Pre-fusion stabilized cchfv GP38-GN-GC glycoprotein complex

Abstract

Provided herein are engineered proteins comprising engineered CCHFV glycoprotein ectodomain. Such engineered CCHFV glycoprotein ectodomains may exhibit improved conformational homogeneity and / or biophysical stability. The ectodomains may be incorporated into nanoparticles or virus-like particles. Methods are also provided for use of the engineered CCHFV glycoprotein ectodomains as diagnostics, in screening platforms, and / or in vaccine compositions.

Description

DESCRIPTIONPRE-FUSION STABILIZED CCHFV GP38-GN-GC GLYCOPROTEIN COMPLEXREFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of United States provisional application number 63 / 573,929, filed April 3, 2024, the entire contents of which are incorporated herein by reference.STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant no. R01 AI152246 and Grant no. U19 AI142777 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on March 31, 2025, is named UTFBP1355WO.xml and is 62,876 bytes in size.BACKGROUND1. Field

[0004] The present disclosure relates generally to the fields of medicine, virology, immunology, and protein engineering. More particular, the disclosure relates to engineered protein complexes comprising Crimean Congo Hemorrhagic Fever Virus (CCHFV) glycoproteins and the use thereof in vaccine formulation, antibody detection, and antibody isolation.2. Description of Related Art

[0005] Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe viremia and hemorrhagic fever in humans with a case fatality rate of up to 40% (WHO, 2020; Ergonul, 2006). The virus is spread by ticks of the Hyalomma genus, which are distributed widely throughout parts of Europe, Africa, and Asia (European Centre for Disease Prevention and Control, 2023). CCHFV has a broad host range and can infect humans as well as diverse species of wild animals. Although transmission to humans mainly results from the bite of infected ticksor contact with infected livestock, human-to-human transmission has also been reported (Hawman & Feldmann, 2023; CDC, 2013). The widening range of the Hyalomma tick vector due to climate change, the ability of CCHFV to infect migratory birds and animals subject to livestock trade, the high case fatality rate in humans, and the possibility of human-to-human transmission have resulted in CCHFV being listed as a priority pathogen by the World Health Organization (WHO, 2018). Despite this, no vaccine or therapeutic has yet been approved to treat or prevent infection with CCHFV, highlighting the need for the development of medical coun termeasures .SUMMARY

[0006] Provided herein are engineered CCHFV glycoprotein ectodomain that comprise GP38 and GnHand Gc, wherein the GP38 and GnHportion comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of positions 248-587 of SEQ ID NO: 1, and wherein the Gc portion comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of positions 1061-1544 of SEQ ID NO: 1, and wherein the engineered ectodomain comprises at least one substitution or substitution set selected from the group consisting of: Q496C / V562C; G371C / Y546C; S370C / Y546C; K404D; G527C / K1162C; R565C / P1161C; R579C / D1214C; and C1354S, with the positions being relative to the sequence of SEQ ID NO: 1. The engineered ectodomain may comprise at least one substitution set selected from the group consisting of: (a) Q496C / V562C and R565C / P1161C; (b) Q496C / V562C and R579C / D1214C; (c) Q496C / V562C and G371C / Y546C; (d) G371C / Y546C and R565C / P1 161C; (e) G371C / Y546C and R579C / D1214C; and (f) R565C / P1161C and R579C / D1214C, with the positions being relative to the sequence of SEQ ID NO: 1. The Gc region may be fused to the C-terminus of the Gn11domain by a flexible linker. The flexible linker may be a glycine-serine linker. The engineered CCHFV glycoprotein ectodomain may comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 250- 1100 of any one of SEQ ID NOs: 10-17 or 31-36.

[0007] Provided herein are engineered proteins comprising an engineered CCHFV glycoprotein ectodomain, which comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequence of positions 248-587 of SEQ ID NO: 1, and which comprise a disulfide bond between the C-terminal [3-strand of GP38 and the N-terminal -strand of the GnHdomain B. The disulfide bond may be between positions Q496C and V562C, with the positions being relative to the sequence of SEQ ID NO: 1. The cleavage site between GP38 and GnHmay be mutated. The engineered CCHFV glycoprotein ectodomain may further comprise a R516S substitution. The engineered CCHFV glycoprotein ectodomain may further comprise Gn domain C region. The engineered CCHFV glycoprotein ectodomain may comprise a sequence at least 90% identical to the sequence of positions 248-663 of SEQ ID NO: 1. The engineered CCHFV glycoprotein ectodomain may further comprise a Gn MPER region. The engineered CCHFV glycoprotein ectodomain may comprise a sequence at least 90% identical to the sequence of positions 248-690 of SEQ ID NO: 1. The engineered CCHFV glycoprotein ectodomain may further comprise a mucin-like domain region. The engineered CCHFV glycoprotein ectodomain may comprise a sequence at least 90% identical to the sequence of positions 25-587 of SEQ ID NO: 1. The engineered CCHFV glycoprotein ectodomain may be fused or conjugated to a trimerization domain. The trimerization domain may be positioned C-terminally relative to the engineered CCHFV glycoprotein ectodomain.

[0008] The cleavage site between the mucin-like domain and GP38 may be mutated. The engineered CCHFV glycoprotein ectodomain may comprise a substitution corresponding to RSKR (residues corresponding to positions 244-247 of SEQ ID NO: 1) to RRRRRR (SEQ ID NO: 18).

[0009] The engineered CCHFV glycoprotein ectodomain may comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of positions 250-589 of SEQ ID NO: 6; positions 250-665 of SEQ ID NO: 7; positions 250-692 of SEQ ID NO: 8; or positions 250-589 of SEQ ID NO: 9.

[0010] The engineered CCHFV glycoprotein ectodomain may be fused or conjugated to a transmembrane domain.

[0011] Provided herein are engineered CCHFV glycoprotein trimers comprising at least one subunit according to any one of the present embodiments.

[0012] Provided herein are nucleic acid molecules comprising a nucleotide sequence that encodes the amino acid sequence of the engineered protein of any one of the presentembodiments. The nucleic acid may comprise a DNA expression vector. The nucleic acid may comprise an mRNA.

[0013] Provided herein are immunogenic compositions comprising the engineered protein, engineered trimer, or nucleic acid molecule of any one of the present embodiments, and a pharmaceutically acceptable carrier. The immunogenic composition may further comprise an adjuvant. The immunogenic composition may be for use in inducing an immune response to a CCHFV glycoprotein ectodomain in a subject.

