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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/14011—Filoviridae
  • C12N2760/14111—Ebolavirus, e.g. Zaire ebolavirus
  • C12N2760/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2760/00011—Details
  • C12N2760/14011—Filoviridae
  • C12N2760/14111—Ebolavirus, e.g. Zaire ebolavirus
  • C12N2760/14134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• Filoviruses such as Zaire Ebola virus (EBOV) and Marburg virus can cause a lethal hemorrhagic fever in humans and nonhuman primates (NHPs).
• the virus glycoprotein, GP mediates cell entry by initiating attachment and membrane fusion.
• Structures of GP-bound neutralizing antibodies (NAbs) from human survivors demonstrated that GP harbors all NAb epitopes known to date and is the sole target for vaccine design.
• NAbs neutralizing antibodies
• filovirus GP is metastable and produces non-functional forms when expressed in various cell lines.
• the invention provides engineered or redesigned Ebolavirus glycoprotein (GP) sequences.
• the engineered sequences contain (a) a substitution of residue W615 in heptad repeat 2 (HR2) with a smaller hydrophobic residue, and (b) one or more proline substitutions in heptad repeat 1 C segment (HRlc).
• the amino acid numbering is based on Zaire Ebolavirus GP sequence that has the ectodomain sequence identified by UniProt ID Q05320 (SEQ ID NO:1).
• the engineered Ebolavirus GP sequences can be modified or derived from the wildtype GP sequence of a Zaire Ebolavirus (EBOV), a Sudan virus (SUDV), a Tai forest virus (TAFV), a Bundibugyo virus (BDBV), or a Reston Ebolavirus (RESTV).
• EBOV Zaire Ebolavirus
• SUDV Sudan virus
• TAFV Tai forest virus
• BDBV Bundibugyo virus
• RESTV Reston Ebolavirus
• residue W615 in the engineered Ebolavirus GP sequences can be replaced with residue L, A, V, I or F.
• the proline substitutions in HRlc include T577P and/or L579P.
• the engineered Ebolavirus GP sequences additionally include a truncation at the C-terminus of the membrane proximal external region (MPER).
• the engineered Ebolavirus GP sequences can further include (a) a deletion of the mucin-like domain (MLD) deleted from the GP1 subunit, and/or (b) a deletion of MPER from the GP2 subunit.
• MLD mucin-like domain
• the invention provides engineered Ebolavirus GP sequences that contain (a) a truncated soluble GP of an Ebolavirus that has MLD deleted from the GP1 subunit and MPER deleted from the GP2 subunit, and (b) one or more modifications in the HR2 and HR1 regions.
• the additional modifications in the HR2 and HR1 regions can be (i) substitution of W615 in heptad repeat 2 (HR2) with a smaller hydrophobic residue, (ii) an extension of HR2 at the C terminus, (iii) one or more proline substitutions in heptad repeat 1 C segment (HRlc), and (iv) one or more engineered inter-GP disulfide bonds.
• the amino acid numbering is based on the Zaire Ebolavirus GP sequence with UniProt ID Q05320 (SEQ ID NO:1).
• these engineered Ebolavirus GP sequences can be derived from Zaire Ebolaviruses (EBOV), Sudan viruses (SUDV), Tai forest viruses (TAFV), Bundibugyo viruses (BDBV), or Reston Ebolaviruses (RESTV).
• Some engineered Ebolavirus GP sequences are based on the truncated soluble GP sequence shown in SEQ ID NO:2 or SEQ ID NO:3, or a conservatively modified or substantially identical variant thereof.
• the engineered GP sequences can have aN-terminal leader sequence.
• the N-terminal leader sequence can contain SEQ ID NO:41 or a conservatively modified variant.
• Some engineered soluble Ebolavirus GP sequences contain a substitution of residue W615.
• residue W615 can be replaced with L, A, V, I or F.
• Some of these engineered Ebolavirus GP proteins contain the amino acid sequence shown in SEQ ID NOs:4, a conservatively modified variant or a substantially identical sequence thereof.
• the engineered soluble Ebolavirus GP proteins can contain an extension of HR2 at the C-terminus.
• the HR2 extension can be (a) extending HR2 C terminus with a N- terminal fragment of adjacent MPER of the Ebolavirus glycoprotein or (b) replacing a HR2 C-terminal fragment with a longer leucine zipper motif.
• HR2 extension in the engineered soluble Ebolavirus GP proteins can include (a) extension of the HR2 C terminus from residue 632 to residue 637 in MPER, i.e., addition of residues KTLPD (SEQID NO:32), (b) extension of the HR2 C terminus from residue 632 to residues 643 in MPER, i.e., addition of residues KTLPDQGDNDN (SEQ ID NO:33), or (c) replacement of residues 617-632 with a GCN4 leucine zipper sequence shown in SEQ ID NO:34.
• Some engineered soluble Ebolavirus GP sequences of the invention contain both a HR2 extension noted above and a W615 substitution in HR2.
• the W615 substitution can be W615L or P612G/W615F double mutation.
• the engineered GP sequences contain (a) W615L substitution and (b) extension of the HR2 C terminus from residue 632 to residue 637 in MPER of the Ebolavirus GP.
• the engineered GP sequences contain (a) P612G/W615F double mutation in HR2 and (b) replacement of residues 617-632 with a GCN4 leucine zipper sequence shown in SEQ ID NO:34.
• Some specific engineered soluble Ebolavirus GP sequences contain an amino acid sequence shown in any one of SEQ ID NOs:5-8, a conservatively modified variant or a substantially identical sequence thereof.
• the engineered GP sequences in some embodiments can also contain a C-terminal trimerization motif.
• the C-terminal trimerization motif can contain SEQ ID NO:29 or SEQ ID NO:30, or a conservatively modified variant or a substantially identical sequence thereof.
• the engineered GP sequences in some embodiments can also contain one or more proline substitutions in the HRlc segment. Any residue in HRlc can be replaced with a proline residue.
• the proline substitution comprises T577P or L579P.
• engineered Ebolavirus GP sequences having a W612 substitution in HR2 and a proline substitution in HRlc contain an amino acid sequence as set forth in any one of SEQ ID NOs:9-16, a conservatively modified variant or a substantially identical sequence thereof.
• Some engineered GP sequences of the invention contain a HR2 extension, a proline substitution in HRlc, and a substitution at residue W615.
• the W615 residue can be replaced with L, A, V, I or F.
• the engineered Ebolavirus GP sequences contain an extension of HR2 C terminus from residue 632 to residue 637 in MPER.
• Some specific examples of such engineered Ebolavirus GP sequences contain an amino acid sequence as set forth in any one of SEQ ID NOs: 17 and 20, a conservatively modified variant or a substantially identical sequence thereof.
• the engineered Ebolavirus GP sequences can further include a C-terminal trimerization motif.
• the engineered Ebolavirus GP proteins can include the C-terminal trimerization motif shown in SEQ ID NO:29 or SEQ ID NO:30, or a conservatively modified variant or a substantially identical sequence thereof.
• Some engineered Ebolavirus GP sequences contain (a) W615L substitution, (b) proline substitution T577P or L579P, and (c) HR2 extension from residue 632 to residue 637 in MPER. Some specific examples of these engineered Ebolavirus GP sequences contain amino acid sequence as set forth in any one of SEQ ID NOs: 18, 19, 21 and 22, a conservatively modified variant or a substantially identical sequence thereof. [0014] Some engineered Ebolavirus GP sequences contain one or more engineered disulfide bonds between two neighboring GP protomers.
• the engineered disulfide bonds scan be created by cysteine substitutions at residue pairs G91/A575 (SS2), F153/Y534 (SSI), T520/A575 (SS3), G157/I532 (SS4), D522/A575 (SS5), and/or K56/G599 (SS6).
• SS2 residue pairs G91/A575
• SSI F153/Y534
• SS3 T520/A575
• G157/I532 SS4
• D522/A575 SS5
• K56/G599 K56/G599
• the invention provides nucleic acid or polynucleotide sequences that encode an engineered full length or truncated Ebolavirus GP protein sequence described herein. Some engineered Ebolavirus GP sequences also contain a leader peptide encoding sequence at the 5’. Also provided in the invention are pharmaceutical compositions that contain an engineered Ebolavirus GP protein or encoding polynucleotide sequence described herein, and a pharmaceutically acceptable carrier.
• the invention provides vaccine compositions that contain an engineered Ebolavirus GP protein that is displayed on the surface of a selfassembling nanoparticle.
• C-terminus of the engineered Ebolavirus GP protein is fused to the N-terminus of subunit of the self-assembling nanoparticle (NP).
• the engineered Ebolavirus GP proteins in the NP vaccine compositions contain an amino acid sequence as set forth in any one of SEQ ID NOs:4-28, a conservatively modified variant or a substantially identical sequence thereof.
• subunit of the self-assembling nanoparticle contains the polypeptide sequence as shown in SEQ ID NO:36 (E2p), SEQ ID NO:37 (I3-01v9), or SEQ ID NO:38 (ferritin), a conservatively modified variant or a substantially identical sequence thereof.
• Some NP scaffolded vaccine compositions of the invention can additionally contain a locking domain (LD) that is fused to the C terminus of the NP subunit sequence.
• the employed LD contains the sequence shown in SEQ ID NO:39 or 40, a conservatively modified variant or a substantially identical sequence thereof.
• Some of the scaffolded vaccines can additionally contain a T-cell epitope that is fused to the C-terminus of the locking domain.
• the employed T-cell epitope contains the sequence shown in SEQ ID NO:31, a conservatively modified variant or a substantially identical sequence thereof.
• Some NP vaccine compositions of the invention contain (1) a polypeptide sequence containing from N terminus to C terminus (a) an engineered Ebolavirus GP protein, linker sequence G4S (SEQ ID NO:43), nanoparticle sequence ferritin, (b) an engineered Ebolavirus GP protein, linker sequence G4S (SEQ ID NO:43), nanoparticle sequence E2p, or (c) an engineered Ebolavirus 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).
