively measure the B cell responses induced by HCV E2 core (E2cm3) and E2cm3-E2p NPs (He et al., Sci. Adv. 6: eaaz6225, 2020). H) and V H The higher frequency of mutations and the wider range of heavy chain complementarity determining region 3 (HCDR3) lengths observed for E2p suggest the existence of a common mechanism in B cells for an effective NP vaccine. Can this mechanism be generalized to EBOV NP vaccines? Here, we applied the same strategy to obtain GP-specific B cell response profiles for different EBOV vaccine platforms. First, we used Avi-tagged GPΔmuc-WL to select GP-specific mouse splenic B cells. 2 P 2 A -1TD0 trimer probe was generated (Figure 7A). Sorted B cells from 25 mice, 5 per group, were subjected to NGS on an Ion GeneStudio S5 (Figure 7B). NGS data were analyzed with a mouse antibody omics pipeline (Figure 7C). Detailed B cell profiles were derived to facilitate comparison of the different vaccine platforms (Figures 7D-F).

[0120] GPΔmuc-WL 2 P 2 We focused mainly on the -foldon and multi-layered E2P groups and compared the B cell responses induced by soluble and NP-presented GPΔmuc. H and V K A similar pattern was observed for genes, i.e., the stabilized GPΔmuc trimer had more V H / V L Activate (9.4 / 9.4) and V H and V L The significant P values ​​for each were 0.0163 and 0.0076, respectively. In contrast, E2p NP modified with 60 HCV E2 cores showed more V H Activated, but V L The extent of somatic hypermutation (SHM) was not significantly different between the two groups. Nevertheless, there was a visible change in SHM distribution in the NP group, with germline V H / V KThe average diversification rate is 6.4% / 2.9%, This was higher than the average of 5.3% / 2.6% for the trimer group. In the analysis of HCDR3, two metrics were calculated for comparison: mean loop length and rms variation (rmsf) of loop length. Unlike the HCV vaccine study, where the rmsf of HCDR3 was found to have a P value of <0.0001 between the E2 core and NP groups, no significant difference was found between the EBOV trimer and NP groups, with E2P-L4P NP inducing antibodies with longer HCDR3 loops. Overall, EBOV and HCV NPs showed different B cell patterns for individual antigens. There was no clear correlation between the B cell profile and vaccine-induced NAb / c13C6-like responses. Our results suggest that antigen size, structure, glycosylation and epitope distribution other than multivalent NP display may also be important for shaping B cell responses. Example 9 Some Exemplary Materials and Methods

[0121] Expression and purification of EBOV GPΔmuc and GPΔmuc-displayed NP: Wild-type and redesigned GPΔmuc constructs were transiently expressed in HEK293F cells (Thermo Fisher) for biochemical, biophysical, and antigenic analyses. Briefly, 293F cells were thawed and incubated with FreeStyle™ 293 Expression Medium (Life Technologies, CA) in a shaking incubator at 37°C, 135 rpm, and 8% CO2. Cells were cultured at 2.0 × 10 6 When the cells reached a density of 1.0 × 10 / ml, expression medium was added for transfection with polyethyleneimine (PEI) (Polysciences) to bring the cell density to 1.0 × 10 6 ml -1The DNA-PEI-MAX complex was then added to 1 L of 293F cells. The culture supernatant was harvested 5 days after transfection, clarified by centrifugation at 2200 rpm for 22 min, and filtered using a 0.45 μm filter (Thermo Scientific). GPΔmuc protein was extracted from the supernatant using a mAb114 antibody column or a mAb100 antibody column. Bound proteins were eluted three times each with 5 ml of 0.2 M glycine (pH = 2.2) and neutralized with 0.5 ml of Tris base (pH = 9.0). Proteins were further purified by size-exclusion chromatography (SEC) on a Superdex 200 Increase 10 / 300 GL column or a HiLoad Superdex 200 16 / 600 column (GE Healthcare). GPΔmuc-displaying nanoparticles were produced in ExpiCHO cells (Thermo Fisher). Briefly, ExpiCHO cells were thawed and incubated in a shaking incubator at 37° C., 135 rpm and 8% CO2 for 1 h. TM The cells were incubated with expression medium (Thermo Fisher). 6 For transfection, the cell density was increased to 6 x 10 ml. 6 ml -1ExpiCHO™ expression medium was added to reduce the total volume of the plasmid to 100 ml. ExpiFectamine™ CHO / plasmid DNA complexes were prepared for 100 ml transfection in ExpiCHO cells according to the manufacturer's instructions. For these nanoparticle constructs, 100 μg of plasmid and 320 μl of ExpiFectamine™ CHO reagent were mixed in 7.7 ml of chilled OptiPRO™ medium (Thermo Fisher). After the first feed on day 1, ExpiCHO cells were cultured in a shaking incubator at 33° C., 115 rpm and 8% CO2, and fed additionally on day 5 according to the Max Titer protocol (Thermo Fisher). Culture supernatants were harvested 13 to 14 days after transfection, clarified by centrifugation at 4000 rpm for 25 min, and filtered using a 0.45 μm filter (Thermo Fisher). Nanoparticles were extracted from the supernatant using a mAb100 antibody column, followed by SEC on a Superose 6 10 / 300 GL column. Protein concentrations for GPΔmuc and GPΔmuc-displaying nanoparticles were determined by UV chromatography. 280 The absorbance was determined based on its theoretical extinction coefficient.