[0014] Provided herein are methods of preventing a CCHFV infection or a disease associated with a CCHFV infection in a subject, comprising administering to the subject an effective amount of the immunogenic composition according to any one of the present embodiments.

[0015] Provided herein is the use of the engineered protein, engineered trimer, or nucleic acid molecule of any one of the present embodiments in the manufacture of an immunogenic composition for inducing an immune response to a CCHFV glycoprotein ectodomain.

[0016] Provided herein are methods of producing antibodies that bind to a CCHFV glycoprotein ectodomain, comprising administering to a subject an effective amount of the immunogenic composition according to any one of the present embodiments.

[0017] Provided herein are compositions comprising the engineered protein or the engineered trimer of any one of the present embodiments bound to an antibody.

[0018] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0020] FIGS. 1A-1C. AlphaFold2 predicts a CCHFV GP38-Gn heterodimer with conserved domain architecture characteristic of class II accompanying proteins. A cartoon representation of (FIG. 1A) ANDV Gn (PDB ID: 6ZJM) and (FIG. IB) an AlphaFold2 prediction of CCHFV GP38-Gn heterodimer (lbArl0200, GenBank AF467768.2) shaded by conserved domains of class II accompanying proteins. These domains comprise the ‘base’ subdomain of the accompanying proteins. Areas without conservation are colored white. (FIG. 1C) CCHFV GP38-Gn heterodimer shaded by GP38, Gn head, Domain C, and MPER domains. The spheres and corresponding inset represent the location of the beneficial cysteine substitutions in the GP38-Gn heterodimer. Sulfur atoms are displayed.

[0021] FIGS. 2A-2C. Addition of Q496C / V562C and truncations of Gn boost expression of the GP38-Gn heterodimer. (FIG. 2A) A schematic of the GP38-Gn constructs in the SDS-PAGEs in FIGS. 2B and 2C. SS represents the native CCHFV M segment signal sequence while H2S represents the HRV3C-cleavable 8x His and Twin-Strep tags. Gray arrows indicate cleavage sites, and black arrows indicate a mutated cleavage site resulting in a linker with the R- S mutation underlined. The amino acid sequence SRLL is SEQ ID NO: 21. The amino acid sequence 6xR is SEQ ID NO: 18. Gray X’s indicate sites of cysteine substitution, black Y’s indicate sites of N-linked glycosylation in the mature purified protein of interest, and black I’s indicate sites of O-linked glycosylation in the mature purified protein of interest. Note that the MLD is highly glycosylated with both N-linked and O-linked glycans, but MLD glycosylation was omitted for simplicity and clarity. (FIG. 2B) An SDS-PAGE of a GP38-Gn ectodomain construct and two Gn-truncated constructs and (FIG. 2C) an SDS-PAGE of a GP38-Gn ectodomain construct and double cysteine mutants in the ectodomain backbone. Molecular weight standards in kDa are on the left. The black arrows indicate bands corresponding to the proteins of interest. Darkness of the band correlates to level of expression.

[0022] FIGS. 3A-3C. The Q496C / V562C disulfide bond forms and increases TmofGP38-GnH. (FIG. 3A) A schematic of the GP38-GnHconstructs in FIGS. 3B and 3C. SSrepresents the native CCHFV M segment signal sequence while H2S represents the HRV3C- cleavable 8x His and Twin-Strep tags. Gray arrows and dotted lines indicate cleavage sites, and black arrows indicate a mutated cleavage site resulting in a covalent linker with the R->S mutation underlined. The amino acid sequence SRLL is SEQ ID NO: 21. The amino acid sequence 6xR is SEQ ID NO: 18. Gray X’s indicate sites of cysteine substitution, black Y’s indicate sites of N-linked glycosylation in the mature purified protein of interest, and black I’s indicate sites of O-linked glycosylation in the mature purified protein of interest. Note that the MLD is highly glycosylated with both N-linked and O-linked glycans, but MLD glycosylation was omitted for simplicity and clarity. (FIG. 3B) An SDS-PAGE of GP38-GnHconstructs. Molecular weight standards in kDa are on the right. Darkness of the band correlates to level of expression. (FIG. 3C) Differential scanning fluorimetry thermal stability analysis of expressed variants in FIG. 3A. The vertical dotted lines and corresponding labels denote the melting temperature for each construct. The construct GP38-6xR-GnH DShas melting temperature corresponding to 57°C; fhe construct GP38-GnH DShas melting temperature corresponding to 60°C.

[0023] FIGS. 4A-4D. A 2.5 A crystal structure of GP38-GS-GnH DS. (FIG. 4A) A schematic of the GP38-GS-GnHconstruct. SS represents the native CCHFV M segment signal sequence while H2S represents the HRV3C-cleavable 8x His and Twin-Strep tags. (GGGS)x2 (SEQ ID NO: 20) represents the eight amino-acid Gly-Ser linker, and black arrows with the corresponding label indicate the amino acid position preceding and proceeding the Gly-Ser linker (CCHFV IbArl0200 numbering). The amino acid sequence 6xR is SEQ ID NO: 18. Gray arrows and dotted lines indicate cleavage sites. Gray X’s indicate sites of cysteine substitution, black Y’s indicate sites of N-linked glycosylation in the mature purified protein of interest, and black I’s indicate sites of O-linked glycosylation in the mature purified protein of interest. Note that the MLD is highly glycosylated with both N-linked and O-linked glycans, but MLD glycosylation was omitted for simplicity and clarity. (FIG. 4B) Side view (upper panel) and top view (lower panel) of GP38-GS-GnH DS. GP38 is colored in light blue, and GnHis colored in orange. N and C indicate the N and C-termini of the protein. (FIG. 4C) Binding interfaces of GP38 and GnHfrom the crystal structure. Interacting side chains are depicted as sticks. Hydrogen bonds are depicted as black dotted lines. (FIG. 4D) A surface view of GP38- GnH DSdisplaying hydrophobicity. The left panel shows GP38 from this crystal structure as a surface view and GnHfrom this crystal structure as a ribbon. The right panel shows a 180° rotated surface view of GnH.