• a polypeptide sequence containing from N terminus to C terminus (a) an engineered Ebolavirus GP protein, linker sequence G4S (SEQ ID NO:43), nanoparticle sequence ferritin, (b) an engineered Ebolavirus GP protein, linker sequence G4S (SEQ ID NO:43), nanoparticle sequence E2p, or (c) an engine
• the engineered Ebolavirus GP sequences contain W615L substitution, proline substitution T577P, and HR2 extension from residue 632 to residue 637 in MPER.
• These engineered Ebolavirus GP sequences can additionally contain a locking domain and/or a T-cell epitope that is fused to the C-terminus of the nanoparticle subunit sequence.
• the vaccine compositions contain (1) a polypeptide sequence containing fromN- terminus to C-terminus (a) the engineered Ebolavirus GP protein shown in SEQ ID NO: 17, nanoparticle subunit sequence shown in SEQ ID NO:36 (E2p), locking domain shown in SEQ ID NO:39 (LD4), and T cell epitope shown in SEQ ID NO:31 (PADRE) or (b) the engineered Ebolavirus GP protein shown in SEQ ID NO: 17, nanoparticle sequence shown in SEQ ID NO:37 (I3-01v9), locking domain shown in SEQ ID NO:40 (LD7), or (2) a conservatively modified variant of the polypeptide sequence.
• a polypeptide sequence containing fromN- terminus to C-terminus (a) the engineered Ebolavirus GP protein shown in SEQ ID NO: 17, nanoparticle subunit sequence shown in SEQ ID NO:36 (E2p), locking domain shown in SEQ ID NO:39 (LD4), and T cell epitope shown in SEQ ID NO:
• Figure 1 shows (A) a schematic structure of Ebolavirus glycoproteins (GPs) and (B) the general redesign strategy for generating engineered Ebolavirus GP vaccine immunogen proteins of the invention.
• Figure 2 shows sequence alignment of the Hl ( SEQ ID NOs:44-48) and HR2 (SEQ ID NOs:49-53) regions of the glycoproteins (GPs) of randomly selected strains from all 5 Ebolavirus species, Bundibugyo Ebolavirus (AYI50316), Zaire Ebolavirus (AER59712), Reston Ebolavirus (ASU06443), Sudan Ebolavirus (ALH21228) and Tai Forest Ebolavirus (AWK96625).
• Figure 3 shows design, screening, and antigenic characterization of EBOV GPAmuc trimers with modified stalk and HRlc.
• A Schematic representation of a mucin-deleted GP (GPAmuc) and three stalk designs (GPAmuc-2WPZ, GPAmuc-L, and GPAmuc-Ext).
• B SEC profiles of four GPAmuc constructs obtained from a Superdex 200 10/300 column following transient expression in 250 ml HEK293 F cells and mAbll4 purification. Left: the SEC curve of GPAmuc shown in black line; Right: three stalk designs shown for GPAmuc-2WPZ, -L, and -Ext, respectively.
• C BN-PAGE of GPAmuc proteins purified by mAbl 14 (left) and mAbl 10 (right) columns.
• GPAmuc construct with the 8-aa HRlc replaced by a flexible (GS)4 loop is included for comparison.
• D ELISA curves of four mAblOO/SEC-purified, stalk- modified EBOV GP/GPAmuc-foldon trimers binding to 10 antibodies.
• E Summary of ECso values (pg/ml) of EBOV GP/GPAmuc-foldon trimers binding to 10 antibodies.
• F ELISA curves of an mAblOO/SEC-purified, stalk/HRl c-modified EBOV GPAmuc- foldon trimer binding to 10 antibodies.
• Figure 4 shows design and characterization of multilayered EBOV GPAmuc-presenting NPs.
• A Locking domains (LD) identified from the Protein Data Bank (PDB) for stabilizing the NP -forming interface. Shown in the figure are the PDB IDs of the identified LDs and their full sequences (SEQ ID NOs:54-62). Residues deleted at the N- and/or C-termini of the original sequences for generating actual LDs used in the studies herein are noted in the sequences.
• B -(C) Antigenic evaluation by ELISA.
• E2p-LD4-PADRE Binding of FR and reengineered E2p (E2p-LD4-PADRE, or E2p-L4P) and 13- 01 (I3-01-LD7-PADRE, I3-01-L7P) NPs that present a stabilized GPAmuc-WL 2 P 2 trimer to 10 antibodies with ELISA curves shown in (B) and EC50 values summarized in (C). All GP-NPs were expressed transiently in ExpiCHO cells, purified by a mAbl 00 column, and further purified by SEC on a Superose 6 10/300 GL column. (D)-(G) Antigenic evaluation by BLI using Octet96.
• BLI sensorgrams were obtained from an Octet RED96 instrument using a series of six concentrations (400-12.5nM by twofold dilution for GPAmuc; 25-0.78nM and 10-0.3 InM by twofold dilution for FR and E2p-L4p/I3-01-L7P, respectively) and quantitation biosensors (see Materials and Methods).
• Figure 5 shows immunogenicity assessment of EBOV GP/GPAmuc trimers and GPAmuc-presenting NPs in BALB/c mice.
• A ELISA binding curves of mouse serum from three trimer groups, in which mice were immunized with WT GP-foldon, GPAMuc-foldon, and GPAMuc-WL 2 P 2 -fold trimers, and three NP groups, in which mice were immunized with FR, E2p-L4P, and I3-01v9-L7P NPs presenting GPAMuc- WL 2 P 2 , to GPAMUC-WL 2 P 2 at w2, w5, w8, and wl 1.
• Ebolavirus-pp (Makona Cl 5) neutralization by 10 Ebolavirus-specific antibodies in TZM-bl cells.
• E MLV-pp neutralization by 10 Ebolavirus-specific antibodies in 293 T cells.
• F MLV-pp neutralization by purified mouse serum IgGs in 293 T cells. Mouse IgGs were collected from all six vaccine groups at wll.
• Figure 6 shows immunogenicity assessment of EBOV GP/GPAmuc trimers and GPAmuc-presenting NPs in rabbits.
• A ELISA binding curves of rabbit serum from two trimer groups, in which rabbits were immunized with GPAMuc-foldon and GPAMuc-WL 2 P 2 -fold trimers, and three NP groups, in which rabbits were immunized with FR, E2p-L4P, and I3-01v9-L7P NPs presenting GPAMuc-WL 2 P 2 , to GPAMuc- WL 2 P 2 at wO, w2, w5, w8, wl 1, and wl3.
• FIG. 7 shows unbiased NGS repertoire analysis of bulk-sorted Ebola GP- specific mouse splenic B cells.
• A SEC profile of a biotinylated Avi-tagged Ebola GPAmuc trimer, termed GPAmuc-WL 2 P 2 -lTD0-Avi-Biot, obtained from aHiLoad Superdex 200 16/600 column. 1TD0 is a trimeric scaffold used to stabilize GPAmuc in a trimeric state and to deselect B cells directed to foldon in the two trimer groups.
• B Summary of Ebola GPAmuc-specific bulk sorting of mouse splenic B cells from five vaccine groups.
• Ebolaviruses can cause severe hemorrhagic fever.
• Ebola virus aka Zaire Ebolavirus; EBOV
• Sudan virus SUDV
• Tai forest virus TAFV
• Bundibugyo virus BDBV
• RESTV Reston Ebolavirus
• EBOV, SUDV, TAFV and BDBV all have pandemic potential in humans.
• EBOV was solely responsible for the largest filovirus outbreak in history during 2013-2016, which spread across nine African countries with 28,600 cases and 11,325 deaths.
• a previous EBOV outbreak led to 2103 deaths and was declared an international emergency on July 17, 2019 by the World Health Organization (WHO).
• WHO World Health Organization
• NAbs neutralizing antibodies
• rVSV-ZEBOV a replication-competent recombinant vesicular stomatitis virus (VSV) expressing a Zaire EBOV glycoprotein (GP)
• VSV vesicular stomatitis virus
• GP Zaire EBOV glycoprotein
• the Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design.
• NAbs neutralizing antibodies
• the present invention is derived in part from the inventors’ studies to rationally redesign the Ebola virus glycoprotein and engineer single-component multilayered nanoparticles as vaccine candidates.
• the inventors explored the causes of EBOV GP metastability and designed multilayered NP vaccines for in vivo evaluation.
• the inventors developed an immunoaffinity column based on mAblOO that is specific for native-like, trimeric GP.
• the inventors first examined the contribution of two regions in GP2, namely, the HR2 (the “stalk”) region and the heptad repeat 1 C (HRlc) segment, to GP metastability in a mucin-deleted Zaire EBOV GP construct (GPAmuc).
• the inventors extended the HR2 stalk to residue 637 and introduced a W615L mutation based on the comparison of EBOV and MARV GPs.
• the inventors assessed the ability of proline mutation in HRlc to prevent GP refolding from pre- to post-fusion conformational changes. While both stalk and HRlc-proline mutations increased the trimer yield, the latter appeared to exhibit a complex effect on GP thermostability.
• Inter-GP molecule disulfide bonds were also found to increase the trimer stability. Crystal structures were determined for two redesigned GPAmuc constructs to validate the stalk and HRlc-proline mutations at the atomic level. The inventors then displayed a redesigned GPAmuc trimer on ferritin, E2p, and 13-01 NPs. Locking domains (LD) and helper T-cell epitopes were incorporated into E2p and 13-01 60-mers to stabilize the NP shell from the inside and to create multilayered NP carriers. [0029] In mice and rabbits, it was observed that the GP trimers and NPs can induce distinct antibody responses.
• LD Locking domains
• helper T-cell epitopes were incorporated into E2p and 13-01 60-mers to stabilize the NP shell from the inside and to create multilayered NP carriers.
• NGS Next-generation sequencing
• HCV hepatitis C virus
• the invention provides novel engineered Ebolavirus GP sequences that contain the various modifications disclosed herein. Some embodiments of the invention are directed to immunogen polypeptides and polynucleotide vaccines that are derived from the redesigned Ebolavirus GP sequences described herein. Also provided in the invention are Ebolavirus vaccine compositions containing a displaying platform, including a self-assembling nanoparticle, that displays one or more of the engineered Ebolavirus GP immunogens. Therapeutic applications of the engineered Ebolavirus GP immunogen polypeptides and the related nanoparticle vaccine compositions, e.g., treating or preventing Ebolavirus infections, are also provided in the invention.