[0122] Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE): EBOV GPΔmuc and GPΔmuc-displaying nanoparticles were analyzed by blue native polyacrylamide gel electrophoresis (BN-PAGE) and stained with Coomassie Blue. The proteins were mixed with sample buffer and G250 loading dye and loaded onto 4-12% Bis-Tris NativePAGE™ gels (Life Technologies). BN-PAGE gels were run for 2-2.5 hours at 150 V using NativePAGE™ running buffer (Life Technologies) according to the manufacturer's instructions.

[0123] Enzyme-Linked Immunosorbent Assay (ELISA): Each well of a Costar™ 96-well assay plate (Corning) was first coated with 50 μl of PBS containing 0.2 μg of the appropriate antigen. The plate was incubated overnight at 4° C. and then washed five times with wash buffer containing PBS and 0.05% (v / v) Tween 20. Each well was then coated with PBS, 40 mg ml -1 Plates were coated with 150 μl of blocking buffer consisting of blotting grade blocker (Bio-Rad) and 5% (v / v) FBS. Plates were incubated with blocking buffer for 1 h at room temperature and then washed five times with washing buffer. For antigen binding, antibodies were added at 10 μg ml -1 For each antibody dilution, serum or plasma was diluted 10-fold for mouse and 50-fold for rabbit in blocking buffer and subjected to a 10-fold dilution series. For each sample dilution, a total volume of 50 μl was added to the appropriate well. Each plate was incubated for 1 hour at room temperature and then washed five times with PBS containing 0.05% Tween 20. For antibody binding, a 1:5000 dilution of goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Inc.) or for animal sample analysis, a 1:2000 dilution of horseradish peroxidase (HRP)-labeled goat anti-mouse or anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories) was then made in wash buffer (PBS with 0.05% Tween 20) and 50 μl of this diluted secondary antibody was added to each well. Plates were incubated with secondary antibody for 1 hour at room temperature and then washed five times with PBS containing 0.05% Tween 20. Finally, wells were developed with 50 μl TMB (Life Sciences) for 3 to 5 minutes and then stopped with 50 μl 2N sulfuric acid. The resulting plate readings were measured at a wavelength of 450 nm. Of note, serum binding in the second week was significantly higher than EC 50A plateau (or saturation) was not reached to allow for accurate determination of titers. Nevertheless, the EC 50 Values ​​were used as a quantitative measure of antibody titers to facilitate comparison of the various vaccine groups at week 2.