[0024] FIGS. 5A-5C. Vaccination of C57BL6 / J mice with GP38-Gn constructs. (FIG. 5 A) Schematic of vaccination schedule. Mouse image generated by BioRender. Black arrows indicate vaccination and gray arrow indicates sera harvest. (FIG. 5B) ELISA of vaccinated mice sera tested for binding to GP38, GP38-GnH DS, and GP38-GnH DS+c. Within the groups for each coating antigen, the immunogens are, from left to right, GP38, GP38-GnH DS, GP38-GnH"DS+C, and PBD. Values shown are the logarithm of the average area under the curve (AUC) for each group. (FIG. 5C) Ability of sera from vaccinated mice to neutralize CCHFV tecVLP infection of Vero cells. Non-neutralizing anti-GP38 antibody ADI-46152 used as a negative control, neutralizing anti-Gc antibody ADI-36145 used as a positive control. Statistical comparison was performed using Turkey’s multiple comparison test (****p < 0.0001, ***p < 0.001, **p < 0.01, *p <0.05, ns p>0.05).

[0025] FIGS. 6A-6G. A 3.4 A cryo-EM structure of GP38-GnH DS-Gc. (FIG. 6A) SS represents the native CCHFV M segment signal sequence while H2S represents the HRV3C- cleavable 8x His and Twin-Strep tags. (GGGS)x3 (SEQ ID NO: 30) represents the 12 aminoacid Gly-Ser linker bordered by the TEV cleavage sites in cyan. Gray arrows and dotted lines indicate cleavage sites, and black arrows indicate a mutated cleavage site resulting in a covalent linker with the R->S mutation underlined. The amino acid sequence SRLL is SEQ ID NO: 21. The amino acid sequence 6xR is SEQ ID NO: 18. Gray X’s indicate sites of cysteine substitution, black Y’s indicate sites of N-linked glycosylation in the mature purified protein of interest, and black I’s indicate sites of O-linked glycosylation in the mature purified protein of interest. Note that the MLD is highly glycosylated with both N-linked and O-linked glycans, but MLD glycosylation was omitted for simplicity and clarity. (FIG. 6B) Side view of a 3D cryo-EM map of GP38-GnII DS-Gc bound by ADI-46152 (VH dark gray, VL light gray) and ADI-36125 (VH gray, VL white) Fabs. GP38 is colored in light blue, GnHis colored in orange, Gc is colored in green. (FIG. 6C) Interfaces of GP38 and GnHwith the fusion loops of Gc and (FIG. 6D) the Domain II tip. (FIG. 6E) Binding interface of Gn11and Gc Domain II base. (FIG. 6F) Binding interface of GP38 and the [3-ribbon domain of Gn11(FIG. 6G) Fusion loops of Gc in the pre-fusion (left, this study) and post-fusion (right, PDB ID: 7A59) conformation. Interacting side chains are depicted as sticks. Hydrogen bonds are depicted as black dotted lines.

[0026] FIGS. 7A-7H. Vaccination of mAb5A3-treated C57BL6 / J mice with GPC constructs. (FIG. 7A) Schematic of vaccination schedule. Mouse image generated byBioRender. The first two arrows indicate vaccination, the third arrow indicates sera harvest, the fourth arrow indicates challenge with IbArl0200. (FIG. 7B) Survival curve of vaccinated mice (n=10). (FIG. 7C) Ability of sera from vaccinated mice (6 weeks post-vaccination) to neutralize lb Ari 0200 infection of VeroE6 cells. Higher value indicates more neutralization. (FIGS. 7D-7H) ELISA of vaccinated mice sera (6 weeks post- vaccination) tested for binding to whole irradiated virus, GP38, Gn, Gc, and GP38-GnH DS-Gc. Values shown are the area under the curve (AUC) for each group. Higher value indicates more binding. Black line reflects the median value. Circle with black X denotes mice that succumbed to challenge. Statistical comparison was performed using 2-way ANOVA with Turkey corrections for multiple comparisons (****p < 0.0001, ***p < 0.001, **p < 0.01, *p <0.05, ns p>0.05).

[0027] FIGS. 8A-8D. Addition of disulfides in GP38-Gn-Gc boost expression and thermostability. (FIG. 8 A) A schematic of the GP38-Gn-Gc base construct in the SDS-PAGEs in panels B and thermostability analysis in panel C. SS represents the native CCHFV M segment signal sequence while H2S represents the HRV3C-cleavable 8x His and Twin-Strep tags. Gray arrows indicate cleavage sites, and black arrows indicate a mutated cleavage site resulting in a linker with the R- S mutation underlined. The amino acid sequence SRLL is SEQ ID NO: 21. The amino acid sequence 6xR is SEQ ID NO: 18. (FIG. 8B) An SDS-PAGEs of a disulfide substitutions and point substitutions in GP38-Gn-Gc. Molecular weight standards in kDa are on the left. Darkness of the band correlates to level of expression. (FIG. 8C) Thermostability analysis of three disulfide substitutions using the UNcle (UNchained Labs). Melting temperature (Tm) is indicated by colored dotted line. (FIG. 8D) Table with Tm for the constructs shown in FIG. 8C.

[0028] FIGS. 9A-9G. Several disulfides and their combinations boost expression, thermostability, and prefusion stability in the GPC ectodomain. (FIG. 9A) A schematic of the GPC base construct. SS represents the native CCHFV M segment signal sequence, and H2S represents the HRV3C-cleavable 8x His and Twin-Strep tags. Gray arrows indicate cleavage sites, and black arrows indicate a mutated cleavage site resulting in a linker with the R^S mutation underlined. Black Ys indicate sites of N-linked glycosylation in the mature purified protein of interest, and black Is indicate sites of O-linked glycosylation in the mature purified protein of interest. The amino acid sequence SRLL is SEQ ID NO: 21. The amino acid sequence 6xR is SEQ ID NO: 18. The amino acid sequence (GGGS)x3 is SEQ ID NO: 30. Note that the MLD is highly glycosylated with both N-linked and O-linked glycans, but MLDglycosylation was omitted for simplicity and clarity. The full amino acid sequence of the GPC base construct is SEQ ID NO: 37. (FIG. 9B) Model illustrating location of beneficial cysteine substitutions. (FIG. 9C) SDS-PAGE gel corresponding to relative expression of the GPC ectodomain constructs. (FIG. 9D) Biolayer interferometry curve showing the binding ability of each construct to a postfusion-specific antibody, ADI-37801. (FIG. 9E) Table outlining the beneficial cysteine substitutions in each combination variant (Cl -6) and their respective melting temperatures (Tm) and binding to ADI-37801. (FIG. 9F) Differential scanning fluorimetry melting temperature profiles of the single disulfide variants and (FIG. 9G) the disulfide combination variants. Dotted line indicates the Tm of the base construct.