• the vaccine immunogens of the invention can all be generated or performed in accordance with the procedures exemplified herein or routinely practiced methods well known in the art. See, e.g., Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos.
• an Env-derived trimer can refer to both single or plural Env-derived trimer molecules, and can be considered equivalent to the phrase “at least one Env-derived trimer.”
• antigen or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject.
• the term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular type directed against that protein.
• vaccine immunogen is used interchangeably with “protein antigen” or “immunogen polypeptide”.
• conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
• conservatively modified variants refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art.
• 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
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
• Epitope refers to an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit 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 noncontiguous amino acids juxtaposed by tertiary folding of a protein.
• Effective amount of a vaccine or other agent that is sufficient to generate a desired response, such as reduce or eliminate a sign or symptom of a condition or disease, such as a viral infection.
• a desired response such as reduce or eliminate a sign or symptom of a condition or disease, such as a viral infection.
• this can be the amount necessary to inhibit viral replication or to measurably alter outward symptoms of the viral infection.
• this amount will be sufficient to measurably inhibit virus (for example, an Ebolavirus) replication or infectivity.
• a dosage When administered to a subject, a dosage will generally be used that will achieve a target concentration that has been shown to be sufficient for in vitro inhibition of viral replication.
• an "effective amount" is one that treats (including prophylaxis) one or more symptoms and/or underlying causes of any of a disorder or disease, for example to treat Ebolavirus infection.
• an effective amount is a therapeutically effective amount.
• an effective amount is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with an Ebolavirus infection.
• a fusion protein is a recombinant protein containing amino acid sequence from at least two unrelated proteins that have been joined together, via a peptide bond, to make a single protein.
• the unrelated amino acid sequences can be joined directly to each other or they can be joined using a linker sequence.
• proteins are unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment(s) (e.g., inside a cell).
• the amino acid sequences of bacterial enzymes such as B. stear other mophilus dihydrolipoyl acyltransferase (E2p) and the amino acid sequences of Ebolavirus GP are not normally found joined together via a peptide bond.
• Ebolavirus refers to members of the family Filoviridae, which are associated with outbreaks of highly lethal hemorrhagic fever in humans and nonhuman primates.
• Human Ebolavirus 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.
• the natural reservoir of the Ebolaviruses is unknown, and there are currently no available vaccines or effective therapeutic treatments for Ebolavirus infections.
• the Ebolavirus genome consists of a single strand of negative sense RNA that is approximately 19 kb in length.
• RNA contains seven sequentially arranged genes that produce 8 mRNAs upon infection.
• Ebola virions contain seven proteins: a surface glycoprotein (GP), a nucleoprotein (NP), four virion structural proteins (VP40, VP35, VP30, and VP24), and an RNA-dependent RNA polymerase (L)
• GP surface glycoprotein
• NP nucleoprotein
• VP40, VP35, VP30, and VP24 four virion structural proteins
• L RNA-dependent RNA polymerase
• Ebolavirus glycoprotein (GP) refers to the only surface antigen of Ebolaviruses that is expressed as atrimer on the viral surface. It is unusual in that it is encoded in two open reading frames. Transcriptional editing is needed to express the transmembrane form that is incorporated into the virion (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 during the course of infection.
• sGP nonstructural secreted glycoprotein
• the GP of Ebolaviruses is a transmembrane glycoprotein (GP) that is responsible for both receptor binding and membrane fusion.
• GP transmembrane glycoprotein
• the GP1 subunit contains the core of the glycoprotein, receptor binding domain (RBD), a glycan cap, and a large mucin-like domain (MLD) which extends around the RBD.
• the GP2 (amino acids 502-676) subunit contains the internal fusion loop (IFL), heptad repeats 1 and 2 (HR1 and HR2), the membrane-proximal external region (MPER), the transmembrane region (TM), and the cytoplasmic tail (CT).
• IFL internal fusion loop
• HR1 and HR2 heptad repeats 1 and 2
• MPER membrane-proximal external region
• TM transmembrane region
• CT cytoplasmic tail
• amino acid residue numbering of the GP from Zaire Ebolavirus strain Mayinga-76 is used herein as the reference, which has a complete genome sequence identified by GenBank ID of AF272001. Its GP ectodomain sequence (UniProt ID Q05320; GenBank ID AAG40168.1) is shown in SEQ ID NO:1 herein.
• corresponding residues refers to residues that occur at aligned loci.
• Related or variant polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches, and include methods such as using manual alignments and by using the numerous alignment programs available (for example, BLASTP) and others known to those of skill in the art.
• BLASTP the numerous alignment programs available
• By aligning the sequences of polypeptides one skilled in the art can identify corresponding residues, using conserved and identical amino acid residues as guides. For example, by aligning the sequences of GP polypeptides from different Ebolavirus 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 guides.
• Corresponding positions also can be based on structural alignments, for example by using computer simulated alignments of protein structure.
• Immunogen refers to a protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen.
• Administration of an immunogen can lead to protective immunity and/or proactive immunity against a pathogen of interest.
• 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.
• the response is specific for a particular antigen (an "antigen-specific response").
• an immune response is a T cell response, such as a CD4+ response or a CD8+ response.
• the response is a B cell response, and results in the production of specific antibodies.
• Immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against virus expressing the immunogenic polypeptide, or induces a measurable B cell response (such as production of antibodies) against the immunogenic polypeptide.
• Sequence identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.
• Two sequences are "substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
• the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
• subject refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. Preferably, the subject is human.
• treating includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., an Ebolavirus infection), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
• Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
• Vaccine refers to a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject.
• the immune response is a protective immune response.
• a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition.
• a vaccine may include a polynucleotide (such as a nucleic acid encoding an Ebolavirus GP polypeptide disclosed herein), a peptide or polypeptide (such as an Ebolavirus GP polypeptide disclosed antigen), a virus, a cell or one or more cellular constituents.
• vaccines or vaccine immunogens or vaccine compositions are expressed from fusion constructs and self-assemble into nanoparticles displaying an immunogen polypeptide or protein on the surface.
• VLP Virus-like particle
• VLPs refers to a non-replicating, viral shell, derived from any of several viruses.
• VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins.
• VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular 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, such as by electron microscopy, biophysical characterization, and the like. See, for example, Baker et al. (1991) Biophys. J.
• VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding.
• cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.
• a self-assembling nanoparticle refers to a ball-shape protein shell with a diameter of tens of nanometers and well-defined surface geometry that is formed by identical copies of a non-viral protein capable of automatically assembling into a nanoparticle 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.
• Selfassembling nanoparticles can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for nanoparticle production, detection, and characterization can be conducted using the same techniques developed for VLPs.
• the invention provides engineered or redesigned Ebolavirus glycoprotein (GP) sequences (polypeptides or polynucleotide sequences) for producing Ebolavirus vaccines.
• GP Ebolavirus glycoprotein
• these engineered GP polypeptides can contain various modifications, esp. in the GP2 subunit.
• the GP2 subunit of Ebolavirus GPs contains the N-terminal peptide, the internal fusion loop, two consecutive two heptad repeat regions (HR1 and HR2), the membrane proximal external region (MPER), and the C-terminal transmembrane domain (see Figure 1 A).
• HR1 is in turn structurally divided into 4 segments, HRIA , HRIB , HRlc and HRID.
• the first two segments, HRIA and HRIB form an a-helix with an approximately 40° kink at Thr565, which delineates HRIA from HRIB.
• HRlc forms an extended coil linker between HRIB and the HRID segment.
• HRID forms an amphipathic helix, the hydrophobic faces of the three helices in the trimer pack together to form the interface of the peplomer.
• HRlc and HRID were first defined to correspond to residues 576-582 and residues 583-598, respectively (Lee et al., Future Virol. 2009; 4: 621-635). Based on a higher-resolution and more complete crystal structure of GP reported in Zhao et al.
• HRlc is redefined herein to encompass aa576-583, and HRID refers to aa 584-598.
• HR2 is a largely alpha-helical section of protein, also termed the “HR2 stalk”, that connects the GP core to the viral membrane. In the EBOV sequence, the HR2 stalk encompasses amino acid residues 599-632.
• the engineered Ebolavirus GP polypeptides contain various mutations or modifications primarily in and around the HRlc and HR2 motifs ( Figure IB). Unless otherwise noted, the organization and amino acid numbering with regard to various domains or regions of Ebolavirus GP is based on GP sequence of Zaire Ebolavirus strain Mayinga-76, which has ectodomain sequence described by GenBank ID AAG40168.1 (SEQ ID NO:1).
• the engineered Ebolavirus GP sequences of the invention typically contain, in addition to the mutations or modifications described herein, structural motifs that correspond to the GP ectodomain without the N-terminal leader, MLD and MPER (see, e.g., SEQ ID NO:2).
• the engineered Ebolavirus GP sequences 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.
• some engineered Ebolavirus GP sequences of the invention can additionally contain one or more of (1) a leader sequence, (2) part or all of MLD, (3) part of all of MPER, (4) part or all of the transmembrane domain, and (5) part or all of the cytoplasmic tail.
• Some embodiments of the invention are directed to the engineered Ebolavirus GP sequences that correspond to full length Ebolavirus GPs with one or more stabilizing modifications or mutations in the HR2 and HR1 regions described herein.
• the engineered Ebolavirus GP sequences can contain substitution at residue W615 in HR2 of a wildtype GP sequence.
• the W615 residue can be replaced with a small hydrophobic residue, e.g., L, A, V, I or F.
• the engineered Ebolavirus GP sequences can have one or more proline substitutions in the HRlc segment.
• any residue in the HRlc segment, T576, T577, E578, L579, R580, T581, F582 and S583, can be substituted with proline.
• the engineered Ebolavirus GP sequences contain T577P and/or L579P substitutions in HRlc.
• further cysteine substitutions can be introduced into the GP sequence to generate inter-GP disulfide bonds as exemplified herein.
• the leader peptide sequence at the N-terminus of the GP sequences can be removed.