[0124] Biolayer Interferometry (BLI): Kinetics of GPΔmuc and GPΔmuc-displaying nanoparticle binding to a panel of 10 antibodies were measured using an Octet Red96 instrument (forteBio, Pall Life Sciences). All assays were performed in forteBio's 1× kinetic buffer with stirring set at 1000 rpm. The final volume of all solutions was 200 μl per well. Assays were performed in solid black 96-well plates (Geiger Bio-One) at 30°C. 5 μg ml ?1 1x kinetic buffer containing 10 ... The best fit was obtained by fitting the experimental data to a binding equation describing a 2:1 interaction. Of note, the binding of GPΔmuc trimers was also measured using AHQ to facilitate comparison of antibody binding to nanoparticles.

[0125] Differential scanning calorimetry (DSC): Thermal melting curves of WT and redesigned GPΔmuc trimers were acquired using a MicroCal VP-capillary calorimeter (Malvern). Purified GPΔmuc produced from 293F cells was buffer exchanged into 1× PBS and concentrated to 27-50 μM prior to analysis on the instrument. Melting was performed over a 90°C·h period from 25°C to 110°C. ?1 Data processing, including buffer correction, normalization, and baseline subtraction, was performed using a standardized protocol from Origin 7.0 software.

[0126] Protein production, crystallization and data collection: Two Zaire EBOV GP Δmuc-foldon constructs, one with the W615L mutation and L extension (up to aa637) and one with an additional T577P mutation, were expressed in HEK293 S cells. Expressed GP was purified using a mAB100 antibody column followed by size-exclusion chromatography (SEC) on a HiLoad Superdex 200 16 / 600 column (GE Healthcare). A sample of freshly purified GP was used for crystallization. Crystallization experiments were performed at The Scripps Research Institute (TSRI) at both 4°C and 20°C on an automated CrystalMation™ robotic system (Rigaku) ​​using the sitting-drop vapor diffusion method (Elsliger et al., Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 1137-1142, 2010). EBOV GP was concentrated to approximately 10 mg / ml in 50 mM Tris-HCl pH 8.0. The reservoir solution contained 12% (w / v) PEG 6000 and 0.1 M sodium citrate, pH 4.5. Diffractable crystals were obtained after 2 weeks at 20°C. EBOV GP crystals were cryoprotected in 25% glycerol, mounted on nylon loops, and flash frozen in liquid nitrogen. GPΔmuc-WL 2 -Foldon and GPΔmuc-WL 2 P 2Data were collected at the Advanced Photon Source (APS) beamline 23IDB for two crystals of GPΔmuc-WL2-foldon, which diffracted to 2.3 Å and 3.2 Å resolution, respectively. The diffraction data sets were processed with HKL-2000. The crystals belong to the rhombohedral H32 and tetragonal P321 space groups, with lattice dimensions a = 114.58 Å, b = 114.58 Å, c = 312.38 Å for GPΔmuc-WL2-foldon and GPΔmuc-WL 2 P 2 -foldons were a = 114.06 Å, b = 114.06 Å, and c = 136.22 Å. The overall completeness of the two data sets was 95.77% and 99%, respectively.

[0127] Structure determination and refinement: The structure of EBOV GP was determined by molecular replacement (MR) using Phaser, a suite of CCP4i with the coordinate system of Zaire Ebola GP (PDB: 5JQ3) and the program MOLREP. The polypeptide chain was converted into electron density with manual adjustments using Coot and structure validation was performed using MolProbity. The final R of the fine structure was cryst and R free The value of GPΔmuc-WL 2 -Foldon and GPΔmuc-WL 2 P 2 The results were 19.2% and 22.9% for the -foldon and 25% and 29.3% for the foldon, respectively. Data processing and refinement parameters are listed in Table S1.

[0128] Electron microscopy (EM) evaluation of nanoparticle constructs: Initial EM evaluation of EBOV GPΔMuc nanoparticles was performed at the Scripps Core Microscopy Facility. Briefly, nanoparticle samples were prepared at a concentration of 0.01 mg / ml. Carbon-coated copper grids (400 mesh) were glow discharged and 8 μL of each sample was adsorbed for 2 min. Excess sample was removed and grids were negatively stained with 2% uranyl formate for 2 min. Excess stain was removed and grids were allowed to dry. Samples were analyzed on a Talos L120C transmission electron microscope (Thermo Fisher) at 80 kV and images were acquired with a CETA 16M CMOS camera.