[0029] FIGS. 10A-10B. R516S boosts expression of GP38-Gn ectodomain, and six consecutive arginine residues increase production of protein of interest. (FIG. 10A) A schematic of the GP38-Gn constructs in FIG. 10B. SS represents the native CCHFV M segment signal sequence while H2S represents the HRV3C-cleavable 8x His and Twin-Strep tags. Gray arrows and dotted lines indicate cleavage sites, and black arrows indicate a mutated cleavage site resulting in a covalent linker with the R->S mutation underlined. Gray X’s indicate sites of cysteine substitution, black Y’s indicate sites of N-linked glycosylation in the mature purified protein of interest, and black I’s indicate sites of O-linked glycosylation in the mature purified protein of interest. The amino acid sequence SRLL is SEQ ID NO: 21. The amino acid sequence 6xR is SEQ ID NO: 18. Note that the MLD is highly glycosylated with both N-linked and O-linked glycans, but MLD glycosylation was omitted for simplicity and clarity. (FIG. 10B) An SDS-PAGE of GP38-GnHconstructs. Molecular weight standards in kDa are on the right. Darkness of the band correlates to level of expression.

[0030] FIG. 11. Electron density of stabilizing disulfide bond, Asn557-GlcNAc, and interfacing residues in the GP38-GnH DScrystal structure. Electron density (2Fo-Fc) is shown as gray mesh. GP38 is shown in light gray and Gn is shown in dark gray. Side chains are depicted as sticks.

[0031] FIG. 12. Sequence alignment of nairo virus Gn (residues 552-562, lb Ari 0200 numbering). Multiple sequence alignment was generated by Clustal Omega. Asterisks indicate positions that have fully conserved residues, colons indicate strong conservation among groups, and periods indicate weak conservation among groups. Gray text indicates N-X-T / S N-linked glycosylation site. The amino acid sequences, from top to bottom, are SEQ ID NOs:

[0032] FIG. 13. ELISA of vaccinated mice sera tested for binding to GP38, GP38-GnH"DS, and GP38-GnH DS+C. The elicited antibody binding titers to GP38, GP38-GnH DSand GP38- GnH-Ds+cwerequantifiedviaELISA. The area under the curve (AUC) values are shown in FIG. 5B.

[0033] FIG. 14. SEC and DSF of GP38-GnH DS-Gc. Size exclusion chromatography for GP38-GnH DS-Gc (left). Differential scanning fluorimetry thermal stability analysis of GP38, Gc, and GP38-GnH DS-Gc (right). The vertical dotted lines and corresponding labels denote the melting temperature for each construct. Gc has a melting temperature of 48°C; GP38-GnH DS- Gc has a melting temperature of 65 °C.

[0034] FIG. 15. Representative cryo-EM processing workflow of GP38-GnH DS-Gc. Flowchart outlining cryo-EM processing of GP38-GnH DS-Gc bound to ADI-46152 and ADI- 36125 Fabs.

[0035] FIG. 16. Validation and fit in map.

[0036] FIG. 17. Hydrophobicity analysis of GP38-GnH DS-Gc.

[0037] FIGS. 18A-18F. Weight loss (FIG. 18 A), clinical score (FIG. 18B), and sera binding titers (FIGS. 18C-18F) of mice immunized with CCHFV immunogens. Within each immunogen on the X-axis, the data represent, from left to right, Pre-vaccinated (day 0), Postprimer (day 21), and Post-boost (day 42). Values shown are the normalized logarithm of the area under the curve (AUC) for each group. Bar reflects the median value of the individual values, shown by points overlay ed on the bar.

[0038] FIGS. 19A-19F. ELISA of vaccinated mice sera (6 weeks post-vaccination) tested for binding to clinically relevant strains of GP38. Values shown are AUC for each group. Higher value indicates more binding. Black line reflects the median value. Circle with black X denotes mice that succumbed to challenge.DETAILED DESCRIPTION

[0039] Provided herein are stabilized complexes of CCHFV glycoproteins that can be used as a vaccine antigen and / or to detect anti-CCHFV antibodies in human sera and / or to isolate anti-CCHFV antibodies from human sera. Such engineered CCHFV glycoproteinectodomains exhibit improved conformational homogeneity and / or biophysical stability, such as, for example, increased expression, thermostability, and prefusion stability.I. Crimean Congo Hemorrhagic Fever Virus (CCHFV) Glycoproteins

[0040] CCHFV belongs to the Orthonairovirus genus within the Nairoviridae family and Bunyavirales order (generally termed nairoviruses and bunyaviruses) (Garrison et al., 2020) and has a negative-sense RNA genome that is made up of small, medium, and large segments. The medium (M) segment encodes for the glycoprotein precursor (GPC), which undergoes several proteolytic processing events in the infected cell to generate immature precursor proteins that are subsequently processed into mature proteins found on the viral envelope (Vincent et al., 2003; Sanchez et al., 2006; Altamura et al., 2007). In addition to a highly glycosylated mucin-like domain (MLD) at the N-terminus, the M segment encodes for three glycoproteins (GP38, Gn, and Gc) and an incompletely understood accessory protein (NSm). The organization of the CCHFV M segment is much more complex than other bunyaviruses, such as hantaviruses, where the M segment encodes for only Gn and Gc (Guardado-Calvo & Fey, 2021). As observed for the Gn and Gc proteins of other bunyaviruses, CCHFV Gn and Gc are presumed to form heterodimers that complex into higher-order assemblies on the viral surface (Punch et al., 2018).