• a sequence that encodes the leader peptide is included at the 5’-end of the engineered Ebolavirus GP polynucleotide sequence.
• the engineered Ebolavirus GP sequences of the invention contain only the ectodomain (i.e., MPER at the C-terminus) or otherwise a soluble portion of Ebolavirus GP proteins along with the alterations in the HR1 and HR2 regions described herein.
• the truncated or altered soluble Ebolavirus GP sequence also has MLD deleted.
• the shortened soluble GP sequence additionally has MPER at the C-terminus of the GP ectodomain removed.
• the expressed and assembled trimer protein also does not contain the leader peptide sequence (SEQ ID NO:41).
• shortened GP soluble sequence based on a Zaire Ebolavirus (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 wildtype ectodomain sequence.
• the shortened soluble GP sequence further contains a T42A mutation in the GP1 base motif (GPAmuc; SEQ ID NO:3).
• engineered soluble Ebolavirus GP sequences of the invention typically contain additional sequence modifications in the HR1 and HR2 regions of a soluble GP sequence (e.g., SEQ ID NO:2 or 3).
• the additional sequence modifications include substitution of residue W615 in HR2, extension of HR2 by (1) adding one or more adjacent residues in the MPER or (2) replacing some C-terminal HR2 residues with a longer heterologous sequence, substitutions of one or more residues in the HRlc segment with proline, and introduction of one or more disulfide bonds.
• these additional sequence modifications alone or in any combination, can promote GP trimer formation, reduce metastability, and stabilize Ebolavirus GP in a native-like trimer conformation.
• the engineered Ebolavirus GP sequences of the invention contain a truncated or shortened soluble GP sequence (e.g., SEQ ID NO:2 or SEQ ID NO:3, or a conservatively modified or substantially identical variant thereof) with additional modifications in HR2.
• a truncated or shortened soluble GP sequence e.g., SEQ ID NO:2 or SEQ ID NO:3, or a conservatively modified or substantially identical variant thereof
• the HR2 motif encompasses residues 599-632.
• the inward-facing residue in the 3-dimentional structure W615 is replaced with an amino acid residue that is smaller and more hydrophobic.
• the W615 residue can be replaced with Leu, Phe, Ala, Vai, or He so as to improve the HR2 stalk packing. These substitutions are intended to stabilize the Ebolavirus GP trimer.
• the sequence substitution is W615L.
• the HR2 can contain a further substitution at residue 612 in additional to W615 substitution.
• the amino acid substitutions are P612G/W615F.
• the engineered soluble Ebolavirus GP sequences can alternatively or additionally contain an extension of the HR2 motif.
• extension of the HR2 motif involves adding one or more adjacent residues in the MPER motif (starting at residue 633) that are naturally present in the Ebolavirus GP sequence.
• the HR2 extension can be addition of its C-terminal adjacent residues in MP ER up to residue 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646 or beyond.
• the HR2 extension include extension to the TACE cleavage site (residue 637) in MPER, i.e., having MPER N-terminal residues KTLPD (SEQ ID NO:32).
• the TACE cleavage site is responsible for GP shedding from the virion surface after the host TACE cleavage.
• the HR2 extension can include 6 additional adjacent residues in the MPER motif, i.e., having MPERN- terminal residues KTLPDQGDNDN (SEQ ID NO:33).
• extension of the HR2 motif involves substitution of some of the HR2 C-terminal residues with a longer heterologous sequence.
• some of the HR2 C-terminal residues can be replaced with a longer GCN4 leucine zipper sequence.
• HR2 can be extended by replacing residues 617-632 in HR2 with a 31 aa sequence (SEQ ID NO:34) from a GCN4 leucine zipper with PDB ID 2WPZ as exemplified herein.
• SEQ ID NO:34 a 31 aa sequence from a GCN4 leucine zipper with PDB ID 2WPZ as exemplified herein.
• Some specific embodiments of engineered Ebolavirus GP proteins containing an HR2 extension are shown in SEQ ID NOs:5-7.
• Specific embodiments of engineered Ebolavirus GP proteins containing both an HR2 extension and a residue W615 substitution are shown in SEQ ID NOs:6 and 8.
• the engineered Ebolavirus GP proteins of the invention can also contain mutations in the C segment of the HR1 region (HRlc).
• the mutations in HRlc contain one or more proline substitutions that can stabilize the GP trimer and reduce metastability.
• the proline substitution can be present at each position of HRlc.
• proline substitution can be introduced at each of residues 576-583 (TTELRTFS; SEQ ID NO:35).
• engineered Ebolavirus GP proteins of the invention can contain one or more HRlc mutations among T576P, T577P, E578P, L579P, R580P, T581P, F582P, and S583P.
• the engineered Ebolavirus GP proteins of the invention contain T577P (P 2 ) or L579P (P 4 ) mutations in HRlc.
• Specific embodiments of engineered Ebolavirus GP sequences containing one or more proline substitutions in the HRlc segment are shown in SEQ ID NOs:9-16.
• the engineered Ebolavirus GP sequences of the invention contain both proline substitution in HRlc and modifications in HR2 noted above.
• the engineered GP proteins can contain a proline substitution, and a W615 substitution and/or a further HR2 extension into MPER.
• the proline substitution can be at any residue in HRlc (e.g., at residue 577 or 579).
• W615 substitution can be any ofW615L, W615F, W615A, W615V, or W615I.
• HR2 extension in these in these engineered GP proteins can be, e.g., extension to residue 637 (i.e. , the TACE cleavage site) in MPER.
• Specific embodiments of engineered Ebolavirus GP sequences containing both a proline substitution in HRlc and also HR2 modifications are shown in SEQ ID NOs: 17-22.
• some engineered Ebolavirus GP sequences of the invention can alternatively or additionally contain one or more engineered cysteine residues for forming inter-GP disulfide bonds.
• these Cys substitutions and resulting engineered disulfide bonds can similarly function to promote trimer formation and to stabilize the GP in a native-like trimer conformation.
• the engineered GP trimer protein can contain one or more engineered inter-protomer SS bonds among G91/A575 (SS2), F153/Y534 (SSI), T520/A575 (SS3), G157/I532 (SS4), D522/A575 (SS5) and K56/G599 (SS6).
• the engineered GP trimer proteins of the invention contains engineered disulfide bond at G91/A575. Specific embodiments of engineered Ebolavirus GP proteins containing such engineered disulfide bonds are shown in SEQ ID NOs:23-28.
• the engineered Ebolavirus GP sequences of the 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.
• Suitable trimerization motifs for the invention include, e.g., T4 fibritin foldon (PDB ID: 4NCV) and 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 in promoting folding and trimerization of fibritin. See, e.g., Papanikolopoulou et al., J.
• the trimerization motif in the engineered GP proteins comprise a foldon sequence shown in SEQ ID NO:29 or the 1TD0 protein sequence shown in SEQ ID NO:30.
• the employed trimerization motif can contain a sequence that is a conservatively modified variant or substantially identical (e.g., at least 90%, 95% or 99% identical) sequence of the exemplified sequence.
• the trimerization motif can be inserted with a short GS linker.
• the linker can contain 1-6 tandem repeats of GS.
• an His6-tag can be added to the C- terminus of the trimerization motif to facilitate protein purification, e.g., by using a Nickel column.
• engineered Ebolavirus GP sequences of the invention also encompass sequences having an amino acid sequence that is substantially identical to one of these sequences, including conservatively modified variant sequences.
• the engineered Ebolavirus GP proteins of the invention 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 among different Ebolavirus species or strains, or substitutions in the nonconserved region or motif of the GP sequence of different Ebolavirus species or strains.
• Ebolaviruses suitable for the invention include any of EBOV, SUDV, TAFV, BDBV and RESTV, as well as any strain of a given Ebolavirus species.
• the engineering Ebolavirus GP proteins are chimeric.
• chimeric Ebolavirus GP proteins or immunogen polypeptides can contain a chimeric GP sequence with different subunits or domains derived from multiple Ebolavirus species or from multiple strains of the same Ebolavirus species.
• the GP1 and GP2 subunit sequences in the chimeric engineered Ebolavirus GP sequences can be derived from two different Ebolavirus species (e.g., GP2 from EBOV and GP1 from SUDV or TAFV) or from two different strains of the same Ebolavirus species (e.g., two different EBOV strains).
• the engineering Ebolavirus GP proteins may be conjugated to a presenting platform (e.g., nanoparticles or VLPs) via various means.
• the conjugation is achieved via covalent linkage, e.g., protein fusions or insertions.
• the protein sequence is fused with the presenting platform sequence via a linker sequence.
• other modifications can also be made to the engineering Ebolavirus GP proteins or the conjugating partner in order to improve stability or antigenicity.
• the various engineered Ebolavirus GP molecules of the invention can be obtained or generated in accordance with the protocols exemplified herein or methods well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3 rd ed., 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 proteins can be purified by any of the routinely practiced procedures. See, e.g., Guide to Protein Purification, Ed. Lieber, Meth.
• a substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure.
• antigenicity and other properties of the engineered Ebolavirus GP proteins can also be readily examined with standard methods, e.g., antigenic profiling using known bNAbs and non-NAbs, differential scanning calorimetry (DSC), electron microscopy, binding analysis via ELISA, Biolayer Interferometry (BLI), Surface Plasmon Resonance (SPR), and co-crystallography analysis as exemplified herein.
• standard methods e.g., antigenic profiling using known bNAbs and non-NAbs, differential scanning calorimetry (DSC), electron microscopy, binding analysis via ELISA, Biolayer Interferometry (BLI), Surface Plasmon Resonance (SPR), and co-crystallography analysis as exemplified herein.
• the invention provides Ebolavirus GP based vaccine compositions that contain a heterologous scaffold presenting or incorporating an engineered Ebolavirus GP protein described herein.
• a heterologous scaffold can be used to present the engineered Ebolavirus GP protein in the construction of the vaccines of the 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), as well as various chemical scaffolds.
• a virus-like particle (VLP) such as bacteriophage Qp VLP and nanoparticles can be used.
• the heterologous scaffold for presenting or displaying the engineered Ebolavirus GP protein is a nanoparticle.
• Various nanoparticle platforms can be employed in generating the vaccine compositions of the invention.