[0129] Mouse immunization and sample collection: Animal subjects tested in the immunization study were followed according to Institutional Animal Care and Use Committee (IACUC) guidelines. Eight-week-old BALB / c mice were purchased from Jackson Laboratory. Mice were housed in ventilated cages in Scripps Research's environmental control room in compliance with approved IACUC protocols and AAALAC guidelines. Mice were immunized a total of four times at weeks 0, 3, 6, and 9. Each immunization was performed via the intraperitoneal (ip) route with 200 μl of antigen / adjuvant mixture containing 50 μg of vaccine antigen (or 20 μg for I3-01v9 NP) and 100 μl of adjuvant, AddaVax or Adju-Phos (InvivoGen). Blood was collected two weeks after each immunization. All bleeds were performed through the facial vein (submandibular bleed) using a lancet (Goldenrod). Intermediate bleeds were collected without anticoagulant, whereas terminal bleeds were collected using EDTA-coated tubes. Serum and plasma were heat inactivated at 56°C for 30 min, spun at 1000 RPM for 10 min, and sterile filtered. Cells were washed once with PBS and then resuspended in 1 ml of ACK red blood cell lysis buffer (Lonza). After two washes with PBS, peripheral blood mononuclear cells (PBMCs) were resuspended in 2 ml of Bambanker Freezing Media (Lymphotec). In addition, spleens were collected and mashed through a 70 μm cell strainer (BD Falcon) to release splenocytes into the cell suspension. Spleen cells were centrifuged, washed in PBS, treated with 5 ml of red blood cell lysis buffer Hybri-Max (Sigma-Aldrich), and frozen in 10% DMSO in FBS. Serum and plasma were used for EBOV neutralization assays: 80% of plasma from individual mice at week 11 was purified using 0.2 ml of a Protein G spin kit (Thermo Scientific) according to the manufacturer's instructions. Purified mouse IgG at week 11 (w11) was evaluated in a pseudovirus neutralization assay.Rabbit immunization and blood collection were outsourced to ProSci (San Diego, CA). Five groups of four female New Zealand White rabbits per group were immunized intramuscularly (im) with 50 μg (20 μg for I3-01v9 NP) vaccine antigen in a total volume of 500 μl, formulated with 250 μl of adjuvant, AddaVax or Adju-Phos (InvivoGen), at weeks 0, 3, 6, and 9. Blood samples of 20 ml each were collected from the auricular artery at days 0 (pre-immunization), 2, 5, 8, and 11. At the last time point (week 13), more than 100 ml of blood was collected by cardiac puncture. Serum was separated from the blood and heat inactivated for ELISA binding assays. Purified rabbit IgG was evaluated in a pseudovirus neutralization assay.

[0130] Pseudovirus Neutralization Assay: Ebola virus pseudovirus particle (Ebolavirus-pp) neutralization assay was utilized to evaluate the neutralization activity of previously reported mAbs and vaccine-induced antibody responses in mice and rabbits. Ebolavirus-pp was generated by co-transfecting HEK293T cells with pNL4-3.lucR-Eplasmid (NIH AIDS Reagent Program) and an expression plasmid encoding the GP gene of EBOV Makona strain (GenBank Accession No.: KJ660346) or BDBV Uganda strain (GenBank Accession No.: KR063673) at a 4:1 ratio by Lipofectamine 3000 (Thermo Fisher Scientific). After 48 to 72 hours, Ebolavirus-pp was harvested from the supernatant by centrifugation at 4000 rpm for 10 min, aliquoted, and stored at -80°C before use. mAbs at a starting concentration of 10 μg / ml or purified IgG from mouse at a starting concentration of 300 μg / ml and rabbit at a starting concentration of 1000 μg / ml were mixed with the supernatant containing Ebola virus-pps and incubated for 1 h at 37°C in white solid-bottom 96-well plates (Corning). Based on recent studies on EBOV infectivity in various cell lines, HEK293T cells or TZM-bl cells were used for Ebola virus-pp neutralization assays. Briefly, 1 × 10 4HEK293T or TZM-bl cells were added to each well and the plates were incubated at 37°C for 48 hours. After incubation, the overlying medium was removed and the cells were lysed. Firefly luciferase signal from infected cells was determined using the Bright-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. Data was extracted from the BioTek microplate reader using Gen 5 software, the average background luminescence from a set of uninfected wells was subtracted from each well, and neutralization curves were generated using GraphPad Prism 8.4.3, comparing values ​​from wells to wells containing Ebola virus-pp alone. A lentiviral vector pseudotyped with the murine leukemia virus (MLV) Env gene, designated MLV-pps, was produced in HEK293T cells and included in the neutralization assay as a negative control. Because the non-NAb c13C6 showed enhanced MLV-pp infection, the MLV-pp assay was used to detect antibody responses that elicited ADE similar to c13C6 in samples from immunized animals.