[0041] CCHFV Gc is a class II viral fusion protein composed of three domains (I, II, and III) that transition from a metastable pre-fusion conformation to a highly stable post-fusion conformation to facilitate membrane fusion and entry. Within the tip of domain II, Gc contains three fusion loops with hydrophobic residues that insert into the host cell membrane during fusion. Previous structural studies on hantaviruses, which are close relatives of nairoviruses, have demonstrated that the Gc fusion loops exist in distinct pre- and post-fusion conformations (Serris et al., 2020). For CCHFV, structures have been determined for the post- fusion Gc trimer as well as a pre-fusion-like Gc monomer in complex with two neutralizing antibodies (Li et al., 2022; Mishra et al., 2022). However, in the monomeric CCHFV Gc structure, the fusion loops were in a post-fusion conformation, suggesting that like hantaviruses, an accompanying glycoprotein stabilizes the Gc fusion loops in the pre-fusion conformation (Serris et al., 2020; Mishra et al., 2022).

[0042] Gn is the accompanying glycoprotein in bunyaviruses, and CCHFV Gn is generated following SKI-I cleavage from the PreGn precursor at an RRLL cleavage sitebetween GP38 and Gn (Vincent et al., 2003). Gn is implicated in receptor-binding for several bunyaviruses, however, the CCHFV entry factor low-density lipoprotein receptor (LDLR) binds a Gc monomer with high affinity but not monomeric Gn (Ganaie et al., 2021 ; Jangra et al., 2018; Xu et al., 2024). Therefore, the function of CCHFV Gn is currently unknown. The structure of CCHFV Gn has not yet been reported, but across the Bunyavirales order, structurally characterized Gn proteins exhibit a common domain architecture that CCHFV is expected to exhibit, based in part on a predicted structure generated by AlphaFold2 (Guardado- Calvo et al., 2021 ; Tunyasuvunakool et al., 2021). For example, hantavirus Gn contains three domains within the ‘head’ of Gn that are predominantly composed of [3-strands (domain A, - ribbon domain, and domain B). The ‘base’ is composed of a P-sheet (domain C) followed by an a-helical membrane-proximal external region (Serris et al., 2020; Guardado-Calvo et al., 2021). CCHFV Gn has high structural similarity to hantavirus Gn but is predicted to lack a canonical domain A fold found in most Gn proteins within the Bunyavirales order. Interestingly, GP38, a protein unique to nairoviruses, has been proposed as a domain A equivalent (Gaurdado-Calvo et al., 2021). GP38 is released from GP160 and GP85 precursors at an RSKR furin cleavage site between GP38 and the MLD. GP38 has been detected in the supernatant of infected cells, suggesting that the protein is secreted (Sanchez et al., 2006). However, there is evidence that suggests GP38 may also localize to the viral surface (Golden et al., 2019). GP38 in combination with Gn would comprise the complete domain architecture observed for the Gn protein of other bunyaviruses, which raises the possibility of GP38 as a stable component of a glycoprotein complex with Gn and Gc.

[0043] The relatively complex glycoprotein organization and processing the CCHFV M segment has hindered the expression of stable proteins and protein complexes for characterization and vaccine development.

[0044] Provided herein are stabilized CCHFV glycoprotein complexes, the first glycoprotein complexes reported for the Nairoviridae family. This was achieved through an iterative approach, starting with engineering and structurally characterizing a highly thermostable, well-expressing GP38-Gn heterodimer construct named GP38-GnH DS. Stabilization of GP38-GnH DSwas facilitated by an engineered disulfide bond between GP38 and Gn that increases expression and thermostability. A 2.5 A resolution X-ray crystal structure of the GP38-Gn heterodimer displays a hydrophobically packed GP38-Gn interface. The GP38-GnH DSdesign was leveraged to obtain a 3.4 A cryo-EM structure of the GP38-GnH DSincomplex with Gc. The complex revealed a heterotrimeric glycoprotein complex fortified by many polar contacts between Gn and Gc, GP38 and Gn, and a contact between GP38 and Gc. The structure of GP38-GnH DS-Gc provides the basis for GP38’s association with the virion and is the first structure of the CCHFV Gc fusion loops in the prefusion conformation, largely restrained by a highly conserved N-linked glycan on Gn. GP38-GnH DS-Gc. Forty percent of mice vaccinated with GP38-GnH DS-Gc survived lethal CCHFV-IbArl0200 challenge and had enhanced antibody responses to GP38 in comparison to mice immunized with GP38 only and GP38 combined with Gc, highlighting its promise as a vaccine antigen for further development.

[0045] In the studies provided herein, the inventors found that GP38 makes a stable complex with Gn and Gc, therefore establishing that GP38 plays a structural role in a CCHFV glycoprotein complex. These data support a model in which GP38 initially folds into a stable component of a heterotrimeric complex before protease cleavage and GP38 dissociation, which is supported by previous studies that detected GP38 in the supernatant of infected cells (Sanchez et al., 2006) as well as localized to the viral envelope (Golden et al., 2019). The structure reported in this study displays GP38’s C-terminal [3-strands (residues 488-499) in an alternative conformation than the previously reported structure of GP38 as a monomer (Mishra et al., 2020), and this conformational change enables the interaction of GP38 with the Gc fusion loops. This interaction may indicate that GP38 contributes to the pre-fusion stability of Gc. However, the functional roles of GP38 on the viral surface and the extent to which GP38 is present on the mature virion remain to be investigated.

[0046] The structures reported in this study establish the foundation for understanding the CCHFV glycoprotein complex, but how the CCHFV glycoprotein complex organizes into a higher-order assembly remains incompletely understood. The evidence to support a higher- order assembly arises from tomographic studies, demonstrating that Hazara Virus, a nairovirus closely related to CCHFV, exhibits an ordered and possibly tetrameric higher-order assembly (Punch et al., 2018). Additionally, the structural homology between CCHFV and hantavirus glycoproteins, which assemble into a tetrameric spike lattice, further supports that nairoviruses may form a tetrameric higher-order assembly (Serris et al., 2020; Guardado-Calvo et al., 2021). Future tomographic studies should be conducted to fully understand the glycoprotein complex organization of CCHFV. An understanding of the higher-order assembly may be required to fully explain results obtained with bi-specific antibodies (Fels et al., 2021), which demonstratedthat only certain combinations of Fabs, in a specific organization in the bi-specific, led to enhanced neutralization.