• the nanoparticles employed in the invention need to be formed by multiple copies of a single subunit.
• the nanoparticles are typically ball-like shaped, and/or have rotational symmetry (e.g., with 3-fold and 5-fold axes), e.g., with an icosahedral structure exemplified herein.
• the amino-terminus of the particle subunit has to be exposed and in close proximity to the 3-fold axis, and the spacing of three amino-termini has to closely match the spacing of the carboxyl-termini of the Ebolavirus GP protein.
• the immunogens comprise selfassembling naoparticles with a diameter of about 20nm or less (usually assembled from 12, 24, or 60 sububits) and 3-fold axes on the particle surface.
• the engineered Ebolavirus GP protein is presented on self-assembling nanoparticles such as self-assembling nanoparticles derived from E2p, I3-01v9 and ferritin (FR) as exemplified herein.
• E2p is a redesigned variant of dihydrolipoyl acyltransferase from Bacillus stear other mophilus that has been shown to self-assemble into thermostable 60-meric nanoparticle. See, e.g., He et al., Nat. Commun. 7:12041, 2016.
• 13-01 is an engineered protein that can selfassemble into hyperstable nanoparticles.
• Ferritin is a globular protein found in all animals, bacteria, and plants.
• the globular form of ferritin is made up of monomeric subunit proteins (also referred to as monomeric ferritin subunits), which are polypeptides having a molecule weight of approximately 17-20 kDa.
• Amino acid sequences of E2p, I3-01v9 and ferritin nanoparticle subunits as exemplified herein are shown in SEQ ID NOs:36-38, respectively.
• E2p sequence shown in SEQ ID NO:36 contains an Ala substitution at residue 92 as underscored in the sequence below.
• Sequences of some other suitable nanoparticle sequences are also known in the art. See, e.g., WO2017/192434, WO2019/089817 and WO19/241483.
• the Ebolavirus nanoparticle vaccines of the invention can employ any of these known nanoparticles, as well as their conservatively modified variants or variants with substantially identical (e.g., at least 90%, 95% or 99% identical) sequences.
• E2p subunit sequence (SEQ ID NO:36)
• nanoparticles or VLPs known in the art may also be used in the practice of the invention. These include, e.g., Aquifex aeolicus lumazine synthase, Thermotoga Maritima encapsulin, Myxococcus xanthus encapsulin, bacteriophage Qbeta virus particle, Flock House Virus (FHV) particle, ORSAY virus particle, and infectious bursal disease virus (IBDV) particle.
• Aquifex aeolicus lumazine synthase Thermotoga Maritima encapsulin, Myxococcus xanthus encapsulin, bacteriophage Qbeta virus particle, Flock House Virus (FHV) particle, ORSAY virus particle, and infectious bursal disease virus (IBDV) particle.
• FHV Flock House Virus
• IBDV infectious bursal disease virus
• molecules that may be used as the presenting platform of the nanoparticle vaccines of the invention include, e.g., molecules with the following PDB IDs: 1 JIG (12-mer Dlp-2 from Bacillus anthracis), 1UVH (12-mer DPS from Myer obacterium smegmatis), 2YGD (24-mer eye lens chaperone aB-crystallin), 3CS0 (24-mer DegP24), 3MH6 and 3MH7 (24-mer HtrA proteases), 3PV2 (12-mer HtrA homolog DegQ WT), 4A8C (12-mer DegQ from A’. Coir). 4A9G (24-mer DegQ from E. Coli.), 4EVE (12-mer HP -NAP from Helicobacter pylori strain YS29), and 4GQU (24-mer HisB from Mycobacterium tuberculosis).
• PDB IDs 1 JIG (12-mer Dlp-2 from Bacillus anth
• the Ebolavirus GP protein to be displayed on a nanoparticle platform may optionally contain a trimerization motif described above, e.g., foldon or SHP.
• Some Ebolavirus nanoparticle vaccine compositions can additionally contain other structural components that function to further enhance stability and antigenicity of the displayed immunogen.
• a locking protein domain can be inserted into the nanoparticle construct, e.g., by covalently fused to the C-terminus of the nanoparticle subunit.
• the locking domain can be any dimeric protein that is capable of forming an interface through specific interactions such as hydrophobic (van der Waals) contacts, hydrogen bonds, and/or salt bridges.
• the locking domain used in the Ebolavirus nanoparticle vaccines of the invention can contain locking domain LD4 or LD7 exemplified herein.
• Ebolavirus nanoparticle vaccines of the invention can also contain a T-cell epitope to promote robust T-cell responses and to steer B cell development towards bNAbs.
• the T-cell epitope can be located at any position in relation to the other structural components as long as it does not impact presentation of the immunogen polypeptides on the nanoparticle surface.
• Any T-cell epitope sequences or peptides known in the art may be employed in the practice of the present invention. They include any polypeptide sequence that contain MHC class-II epitopes and can effectively activate CD4+ and CD8+ T cells upon immunization, e.g., T-helper epitope that activates CD4+ T helper cells.
• the T cell epitope inserted into the Ebolavirus nanoparticle vaccine construct is a universal pan DR epitope peptide (PADRE). See, e.g., Hung et al., Mole. Ther. 15: 1211-19, 2007; Wu et al., J.
• the employed PADRE peptide contains a sequence AKFVAAWTLKAAA (SEQ ID NO:31), a conservatively modified variant or substantially identical (e.g., at least 90%, 95% or 99% identical) sequence thereof.
• the PADRE epitope is inserted at the C-terminus of a locking domain, e.g., LD4 or LD7 as exemplified herein.
• a GS restriction site can be added between the LD and the PADRE epitope.
• the scaffolded Ebolavirus vaccine compositions of the invention can be constructed in accordance with standard recombinant techniques and the methods described herein (see, e.g., the Examples herein) and/or other methods that have been 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(l l):eaau6769, 2018; and PCT publications WO2017/192434, WO2019/089817 and WO19/241483.
• nanparticle displaying any of the engineered Ebolavirus GP proteins can be constructed by fusing the Ebolavirus GP polypeptide to the subunit of the nanoparticle (e.g., E2p subunit).
• C-terminus of the Ebolavirus GP polypeptide is fused to the N-terminus of the nanoparticle subunit.
• a short peptide spacer can be used to connect the Ebolavirus GP polypeptide and the nanoparticle.
• the spacer can contain a GS restriction site and/or a longer G4S (SEQID NO:43) or (G4S)2 (SEQID NO:42) linker as exemplified herein.
• Some embodiments of the invention are directed to nanoparticles displaying engineered Ebolavirus GP protein GPAmuc-WL 2 P 2 protein (SEQ ID NO: 17) with different combination of nanoparticle subunit sequence, locking domain sequence and/or T-cell epitope.
• Some specific examples of such nanoparticle vaccine compositions have a sequence structure of GPAmuc-WL 2 P 2 -AS-G4S-ferritin, GPAmuc- WL 2 P 2 -AS-E2p-LD4-PADRE, or GPAmuc-WL 2 P 2 -AS-(G4S) 2 -I3-01v9-LD7-PADRE.
• the antigeniciy and structural integrity of the nanoparticle displayed Ebolavirus GP polypeptides can be readily analyzed via standard assays, e.g., antibody binding assays, biolayer interferometry, and negative-stain electron microscopy (EM).
• EM negative-stain electron microscopy
• the various fusion molecules can all self-assemble into nanoparticles that display immunogenic epitopes of the Ebolavirus GP proteins. By eliciting a robust neutralizing antibody response, these nanoparticles are useful for vaccinating individuals against a broad range of Ebolavirus infections.
• the engineered Ebolavirus GP proteins and the nanoparticle vaccine compositions of the invention are typically produced by first generating expression constructs (i.e. , expression vectors) that contain operably linked coding sequences of the various structural components described herein.
• the invention provides polynucleotides (DNA or RNA) that encode the engineered Ebolavirus GP proteins or polypeptides, and that encode the subunit of nanoparticles displayed the engineered Ebolavirus GP polypeptides as described herein, as well as expression vectors that harbor such polynucleotides and host cells for producing the Ebolavirus GP immunogen polypeptides and the vaccine compositions (e.g., HEK293 F cells and ExpiCHO cells exemplified herein).
• the fusion polypeptides encoded by the polynucleotides or expressed from the vectors are also included in the invention.
• polynucleotides and related vectors can be readily generated with standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfecting, transient gene expression and obtaining stable transfected cell lines are described in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3 rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou edition, 2003).
• 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.
• the selection of a particular vector depends upon the intended use of the fusion polypeptides.
• the selected vector must be capable of driving expression of the fusion polypeptide in the desired cell type, whether that cell type be prokaryotic or eukaryotic.
• Many vectors contain sequences allowing both prokaryotic vector replication and eukaryotic expression of operably linked gene sequences.
• Vectors useful for the invention may be autonomously replicating, that is, the vector exists extrachromosomally and its replication is not necessarily directly linked to the replication of the host cell's genome.
• the replication of the vector may be linked to the replication of the host's chromosomal DNA, for example, the vector may be integrated into the chromosome of the host cell as achieved by retroviral vectors and in stably transfected cell lines.
• Both viral-based and nonviral expression vectors can be used to produce the immunogens in a mammalian host cell.
• Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet. 15:345, 1997).
• Useful viral vectors include vectors based on lentiviruses or other retroviruses, adenoviruses, adenoassociated viruses, cytomegalovirus, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.
• the host cell can be any cell into which recombinant vectors carrying a fusion of the invention may be introduced and wherein the vectors are permitted to drive the expression of the fusion polypeptide is useful for the invention. It may be prokaryotic, such as any of a number of bacterial strains, or may be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells including, for example, rodent, simian or human cells. Cells expressing the fusion polypeptides of the invention may be primary cultured cells or may be an established cell line.
• cell lines exemplified herein e.g., CHO cells
• a number of other host cell lines capable well known in the art may also be used in the practice of the invention. These include, e.g., various Cos cell lines, HeLa cells, HEK293, AtT20, BV2, and N18 cells, myeloma cell lines, transformed B-cells and hybridomas.