[0131] Bulk sorting of EBOV GPΔmuc-specific mouse B cells: Spleens were harvested from immunized mice 15 days after the last immunization and cell suspensions were prepared. Cells were stained as follows: Dead cells were excluded by staining with Fixable Aqua Dead Cell Stain kit (Thermo Fisher L34957). Receptors FcγIII (CD16) and FcγII (CD32) were blocked by adding 20 μl of 2.4G2 mAb (BD Pharmigen N553142). Cells were then incubated with 10 μg / ml of biotinylated GPΔmuc, WT or UFOg. 2Briefly, GPΔmuc was incubated with EBOV GPΔmuc and the excess biotin generated upon biotinylation of each Avi-tagged EBOV GPΔmuc was removed by SEC on a HiLoad Superdex 200 16 / 600 column (GE Healthcare) using biotin ligase BirA according to the manufacturer's instructions (Avidity LLC). In the SEC profile, the Avi-tagged GPΔmuc peak was centered at 14.5 ml, whereas the broader biotin ligase peak was centered at 65 to 70 ml (WT) or 60 to 65 ml (UFOg). 2 ) can be found in the following table. The cells and biotinylated proteins were incubated for 5 min at 4°C, followed by the addition of 2.5 μl of anti-mouse IgG fluorescently labeled with FITC (Jackson ImmunoResearch 115-095-071) and incubation for 15 min at 4°C. Finally, 5 μl of premium grade streptavidin labeled with allophycocyanin (APC) was added to the cells and incubated for 15 min at 4°C. At each step, the cells were washed with DPBS, but the sorting buffer was 0.5 ml of FACS buffer. FITC + APC + GPΔmuc-specific B cells were sorted using a BD FACSAria II into Eppendorf tubes containing 500 μl FACS buffer.

[0132] Next-generation sequencing (NGS) and bioinformatics analysis of mouse B cells: A protocol for 5'-rapid amplification of cDNA ends (RACE) has been reported to unbiasedly sequence the mouse B cell repertoire. Here, this protocol was applied to bulk-sorted E2-specific mouse splenic B cells. Briefly, 5'-RACE cDNA was obtained from bulk-sorted splenic B cells of each mouse using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (TaKaRa). Immunoglobulin PCR was set up with Platinum Taq High-Fidelity DNA Polymerase (Life Technologies) in a total volume of 50 μl using 5 μl of cDNA as template, 1 μl of 5'-RACE primer and 1 μl of 10 μM reverse primer. The 5'-RACE primer had a PGM / S5 P1 adaptor, while the reverse primer had a PGM / S5 A adaptor. As a reverse primer for 5'-RACE-PCR processing of the heavy and light chains (κ), mouse 3'-C γ 1-3 / 3'-C μ Internal primer and 3'-mC κExternal primers were used. A total of 25 cycles of PCR were performed and the expected PCR products (500 to 600 bp) were gel purified (Qiagen). NGS was performed on an Ion S5 GeneStudio system. Briefly, heavy and light chain (kappa) libraries from the same mouse were quantified using a Qubit® 2.0 Fluorometer with Qubit® dsDNA HS Assay Kit, then mixed using a 3:1 ratio and pooled with antibody libraries from other mice in equal ratios for sequencing. Template preparation and (Ion 530) chip loading were performed on Ion Chef using the Ion 520 / 530 Ext Kit, followed by sequencing on an Ion S5 system with default settings. The Mouse Antibodyomics pipeline was used to process the raw data to determine germline gene usage, somatic hypermutation (SHM), germline diversification and H / KCDR3 loop length distribution.