[0047] The heterotrimeric structure of GP38-GnH DS-Gc reveals that GnHis almost completely masked by GP38 and Gc. Although previous studies have attempted to isolate Gn- specific antibodies from immunized mice or convalescent donors (Fels et al., 2021; Bertolotti- Ciarlet et al., 2005), the structural integrity and topological organization of the constructs used in these experiments have been suggested as rationales for why no Gn-specific antibodies were discovered. Based on the heterotrimeric structure provided herein, probes that include GP38 with Gn for antibody isolation are unlikely to isolate Gn-specific antibodies due to the masking of Gn11, rationalizing the results from the previous studies (Fels et al., 2021 ; Bertolotti-Ciarlet et al., 2005). This hypothesis is further supported by the vaccination experiment from this study showing that vaccination with GP38-GnH DS-Gc did not elicit any detectable Gn-specific antibodies. Additionally, GP38 may mask Gn11to some extent on the mature virion, which could suggest that if Gn-specific antibodies are raised during natural infection, their abundance is lower in comparison to GP38- or Gc-specific antibodies. In contrast, antibody isolation experiments for hantaviruses have yielded many Gn-specific antibodies (Stass et al., 2023; Rissanen et al., 2021; Mittler et al., 2023). For CCHFV, antibodies targeting GP38 have been isolated (Golden et al., 2019; Fels et al., 2021 ; Durie et al., 2022; Shin et al., 2024), and GP38 in combination with Gn would compose an accompanying protein that is similar in size, organization, and immunogenicity to the Gn protein of hantaviruses.

[0048] A wide variety of vaccine strategies targeting the CCHFV glycoproteins, including inactivated virus, viral vectored, virus-like particle, nucleic acid, and subunit vaccine strategies, have been investigated in several different immunosuppressed animal models (Garrison et al., 019; Bereczky et al., 2010; Rodriguez et al., 2019; Zivcec et al., 2013; Garrison et al., 2017; Appelberg et al., 2022; Kortekaas et al., 2015; Dowall et al., 2016; Hinkula et al., 2017). The variance of vaccine type, vaccination regimen, animal model used, and quantity and strain of virus used for challenge limits direct comparison of the vaccination experiment provided herein with previous studies. The majority of investigations exploring the efficacy of a vaccine containing the CCHFV glycoproteins have utilized either the GPC (encoded by the full M segment) or Gn-Gc as the immunogens (Ahata et al., 2023). Of the all the CCHFV glycoproteins, the present results support that GP38 is a key immunogen, as no survival advantage was observed when including Gn or Gc in the vaccinations. These data agree withseveral studies that have established GP38 as immunorelevant in mouse vaccination studies (Suschak et al., 2021 ; Scher et al., 2023). Likely due to the challenge of protein expression, few subunit vaccination experiments have been conducted for CCHFV. Notably, Kortekaas et al. demonstrated that a subunit vaccination with the Gc ectodomain elicits neutralizing antibodies but does not protect mice against lethal challenge (Kortekaas et al., 2015). However, the dose of Gc administered in those experiments was roughly seven-fold less than what was administered in the present study, potentially explaining the increased protection observed herein. Although humoral immune responses observed across mice in the present vaccination experiment were varied, the construct design and the mutations incorporated into GP38-GnH"DS-Gc may lend itself to improvement of a nucleic acid or viral vectored vaccine by improving the stability and expression of the CCFHV glycoproteins, as well as stabilizing GP38 in the glycoprotein complex.II. Definitions

[0049] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term “portion” can include part of a moiety or the entire moiety.

[0050] As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0051] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the disclosed subject matter. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0052] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01 %. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0053] The term “antibody” refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. An “antibody” is a species of an antigen binding protein. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be “chimeric,” that is, different portions of the antibody can be derived from two different antibodies as described further below. The antigen binding proteins, antibodies, or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0054] Naturally occurring antibody structural units typically comprise a tetramer. Each such tetramer typically is composed of two identical pairs of polypeptide chains, each pair having one full-length “light” (in certain embodiments, about 25 kDa) and one full-length “heavy” chain (in certain embodiments, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that can he responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full- length light and heavy chains, typically, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair typically form the antigen binding site.

[0055] The term “variable region” or “variable domain” refers to a portion of the light and / or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target.

[0056] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprisethe domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987) or Chothia et al., Nature, 342:878-883 (1989).

[0057] In certain embodiments, an antibody heavy chain binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody light chain binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody light chain. In certain embodiments, an antibody binding region binds to an antigen in the absence of an antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

[0058] In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition and the contact definition.

[0059] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g. , Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al. , Proc Natl Acad Sci (USA), 86:9268- 9272 (1989); “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary' sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and GeneticsSuppl., 3:194-198 (1999). The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum el al. , J. Mol. Biol., 5:732-45 (1996).

[0060] By convention, the CDR regions in the heavy chain are typically referred to as Hl, H2, and H3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus. The CDR regions in the light chain are typically referred to as LI, L2, and L3 and are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

[0061] The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0062] The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CHI, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxyl-terminus, with the CH3 being closest to the carboxy-terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (including IgAl and IgA2 subtypes), IgM and IgE.

[0063] A bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g. , Songsivilai el al. , Clin. Exp. Immunol., 79: 315-321 (1990); Kostelny el al., J. Immunol., 148: 1547-1553 (1992).

[0064] The term “antigen” refers to a substance capable of inducing adaptive immune responses. Specifically, an antigen is a substance which serves as a target for the receptors of an adaptive immune response. Typically, an antigen is a molecule that binds to antigen-specific receptors but cannot induce an immune response in the body by itself. Antigens are usually proteins and polysaccharides, less frequently also lipids. As used herein, antigens also include immunogens and haptens.

[0065] An “Fc” region comprises two heavy chain fragments comprising the CHI and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0066] The “Fv region” comprises the variable regions from both the heavy and light chains but lacks the constant regions.

[0067] An antibody that “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. For example, the CCHFV glycoprotein ectodomain specific antibodies of the present invention are specific to CCHFV glycoprotein ectodomain. In some embodiments, the antibody that binds to CCHFV glycoprotein ectodomain has a dissociation constant (Kd) of ^100 nM, S=10 nM, = I nM, ^0.1 nM, ^0.01 nM, or ^0.001 nM (e.g., 10-sM or less, e.g., from 10“sM to 10-13M, e.g., from 10“9M to 10“13M).