• fusion polypeptide-expressing vectors may be introduced to the selected host cells by any of a number of suitable methods known to those skilled in the art. For the introduction of fusion polypeptide-encoding vectors to mammalian cells, the method used will depend upon the form of the vector.
• DNA encoding the fusion polypeptide sequences may 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 to eukaryotic, and particularly mammalian cells in culture. For example, LipofectAMINETM (Life Technologies) or LipoTaxiTM (Stratagene) kits are available.
• fusion polypeptide-encoding sequences controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers.
• expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• selectable marker in the recombinant vector confers resistance to the selection and allows cells to stably integrate the vector into their chromosomes.
• selectable markers include neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J. Mol.
• the transfected cells can contain integrated copies of the fusion polypeptide encoding sequence.
• the invention provides pharmaceutical or immunogenic compositions and related methods of using the engineered Ebolavirus GP sequences and nanoparticles displaying the GP proteins as described herein for preventing and treating Ebolavirus infections.
• an engineered Ebolavirus GP sequence (a polypeptide or a polynucleotide sequence) or a nanoparticle displaying an engineered protein is included in a pharmaceutical composition.
• the pharmaceutical composition can be either a therapeutic formulation or a prophylactic formulation.
• the composition additionally includes one or more pharmaceutically acceptable vehicles and, optionally, other therapeutic ingredients (for example, antibiotics or antiviral drugs).
• Various pharmaceutically acceptable additives can also be used in the compositions.
• compositions of the invention are vaccines.
• appropriate adjuvants can be additionally included.
• suitable adjuvants include, e.g., aluminum hydroxide, lecithin, Freund's adjuvant, MPLTM and IL- 12.
• the engineered Ebolavirus GP sequences and related vaccines as disclosed herein can be formulated as a controlled- release or time-release formulation. This can be achieved in a composition that contains a slow release polymer or via a microencapsulated delivery system or bioadhesive gel.
• the various ppharmaceutical compositions can be prepared in accordance with standard procedures well known in the art. See, e.g., Remington’s Pharmaceutical Sciences, 19. sup.
• Therapeutic methods of the invention involve administering an engineered Ebolavirus GP sequence of the invention or a pharmaceutical composition containing the polypeptide to a subject having or at risk of developing an Ebolavirus infection (e.g., EBOV infection).
• a pharmaceutical composition of the invention is employed in therapeutic or prophylactic applications for treating Ebolavirus infections or eliciting an protective immune response against an Ebolavirus species or strain in a subject.
• the composition can be administered to a subject to induce an immune response to an Ebolavirus species, e.g., to induce production of broadly neutralizing antibodies to the Ebolavirus species.
• a vaccine composition of the invention can be administered to provide prophylactic protection against viral infection.
• the pharmaceutical compositions of the invention can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes.
• 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 symptom(s) thereof.
• Symptoms of Ebolavirus exposure or infection include, e.g., inflammation of the liver, decreased appetite, fatigue, abdominal pain, jaundice, flu-like symptoms, itching, muscle pain, joint pain, intermittent low-grade fevers, sleep disturbances, nausea, dyspepsia, cognitive changes, depression headaches and mood changes.
• the immunogenic composition of the invention is administered in an amount sufficient to induce an immune response against an Ebolavirus.
• the compositions should contain a therapeutically effective amount of an engineered Ebolavirus GP sequence or nanoparticle vaccine composition described herein.
• the compositions should contain a prophylactically effective amount of the engineered Ebolavirus GP sequence or a nanoparticle displaying a GP protein.
• the appropriate amount of the polypeptide immunogen or the nanoparticle composition can be determined based on the specific disease or condition to be treated or prevented, severity, age of the subject, and other personal attributes of the specific subject (e.g., the general state of the subject's health and the robustness of the subject's immune system). Determination of effective dosages is additionally guided with animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject.
• the immunogenic composition is provided in advance of any symptom, for example in advance of infection.
• the prophylactic administration of the immunogenic compositions serves to prevent or ameliorate any subsequent infection.
• a subject to be treated is one who has, or is at risk for developing, an Ebolavirus infection, for example because of exposure or the possibility of exposure to the Ebolavirus.
• the subject can be monitored for Ebolavirus infection, symptoms associated with Ebolavirus infection, or both.
• the immunogenic composition is provided at or after the onset of a symptom of disease or infection, for example after development of a symptom of an Ebolavirus infection, or after diagnosis of an Ebolavirus infection.
• the immunogenic composition can thus be provided prior to the anticipated exposure to Ebolaviruses in order to attenuate the anticipated severity, duration or extent of an infection and/or associated disease symptoms, after exposure or suspected exposure to the virus, or after the actual initiation of an infection.
• composition of the invention can be combined with other agents known in the art for treating or preventing Ebolavirus infections.
• Administration of the pharmaceutical compositions and the known anti-viral agents can be either concurrently or sequentially.
• compositions containing an engineered Ebolavirus GP protein or nanoparticle vaccine of the invention can be provided as components of a kit.
• a kit includes additional components including packaging, instructions and various other reagents, such as buffers, substrates, antibodies or ligands, such as control antibodies or ligands, and detection reagents.
• An optional instruction sheet can be additionally provided in the kits.
• Truncated EBOV GPECTO sequence (with leader/MLD/MPER deleted) (SEQ ID NO:2): PLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRW GFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVH KVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDF FSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRF TPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRS EELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQP KCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLA NETTQAL
• GPAmuc (GPECTO with leader/MLD/MPER deleted +T42A) (SEQ ID NO:3) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKR WGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYV HKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKK DFF S SHPLREPVNATEDPS S GYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLES RFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKI RSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNA QPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQ LANETTQAL
• GPAmuc-W615L-L-P4 (or termed GPAmuc-WL 2 P 4 ) (SEQ ID NO:20) PLGVIHNSALQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKR WGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYV HKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKK DFF S SHPLREPVNATEDPS S GYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLES RFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKI RSEELSFTVVSTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNA QPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDG
• Locking domain LD4 (SEQ ID NO: 39):
• EBOV GP contains a heavily glycosylated mucin-like domain (MLD), which shields the glycan cap and neutralizing epitopes in GP1 and GP2.
• MLD mucin-like domain
• a soluble, mucin-deleted form of Zaire EBOV GP (GPAmuc) that produced a high-resolution crystal structures was used as a basic construct to investigate GP metastability.
• IAC immunoaffinity chromatography
• mAbl 14 which targets the receptor binding site (RBS), and mAblOO, which interacts with the GP1/GP2 interface and internal fusion loop (IFL) of two GP subunits (Science 351, 1343-1346, 2016).
• both GPAmuc samples showed aggregate ( ⁇ 9ml), dimer ( ⁇ 12ml), and monomer (-13.5- 14ml) peaks in the SEC profiles, but only GPAmuc-foldon showed a visible trimer peak (-10.5-11ml) in SEC and a faint band of slightly less than 440 kD on the BN gel.
• GPAmuc showed overall low yield, whereas GPAmuc-foldon demonstrated high trimer purity with no dimer and monomer peaks. Consistently, GPAmuc-foldon registered a single trimer band on the BN gel.
• mAblOO offers an effective IAC method for purifying native-like GP trimers due to its recognition of a quaternary GP epitope.
• trimer yield suggests a strong tendency for trimer dissociation.
• EBOV GP possesses a long, extended HR2 stalk. Even in the high- resolution GPAmuc structures, the HR2 stalk still contains less helical content than most coiled-coils in the database, ⁇ 15 aa vs ⁇ 30 aa, and becomes unwounded towards the C terminus, suggesting an inherent instability in HR2.
• King et al. solved a 3.17A-resolution structure for the MARV GPAmuc trimer in complex with a therapeutic human mAh, MR191 (Cell Host Microbe 23, 101-109. el04, 2018).
• the MARV HR2 stalk adopted a well-formed coiled-coil with canonical sidechain packing along the three-fold axis.
• EBOV and MARV GP sequences from the Virus Pathogen Database and Analysis Resource (ViPR).
• ViPR Virus Pathogen Database and Analysis Resource
• Double mutation P612G/W615F was introduced to the GPAmuc- 2WPZ construct between the CXeCC motif and the coiled-coil to reduce any structural strain in this region.
• These constructs were characterized by SEC and BN-PAGE following transient expression in 250-ml 293 F cells and mAbll4 purification. Indeed, all three designs increased trimer yield with GPAmuc-2WPZ showing the most visible trimer peak in SEC (Fig. 3B). Consistently, trimer bands were observed for all three constructs on the BN gel, albeit with less intensity for GPAmuc-L and -Ext (Fig. 3C, left). Upon mAh 100 purification, all three GPAmuc variants showed more visible trimer bands than wildtype GPAmuc (Fig. 3C, right), supporting the notion that the HR2 stalk is critical to GP trimer stability.
• Thermostability was assessed by differential scanning calorimetry (DSC) for two purified GP trimers.
• the thermal denaturation midpoint (Tm) value of the stalk-stabilized trimer was 3 °C higher than that of the wildtype trimer (67 °C vs 64 °C). Consistently, stalk stabilization also increased the onset temperature (Ton) from 52.4 °C to 62.5 °C, with a narrower half width of the peak (AT 1/2) than the wildtype trimer (3.8 °C vs. 5.1 °C).
• Antigenicity was assessed for four mAblOO/SEC-purified EBOV GP trimers in enzyme-linked immunosorbent assay (ELISA) (Fig. 3D-E).
• a panel of 10 antibodies was used, including three NAbs targeting the base (KZ52, c2G4 and c4G7), two human NAbs - mAblOO (IFL) and mAbl 14 (RBS), a non-NAb directed to the glycan cap (cl3C6), and four pan-Ebolavirus bNAbs targeting the HR2-MPER epitope (BDBV223) and IFL (ADI-15878, ADI-15946, and CA45).
• the GPAmuc trimer showed notably improved antibody binding with respect to the GPECTO trimer with an up to 7.6-fold difference in half maximal effective concentration (EC 50), indicating that MLD can effectively shield GP from antibody recognition.
• HRlc is essential to EBOV GP metastability. Since HRlc in wildtype EBOV GP is equivalent in length (8 aa) to a truncated HRIN in the prefusion-optimized HIV-1 Env, metastability in EBOV GP may not be sensitive to the HRlc length and likely requires a different solution.