[0133] Thus, the invention has been broadly disclosed and illustrated with reference to the exemplary embodiments set forth above, it being understood that various modifications can be made thereto without departing from the spirit and scope of the invention.

[0134] It should be further noted that all publications, sequence accession numbers, patents, and patent applications cited herein are expressly incorporated herein by reference in their entirety for all purposes as if each was individually so indicated. Definitions contained in the text incorporated by reference are excluded to the extent that they conflict with definitions in this disclosure.

Claims

1. (a) substitution of residue W615 in heptad repeat 2 (HR2) with L, A, V, I or F, and (b) substitution of residue W615 in heptad repeat 1C segment (HR1 Ｃ ) and the amino acid numbering is based on the sequence of GP of Zaire Ebola virus having UniProt ID Q05320 (SEQ ID NO: 1).

2. The Ebola virus is Zaire Ebola virus (EBOV), Sudan virus (SUDV), Tai Forest virus (TAFV), Bundibugyo virus (BDBV) or Reston Ebola virus (RESTV); 2. The engineered Ebola virus GP protein of claim 1.

3. The proline substitution comprises T577P or L579P.

2. The engineered Ebola virus GP protein of claim 1.

4. Further comprising a truncation at the C-terminus of its membrane proximal external region (MPER); 2. The engineered Ebola virus GP protein of claim 1.

5. (a) a deletion of the mucin-like domain (MLD) from the GP1 subunit, and / or (b) further comprising a deletion of MPER from the GP2 subunit; 5. The engineered Ebola virus GP protein of claim 4.

6. (a) a truncated soluble Ebola virus GP lacking the mucin-like domain (MLD) from the GP1 subunit and the membrane proximal external region (MPER) from the GP2 subunit; (b) in heptad repeat 2 (HR2), a substitution of residue W615 by L, A, V, I or F; An engineered Ebola virus glycoprotein (GP) protein based on the sequence of Zaire Ebola virus GP, where the amino acid numbering corresponds to UniProt ID Q05320 (SEQ ID NO:1).

7. (i) an extension of HR2 at the C-terminus, (ii) the heptad repeat 1C segment (HR1 Ｃ ) one or more proline substitutions in the GP; and (iii) one or more engineered disulfide bonds between the GP.

7. The engineered Ebola virus GP protein of claim 6, further comprising one or more modifications selected from:

8. The truncated soluble GP comprises SEQ ID NO:2 or SEQ ID NO:3 or a variant having at least 99% sequence identity thereto; 7. The engineered Ebola virus GP protein of claim 6.

9. The sequence of SEQ ID NO: 4 or a variant thereof having at least 99% sequence identity, 7. The engineered Ebola virus GP protein of claim 6.

10. Further comprising an extension of HR2 at the C-terminus, 7. The engineered Ebola virus GP protein of claim 6.

11. the extension of HR2 comprises (a) extending the C-terminus of HR2 with an N-terminal fragment of the adjacent membrane proximal external region (MPER) of the Ebola virus glycoprotein, or (b) replacing the C-terminal fragment of HR2 with a longer leucine zipper motif.

11. The engineered Ebola virus GP protein of claim 10.

12. The extension of HR2 comprises (a) extending the C-terminus of HR2 from residue 632 to residue 637 in MPER, (b) extending the C-terminus of HR2 from residue 632 to residue 643 in MPER, or (c) replacing residues 617 to 632 with the GCN4 leucine zipper sequence represented by SEQ ID NO:

34.