[0068] The term “compete” when used in the context of antigen binding proteins e.g., atnibody or antigen-binding fragment thereof) that compete for the same epitope means competition between antigen binding proteins as determined by an assay in which the antigen binding protein e.g., antibody or antigen-binding fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g., CCHFV glycoprotein ectodomain or a fragment thereof). Numerous types of competitive binding assays can be used to determine if one antigen binding protein competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland el al., 1986, J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g. , Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. I. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabelled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label boundto the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein is present in excess, it will inhibit (<?.g. , reduce) specific binding of a reference antigen binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more. In some instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0069] The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. The epitope can be either linear epitope or a conformational epitope. A linear epitope is formed by a continuous sequence of amino acids from the antigen and interacts with an antibody based on their primary structure. A conformational epitope, on the other hand, is composed of discontinuous sections of the antigen’s amino acid sequence and interacts with the antibody based on the 3D structure of the antigen. In general, an epitope is approximately five or six amino acid in length. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.

[0070] The term “host cell” means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.

[0071] The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology,(Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0072] In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1 / 10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al. , 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[0073] Examples of parameters that can be employed in determining percent identity for polypeptides or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J. Mol. Biol. 48:443-453.

[0074] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 or other number of contiguous amino acids of the target polypeptide.

[0075] The term “link” as used herein refers to the association via intramolecular interaction, e.g., covalent bonds, metallic bonds, and / or ionic bonding, or inter-molecular interaction, e.g., hydrogen bond or noncovalent bonds.

[0076] The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter that is operably linked to a coding sequence will direct the expression of the coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0077] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0078] The term “polynucleotide” or “nucleic acid” includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and intemucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

[0079] The terms “polypeptide” or “protein” means a macromolecule having the amino acid sequence of a native protein, that is, a protein produced by a naturally-occurring and nonrecombinant cell; or it is produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally occurring amino acid and polymers. The terms “polypeptide” and “protein” specifically encompass CCHFV glycoprotein ectodomain bindingproteins, antibodies, or sequences that have deletions from, additions to, and / or substitutions of one or more amino acid of antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein. In certain embodiments, fragments are about five to 500 amino acids long. For example, fragments can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of a CCHFV glycoprotein ectodomain-binding antibody, useful fragments include but are not limited to a CDR region, a variable domain of a heavy and / or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

[0080] “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to induce an immune response when administered to a subject. In some embodiments, an engineered CCHFV glycoprotein ectodomain comprises from 1-10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10) conservative substitutions compared to a corresponding native CCHFV glycoprotein ectodomain, respectively. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Thus, a conservative substitution does not alter the basic function of a protein of interest. Non-conservative substitutions are those that reduce an activity or function of the protein, such as the ability to induce an immune response when administered to a subject. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity.

[0081] Ferritin nanoparticle: A multi- subunit, globular shaped protein complex. In nature, native ferritin proteins self-assemble into a globular structure that stores iron and releases it in a controlled fashion. Production and expression of ferritin nanoparticles based on monomeric ferritin subunits that are linked to influenza HA ectodomains have been previously described (see, e.g., Kanekiyo et al., Nature 499, 102-106, 2013 and Zhang, Y. Int. J. Mol. Sci., 12:5406-5421, 2011, which are incorporated by reference herein).

[0082] Fusion Protein: A single polypeptide chain including the sequence of two or more heterologous proteins, often linked by a peptide linker.

[0083] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen-specific response”). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.

[0084] As used herein, reference to “at least 90% identity” refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

[0085] The pharmaceutically acceptable carriers useful in this invention are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically- neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0086] As used herein, the term “subject” refers to a human or any non-human animal e.g. , mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0087] The term “therapeutically effective amount” or “effective dosage” as used herein refers to the dosage or concentration of a drug effective to treat a disease or condition.For example, with regard to the use of the monoclonal antibodies or antigen-binding fragments thereof disclosed herein to treat viral infection.

[0088] “Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.

[0089] As used herein, a “vector” refers to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. A vector may also include one or more therapeutic genes and / or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.III. Pharmaceutical Formulations

[0090] The present disclosure provides pharmaceutical compositions comprising an engineered CCHFV glycoprotein ectodomain; a protein nanoparticle or virus-like particle that includes an engineered CCHFV glycoprotein ectodomain; a nucleic acid molecule encoding an engineered CCHFV glycoprotein ectodomain; and viral vector comprising an engineered CCHFV glycoprotein ectodomain and / or encoding the engineered CCHFV glycoprotein ectodomain in its genomic material. Such compositions can be used for stimulating an immune response, such as part of a vaccine formulation.

[0091] In the case that a nucleic acid molecule encoding an engineered CCHFV glycoprotein ectodomain is used in a pharmaceutical composition, the nucleic acid molecule may comprise or consist of deoxyribonucleotides and / or ribonucleotides, or analogs thereof, covalently linked together. A nucleic acid molecule as described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively,mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. A nucleic acid molecule may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non- nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A nucleic acid molecule is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “nucleic acid sequence” is the alphabetical representation of a nucleic acid molecule. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0092] In some embodiments, the nucleic acids of the present disclosure comprise one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties. In some embodiments, modified sugar moieties are substituted sugar moieties. In some embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.

[0093] In some embodiments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2' and / or 5' positions. Examples of sugar substituents suitable for the 2'- position, include, but are not limited to: 2’-F, 2’-OCH3("OMe" or "O-methyl"), and 2'- O(CH2)2OCH3("MOE"). In certain embodiments, sugar substituents at the 2' position are selected from allyl, amino, azido, thio, O-allyl, O— Ci-Cio alkyl, O— Ci-Cio substituted alkyl;OCF3, O(CH2)2SCH3, O(CH2)2— O— N(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. Examples of sugar substituents at the 5'-position, include, but are not limited to: 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy. In some embodiments, substituted sugars comprise more than one nonbridging sugar substituent, for example, T-F-5'-methyl sugar moieties (see, e.g., PCT International Application WO 2008 / 101157, for additional 5’,2’-bis substituted sugar moieties and nucleosides).

[0094] Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'- substituted nucleosides. In some embodiments, a 2’-substituted nucleoside comprises a 2'- substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2-O-N(Rm)(Rn) or O-CH2- C(=O)— N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted Cl -CIO alkyl. These 2'-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

[0095] In some embodiments, a 2'-substituted nucleoside comprises a 2'-substituent group selected from F, NH2, N3, OCF3, O-CH3, O(CH2)3NH2, CH2— CH=CH2, O-CH2— CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O— (CH2)2— O— N(Rm)(Rn),O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O-CH2-C(=O)-N(Rm)(Rn) where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted Cl -CI O alkyl.