• a proline mutation in HRlc termed P 1 ' 8 , may rigidify the HRlc bend and improve the EBOV GP trimer stability.
• the T577P mutation (P 2 ) was incorporated into the GPAmuc-WL 2 - foldon construct, resulting in a construct named GPAmuc-WL 2 P 2 -foldon.
• This construct was expressed transiently in 1 -liter 293 F cells and purified using an mAblOO column for SEC characterization on aHiLoad Superdex 200 16/600 GL column.
• GPAmuc-WL 2 P 2 -foldon generated a trimer peak that was two- and four-fold higher than GPAmuc-WL 2 -foldon and wildtype GPAmuc-foldon, respectively, with an average yield of 2.6 mg after SEC.
• the antigenicity of GPAmuc-WL 2 P 2 -foldon was assessed using the same panel of 10 antibodies by ELISA (Fig. 3F-G) and bio-layer interferometry (BLI).
• the T577P mutation (P 2 ) appeared to improve GP binding to most antibodies with respect to GPAmuc-WL 2 -foldon (Fig. 3G), with a 40% reduction in ECso observed for bNAb BDBV223, which targets HR2-MPER.
• the remaining six were divided into three groups: the IFL-head group (SS 1/2/4), the IFL-NHR group (SS/5), and the HR2 group (SS6).
• Six GPAmuc- SS constructs were designed and then characterized by SEC following transient expression in 250-ml 293 F cells and mAbll4 purification. Diverse SEC profiles were observed, with SS2 showing a substantial trimer peak, consistent with a band of slightly below 440 kD on the BN gel.
• the mAblOO-purified materials were also analyzed by BN-PAGE, with trimer bands observed for SS2, SS3, and SS5. Antigenicity was assessed for the three SS mutants in ELISA using six antibodies.
• Example 5 Crystallographic analysis of redesigned EBOV GPAmuc trimers [00105] To understand how the stalk and HRlc mutations affect EBOV GP, we solved crystal structures for an unliganded GPAmuc-foldon trimer with WL 2 and WL 2 P 2 at 2.3 A and 3.2 A, respectively. Both proteins showed a three-lobed, chalice- like structure, with Ca root-mean-square deviations (r.m.s.d.) of 0.92-1.14 A to a high- resolution structure (PDB: 5JQ3) at a single subunit level.
• PDB 5JQ3
• WL 2 P 2 yielded a more complete structure than WL 2 at the glycan cap (R302-V310) and the HR2 stalk (1627- D637).
• the glycan cap covers the RBS with glycan moieties visible for N238/N257/N268 in GP1 and for N563 in GP2, while in the WL 2 structure forN238/N257 in GP1 and N563/N618 in GP2.
• GP1 consists mainly of [3-strands which form a broad semi-circular groove that clamps the a3 helix and the (319-J320 strands in GP2.
• the T577P mutation appeared to have a minimum effect on the conformation of the HRlc bend, as indicated by a Ca r.m.s.d. of 0.19 A for this 8-aa segment.
• the backbone carbonyl (CO) groups of R574, A575, and T576 in one subunit formed moderate-to-strong hydrogen bonds with the head group of R164 side chain in an adjacent subunit, whereas only one CO-NH distance was within the 3.5 A cutoff in wildtype GPAmuc.
• the W615L mutation exerted a visible structural effect on the HR2 stalk. We have speculated that a bulky, inwardfacing W615 at the top of the coiled-coil destabilizes the HR2 stalk and a W615L mutation would improve its packing.
• the WL 2 P 2 structure was then compared to a recently reported Makona GPAmuc structure (PDB: 6VKM) containing the T577P/K588F mutation. In total, 353 of 398 residues in the WL 2 P 2 structure matched with the double mutant with a Ca r.m.s.d. of 0.89 A. A more complete cathepsin cleavage loop was modeled in WL 2 P 2 than previous structures (aa 197-210 vs. aa 193-213), suggesting that this loop bridges over the IFL and interacts with IFL-directed NAbs such as mAblOO (42).
• WL 2 P 2 showed more favorable hydrogen bonding patterns with a Ca r.m.s.d of 0.26 A.
• a Ca r.m.s.d of 1.7 A was obtained for the IFL region between the two structures.
• the WL 2 P 2 structure was docked into a panel of known GP/antibody complexes. Overall, WL 2 P 2 has preserved all critical GP-antibody contacts.
• the mAblOO/GP complex is of most interest as mAblOO has been used for GP purification with substantial purity.
• NPs provide an alternative to recombinant VLPs for vaccine development.
• GPAmuc trimers wildtype and WL 2 P 2 , on FR, E2p, and 13-01 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 mAblOO purification and SEC on a Superose 6 10/300 GL column.
• WL 2 P 2 outperformed wildtype GPAmuc with greater NP yield and purity. Based on molecular weight (m.w.), the SEC peaks centered at 15 ml could be unassembled GP-NP species, suggesting an inherent instability for wildtype E2p and 13-01.
• the mAblOO-purified GPAmuc-WL 2 P 2 -presenting NP samples were further analyzed by negative stain EM, showing NPs mixed with impurity.
• LD can stabilize the non-covalent NP-forming interface and the T-cell epitopes can form a cluster at the NP core to elicit a strong T-cell response upon vaccination.
• PDB protein database
• LD4 and LD7 increased the NP peak (UV280 value) by 5- and 2.5-fold for E2p and 13-01, respectively, with substantially improved NP purity. Further incorporation of a T-cell epitope, PADRE, didn’t alter or slightly improved the NP yield and purity.
• NP display appeared to improve antibody binding to the RBS and glycan cap in GP1 and reduce antibody binding for bNAbs targeting the base and IFL at the GP1/GP2 interface and the GP2 stalk.
• Example 7 Immunogenicity of EBOV GP trimers and NPs in BALB/c mice
• mice were immunized with 20pg mAblOO-purified instead of 50pg mAblOO/SEC- purified protein due to the low yield of this NP.
• mice in the NP groups would receive significantly less GP antigen than mice in the trimer group.
• Ebolavirus pseudoparticle (Ebolavirus-pp) neutralization assay using a panel of 10 antibodies against EBOV-Makona and a BDBV strain in 293 T cells.
• Ebolavirus-pp Ebolavirus pseudoparticle neutralization assay
• early EBOV NAbs KZ52, c2G4, and c4G7 neutralized EBOV but not BDBV.
• NAbs mAblOO and mAbl 14 neutralized both Ebolavirus species with different potencies.
• MLV murine leukemia virus
• a cl3C6-like antibody response was observed for 2-3 mice in the FR group and for all mice in the 13-01 v9- L7P group. Since cl3C6 but not any of the NAbs/bNAbs reacted with MLV-pps (Fig. 5E), we sought to use the MLV-pp assay to “gauge” cl3C6-like antibody responses induced by different vaccines (Fig. 5F). Indeed, we observed enhanced MLV-pp infection in excellent agreement with the ADE effect observed in the Ebolavirus-pp assay. The MLV-pp assay also indicated that the E2p-L4P NP induced a minimum cl3C6-like response comparable to GPECTO with the MLD shielding. The high level of ADE effect observed for the GPAmuc and I3-01v9-L7P groups appeared to be associated with the presence of open trimers and unassembled GP-NP species, respectively.
• the mouse data offered critical insights into the effect of various GP forms and NP carriers on vaccine-induced antibody response.
• a multilayered E2p NP with 20 closed GPAmuc trimers on the surface provides a promising vaccine candidate.
• the benefit of NP display may not be fully reflected by binding antibody titers and should be judged by the type of antibodies elicited.
• the cl3C6-like antibodies that target the glycan cap and cross-react with small secreted GP (ssGP) may cause adverse effects in vaccination.
• Two GPAmuc trimers, wildtype and WL 2 P 2 , and three NPs presenting the WL 2 P 2 trimer were also assessed in rabbits with four injections over three-week intervals. Rabbit sera collected at six timepoints during immunization were analyzed by ELISA using the same trimer probe (Fig. 6A-B). Of note, rabbits were immunized with 20pg of mAblOO/SEC-purified I3-01v9-L7P NP to reduce the cl3C6-like responses.
• the WL 2 P 2 group showed higher ECso titers for all six time points except for wl 1, with a significant P value of 0.0229 obtained for w5 (two weeks after the second injection).
• the three NP groups a consistent pattern was observed, with the I3-01v9 group showing the highest ECso tier and the FR group the lowest throughout the immunization.
• a significant difference was found between the I3-01v9-L7P group and the E2p-L4P group for w8, wl 1, and wl3 (four weeks after the last injection), with a P value of 0.0021-0.0053.
• the I3-01v9-L7P NP group showed higher ECso tiers for all six time points, with significant P values obtained for w8, wl 1, and wl3.
• the FR and E2p-L4P groups yielded lower ECso titers than their respective trimer group at w2 and w5, but this pattern was reversed at w8 and wl 1 with significant P values (0.0421 and 0.0492 for FR and E2p, respectively) at w8.
• the advantage of these two NP groups diminished at wl 1, showing lower ECso tiers than the trimer group at the last time point, wl3.
• the I3-01v9-L7P NP group yielded higher average IC50 (50% inhibitory concentration) titers, at 211.3 pg/ml and 11.72 pg/ml for EBOV and BDBV, respectively, than other groups, confirming that the unassembled GP-NP species - not the NP itself - were the cause of ADE in mouse immunization.
• IC50 50% inhibitory concentration
• all vaccine groups at wl 1 showed no enhancement of MLV- pp infection, in contrast to the mouse data (Fig. 5F). Therefore, cl3C6-like antibodies appeared to be absent in serum toward the end of rabbit immunization.
• rabbit IgGs obtained from the earlier time points demonstrated an increasing NAb response accompanied by a declining cl3C6-like antibody response (Fig. 6C-F).
• ADE was first observed for the two trimer groups, the FR group, and the two multilayered NP groups at w2, w5, and w8, but then disappeared at w5, w8, and wl 1, respectively.