11. The engineered Ebola virus GP protein of claim 10.

13. The W615 substitution includes a W615L substitution or a P612G / W615F double mutation; 11. The engineered Ebola virus GP protein of claim 10.

14. (a) a W615L substitution; and (b) an extension of the C-terminus of HR2 from residue 632 to residue 637 in the MPER of Ebola virus GP.

11. The engineered Ebola virus GP protein of claim 10.

15. (a) a P612G / W615F double mutation in HR2, and (b) a substitution of residues 617 to 632 with the GCN4 leucine zipper sequence set forth in SEQ ID NO:34; 11. The engineered Ebola virus GP protein of claim 10.

16. The present invention relates to a method for producing a nucleic acid sequence comprising the steps of:

11. The engineered Ebola virus GP protein of claim 10.

17. Further comprising one or more proline substitutions in the HR1C segment; 7. The engineered Ebola virus GP protein of claim 6.

18. The present invention relates to a method for producing a nucleic acid sequence comprising the steps of:

18. The engineered Ebola virus GP protein of claim 17.

19. (a) a W615L substitution; (b) a T577P or L579P proline substitution; and (c) an extension of HR2 from residue 632 to residue 637 in MPER.

18. The engineered Ebola virus GP protein of claim 17.

20. Further comprising an engineered disulfide bond between two adjacent GP promoters; 7. The engineered Ebola virus GP protein of claim 6.

21. the engineered disulfide bond is formed between residues G91 / A575 (SS2), F153 / Y534 (SS1), T520 / A575 (SS3), G157 / I532 (SS4), D522 / A575 (SS5), or K56 / G599 (SS6); 21. The engineered Ebola virus GP protein of claim 20.

22. 7. A method for the preparation of a vaccine comprising administering to a patient a therapeutically acceptable carrier comprising administering to said patient a therapeutically acceptable carrier comprising administering to said patient a therapeutically acceptable carrier. Pharmaceutical compositions.

23. 7. The engineered Ebola virus GP protein of claim 6, which is displayed on the surface of a self-assembling nanoparticle. Vaccine compositions.

24. (1) From the N-terminus to the C-terminus, (a) engineered Ebola virus GP protein, linker sequence G4S (SEQ ID NO: 43), ferritin nanoparticle sequence; (b) an engineered Ebola virus GP protein, the linker sequence G4S (SEQ ID NO: 43), the nanoparticle sequence E2p, or (c) engineered Ebola virus GP protein, linker sequence (G4S)2 (SEQ ID NO:42), nanoparticle sequence I3-01v9; A polypeptide sequence comprising where The engineered Ebola virus GP protein comprises a W615L substitution, a T577P proline substitution, and an extension of HR2 from residue 632 to residue 637 in MPER.

24. The vaccine composition of claim 23.

25. Further comprising a locking domain and / or a T cell epitope fused to the C-terminus of the sequence of the nanoparticle subunit.

25. The vaccine composition of claim 24.

26. (1) From the N-terminus to the C-terminus, (a) an engineered Ebola virus GP protein as set forth in SEQ ID NO: 17, a nanoparticle subunit sequence (E2p) as set forth in SEQ ID NO: 36, a locking domain (LD4) as set forth in SEQ ID NO: 39, and a T cell epitope as set forth in SEQ ID NO: 31, or (b) an engineered Ebola virus GP protein as set forth in SEQ ID NO: 17, a nanoparticle sequence as set forth in SEQ ID NO: 37 (I3-01v9), a locking domain as set forth in SEQ ID NO: 40 (LD7); or a polypeptide sequence comprising (2) a variant of said polypeptide sequence having at least 99% sequence identity; Including, 26. The vaccine composition of claim 25.

27. A polynucleotide encoding the engineered Ebola virus GP protein of claim 1 or 6.

28. 24. The vaccine composition of claim 23, wherein the engineered Ebola virus GP protein and a polynucleotide encoding a nanoparticle subunit on the surface of which the engineered Ebola virus GP protein is fused.

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