• Our analysis thus revealed a unique pattern for vaccine-induced cl3C6-like antibodies in rabbits, which might change epitope specificity through the mechanism of “gene conversion” upon repeated antigen stimulation.
• Example 9 B cell response profiles associated with EBOV GP trimers and NPs
• HCV E2 core E2mc3
• E2mc3-E2p NP He et al., Sci. Adv. 6: eaaz6225, 2020.
• VH heavy-chain variable gene
• HCDR3 heavy chain complementarity determining region 3
• Example 9 Some exemplified materials and methods
• plasmid in 25 ml of Opti-MEM transfection medium (Life Technologies, CA) was mixed with 5 ml of PEI-MAX (1.0 mg/ml) in 25 ml of Opti-MEM. After 30-min incubation, the DNA-PEI-MAX complex was added to IL 293 F cells. Culture supernatants were harvested five days after transfection, clarified by centrifugation at 2200 rpm for 22 min, and filtered using a 0.45 pm filter (Thermo Scientific). GPAmuc proteins were extracted from the supernatants using an mAbll4 antibody column or an mAblOO antibody column.
• ExpiCHOTM Expression Medium was added to reduce cell density to 6x 10 6 ml 4 for transfection.
• the ExpiFectamineTM CHO/plasmid DNA complexes were prepared for 100-ml transfection in ExpiCHO cells following the manufacturer’s instructions.
• 100 pg of plasmid and 320 pl of ExpiFectamineTM CHO reagent were mixed in 7.7 ml of cold OptiPROTM medium (Thermo Fisher).
• ExpiCHO cells were cultured in a shaker incubator at 33 °C, 115 rpm and 8% CO2 following the Max Titer protocol with an additional feed on day five (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 pm filter (Thermo Fisher).
• the mAblOO antibody column was used to extract nanoparticles from the supernatants, which was followed by SEC on a Superose 6 10/300 GL column.
• protein concentration was determined using UV280 absorbance with theoretical extinction coefficients.
• BN-PAGE Blue Native Polyacrylamide Gel Electrophoresis: EBOV GPAmuc and GPAmuc-presenting 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 added to a 4-12% TM
• Enzyme-Linked Immunosorbent Assay Each well of a CostarTM 96-well assay plate (Coming) was first coated with 50 pl PBS containing 0.2 pg of the appropriate antigens. The plates were 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 150 pl of a blocking buffer consisting of PBS, 40 mg ml 4 blotting-grade blocker (Bio-Rad), and 5% (v/v) FBS. The plates were incubated with the blocking buffer for 1 hour at room temperature, and then washed five times with wash buffer.
• antibodies were diluted in the blocking buffer to a maximum concentration of 10 pg ml 4 followed by a 10-fold dilution series. For each antibody dilution, a total of 50 pl volume was added to the appropriate wells.
• serum or plasma was diluted by 10-fold for mouse and 50-fold for rabbit in the blocking buffer and subjected to a 10-fold dilution series.
• sample dilution a total of 50 pl volume was added to the wells. Each plate was incubated for 1 hour at room temperature, and then washed 5 times with PBS containing 0.05% Tween 20.
• Bio-Layer interferometry The kinetics of GPAmuc and GPAmuc- presenting nanoparticle binding to a panel of 10 antibodies was measured using an Octet Red96 instrument (forteBio, Pall Life Sciences). All assays were performed with agitation set to 1000 rpm in forteBio 1 x kinetic buffer. The final volume for all the solutions was 200 pl per well. Assays were performed at 30 °C in solid black 96-well plates (Geiger Bio-One).
• DSC Differential scanning calorimetry
• the crystallization experiments were carried out using the sitting drop vapor diffusion method on an automated CrystalMationTM robotic system (Rigaku) at both 4 °C and 20 °C at The Scripps research Institute (TSRI) (Elsliger et al., Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 1137-1142, 2010).
• TSRI Scripps research Institute
• EBOV GP was concentrated to ⁇ 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.
• the diffractable crystals were obtained after two weeks at 20 °C.
• the crystals of EBOV GP were cryoprotected with 25% glycerol, mounted in a nylon loop and flash frozen in liquid nitrogen.
• the data were collected for two crystals of GPAmuc-WL 2 -foldon and GPAmuc-WL 2 P 2 -foldon at Advanced Photon Source (APS) beamline 23IDB, which were diffracted to 2.3 A and 3.2 A resolution, respectively.
• the diffraction data sets were processed with HKL-2000.
• the overall completeness of the two data sets was 95.77% and 99%, respectively.
• Electron microscopy (EM) assessment of nanoparticle constructs The initial EM assessment of EBOV GPAMuc nanoparticles was conducted at the Scripps Core Microscopy Facility. Briefly, nanoparticle samples were prepared at the concentration of 0.01 mg/ml. Carbon-coated copper grids (400 mesh) were glow- discharged and 8 pL of each sample was adsorbed for 2 minutes. Excess sample was wicked away and grids were negatively stained with 2% uranyl formate for 2 minutes. Excess stain was wicked away and the grids were allowed to dry. Samples were analyzed at 80kV with a Talos L120C transmission electron microscope (Thermo Fisher) and images were acquired with a CETA 16M CMOS camera.
• EM Electron microscopy
• mice were purchased from The Jackson Laboratory. Mice were housed in ventilated cages in environmentally controlled rooms at Scripps Research, in compliance with an approved IACUC protocol and AAALAC guidelines. Mice were immunized at weeks 0, 3, 6 and 9 for a total of four times.
• IACUC Institutional Animal Care and Use Committee
• Each immunization consisted of 200 pl of antigen/ adjuvant mix containing 50 pg of vaccine antigen (or 20 pg for the I3-01v9 NP) and 100 pl of adjuvant, AddaVax or Adju-Phos (InvivoGen), via the intraperitoneal (i.p.) route. Blood was collected two weeks after each immunization. All bleeds were performed through the facial vein (submandibular bleeding) using lancets (Goldenrod). While intermediate bleeds were collected without anticoagulant, 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. The cells were washed once in PBS and then resuspended in 1 ml of ACK Red Blood Cell lysis buffer (Lonza). After two rounds of washing with PBS, peripheral blood mononuclear cells (PBMCs) were resuspended in 2 ml of Bambanker Freezing Media (Lymphotec). In addition, spleens were also harvested and grounded against a 70-pm cell strainer (BD Falcon) to release the splenocytes into a cell suspension.
• PBMCs peripheral blood mononuclear cells
• Splenocytes were centrifuged, washed in PBS, treated with 5 ml of Red Blood Cell Lysis Buffer Hybri-Max (Sigma- Aldrich), and frozen with 10% of DMSO in FBS. While serum and plasma were used in EBOV neutralization assays, 80% of the plasma from individual mice at week 11 was purified using a 0.2-ml protein G spin kit (Thermo Scientific) following the manufacturer’s instructions. Purified mouse IgGs at week 11 (wl 1) were assessed in pseudovirus neutralization assays. Rabbit immunization and blood sampling were carried out under a subcontract at ProSci (San Diego, CA).
• Ebolavirus pseudoviral particle (Ebolavirus-pp) neutralization assays were utilized to assess the neutralizing activity of previously reported mAbs and vaccine-induced antibody responses in mice and rabbits.
• Ebolavirus-pps were generated by co-transfection of HEK293 T cells with the pNL4- 3.1ucR-E- plasmid (NIH AIDS reagent program) and the expression plasmid encoding the GP gene of an EBOV Makona strain (GenBank Accession number: KJ660346) or a BDBV Kenya strain (GenBank Accession number: KR063673) at a 4: 1 ratio by lipofectamine 3000 (Thermo Fisher Scientific). After 48 to 72 h, Ebolavirus-pps were collected 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 pg/ml, or purified IgGs at a starting concentration 300 pg/ml for mouse and 1000 pg/ml for rabbit were mixed with the supernatant containing Ebolavirus-pps and incubated for 1 h at 37°C in white solid-bottom 96-well plate (Coming).
• HEK293 T cells or TZM-bl cells were used for Ebolavirus-pp neutralization assays. Briefly, HEK293 T cells or TZM-bl cells at IxlO 4 were added to each well and the plate was incubated at 37°C for 48 h.
• MLV-pps Lentiviral vectors pseudotyped with the murine leukemia virus (MLV) Env gene, termed MLV-pps, were produced in HEK293 T cells and included in the neutralization assays as a negative control. As non- NAb cl3C6 exhibited enhanced MLV-pp infection, the MLV-pp assay was also used to detect the cl3C6-like, ADE-causing antibody response in immunized animal samples. [00131] Bulk sorting of EBOV GPAmuc-specific mouse B cells: Spleens were harvested from immunized mice 15 days after the last immunization and cell suspension was prepared.
• Biotin excess was removed by SEC on a HiLoad Superdex 200 16/600 column (GE Healthcare).
• the Avi- tagged GPAmuc peak is centered at 14.5 ml, while a broader peak of biotin ligase can be found at 65-70 ml (WT) or 60-65 ml (UFOg 2 ).
• WT 65-70 ml
• UFOg 2 60-65 ml
• Cells and biotinylated proteins were incubated for 5 min at 4 °C, followed by the addition of 2.5 pl of anti-mouse IgG fluorescently labeled with FITC (Jackson ImmunoResearch 115-095-071) and incubated for 15 min at 4 °C.
• 5 '-RACE cDNA was obtained from bulk-sorted splenic B cells of each mouse with SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (TaKaRa).
• the immunoglobulin PCRs were set up with Platinum Taq High-Fidelity DNA Polymerase (Life Technologies) in a total volume of 50 pl, with 5 pl of cDNA as template, 1 pl of 5 '-RACE primer, and 1 pl of 10 pM reverse primer.
• the 5 ’-RACE primer contained a PGM/S5 Pl adaptor, while the reverse primer contained a PGM/S5 A adaptor.
• Template preparation and (Ion 530) chip loading were performed on Ion Chef using the Ion 520/530 Ext Kit, followed by sequencing on the Ion S5 system with default settings.
• the mouse Antibodyomics pipeline was used to process the raw data and determine distributions for germline gene usage, somatic hypermutation (SHM), germline divergence, and H/KCDR3 loop length.

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