[0096] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2’-substituent group selected from F, OCF3, O-CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2-O-N(CH3)2, -O(CH2)2O(CH2)2N(CH3)2, and O-CH2- C(=O)-N(H)CH3.

[0097] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, O-CH3, and OCH2CH2OCH3.

[0098] In some embodiments, nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present disclosure comprise one or more modified nucleobases.

[0099] In some embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3- deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][l,4]benzoxazin-2(3H)- one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4- 13][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2- pyridone. Further nucleobases include those disclosed in U.S. Patent 3,687,808 and those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859.

[0100] Representative United States Patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Patents 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121 ; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of which is herein incorporated by reference in its entirety.

[0101] Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. For example, one additional modification of the ligand conjugated oligonucleotides of the present disclosure involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di -hexadecyl -rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. For some aspects, a nucleic acid molecule encoding an engineered CCHFV glycoprotein ectodomain is a modified RNA, such as, for example, a modified mRNA. Modified (m)RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1 -methyl-pseudouridine (Nlm ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. In some embodiments, the (m)RNA molecules used herein may have the uracils replaced with psuedouracils such as l-methyl-3 '-pseudouridylyl bases. In some embodiments, some of the uracils are replaced, but in other embodiments, all of the uracils have been replaced. The (m)RNA may comprise a 5' cap, a 5' UTR element, an optionally codon optimized open reading frame, a 3' UTR element, and a poly (A) sequence and / or a poly adenylation signal.

[0102] The nucleic acid molecule, whether native or modified, may be delivered as a naked nucleic acid molecule or in a delivery vehicle, such as a lipid nanoparticle. A lipid nanoparticle may comprise one or more nucleic acids present in a weight ratio to the lipid nanoparticles from about 5: 1 to about 1 : 100. In some embodiments, the weight ratio of nucleic acid to lipid nanoparticles is from about 5: 1, 2.5: 1, 1 :1, 1 :5, 1:10, 1: 15, 1:20, 1 :25, 1:30, 1 :35, 1 :40, 1:45, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, or 1 :100, or any value derivable therein.

[0103] In some embodiments, the lipid nanoparticles used herein may contain one, two, three, four, five, six, seven, eight, nine, or ten lipids. These lipids may include triglycerides,phospholipids, steroids or sterols, PEGylated lipids, or a group with an ionizable group such as an alkyl amine and one or more hydrophobic groups such as C6 or greater alkyl groups.

[0104] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms.

[0105] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more PEGylated lipids (or PEG lipid). In some embodiments, the present disclosure comprises using any lipid to which a PEG group has been attached. In some embodiments, the PEG lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group. In other embodiments, the PEG lipid is a compound which contains one or more C6-C24 long chain alkyl or alkenyl group or a C6-C24 fatty acid group attached to a linker group with a PEG chain. Some non-limiting examples of a PEG lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified l,2-diacyloxypropan-3-amines, PEG modified diacylglycerols and dialkylglycerols. In some embodiments, PEG modified diastearoylphosphatidylethanolamine or PEG modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1 ,200 to about 3,000. The molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about 15,000. Some non-limiting examples of lipids that may be used in the present disclosure are taught by U.S. Patent 5,820,873, WO 2010 / 141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.

[0106] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more phospholipids. In some embodiments, any lipid which also comprises a phosphate group. In some embodiments, the phospholipid is a structure which contains one or two longchain C6-C24 alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionally, a small organic molecule. In some embodiments, the small organic molecule is an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipid defines lipid and lipid-like molecules with both a positive charge and a negative charge.

[0107] In some aspects of the present disclosure, lipid nanoparticle containing compounds containing lipophilic and cationic components, wherein the cationic component is ionizable, are provided. In some embodiments, the cationic ionizable lipids contain one or more groups that are protonated at physiological pH but may be deprotonated and have no charge at a pH above 8, 9, 10, 11, or 12. The ionizable cationic group may contain one or more protonatable amines which are able to form a cationic group at physiological pH. The cationic ionizable lipid compound may also further comprise one or more lipid components such as two or more fatty acids with C6-C24 alkyl or alkenyl carbon groups. These lipid groups may be attached through an ester linkage or may be further added through a Michael addition to a sulfur atom. In some embodiments, these compounds may be a dendrimer, a dendron, a polymer, or a combination thereof.

[0108] In some aspects of the present disclosure, composition containing compounds containing lipophilic and cationic components, wherein the cationic component is ionizable, are provided. In some embodiments, ionizable cationic lipids refer to lipid and lipid-like molecules with nitrogen atoms that can acquire charge (pKa). These lipids may be known in the literature as cationic lipids. These molecules with amino groups typically have between 2 and 6 hydrophobic chains, often alkyl or alkenyl such as C6-C24 alkyl or alkenyl groups, but may have at least 1 or more than 6 tails.

[0109] In some embodiments, the amount of the lipid nanoparticle with the nucleic acid molecule encapsulated in the pharmaceutical composition is from about 0.1% w / w to about 50% w / w, from about 0.25% w / w to about 25% w / w, from about 0.5% w / w to about 20% w / w, from about 1% w / w to about 15% w / w, from about 2% w / w to about 10% w / w, from about 2% w / w to about 5% w / w, or from about 6% w / w to about 10% w / w. In some embodiments, the amount of the lipid nanoparticle with the nucleic acid molecule encapsulated in thepharmaceutical composition is from about 0.1% w / w, 0.25% w / w, 0.5% w / w, 1% w / w, 2.5% w / w, 5% w / w, 7.5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w, 55% w / w, 60% w / w, 65% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, to about 95% w / w, or any range derivable therein.

[0110] In some aspects, the present disclosure comprises one or more sugars formulated into pharmaceutical compositions. In some embodiments, the sugars used herein are saccharides. These saccharides may be used to act as a lyoprotectant that protects the pharmaceutical composition from destabilization during the drying process. These water- soluble excipients include carbohydrates or saccharides such as disaccharides such as sucrose, trehalose, or lactose, a trisaccharide such as fructose, glucose, galactose comprising raffinose, polysaccharides such as starches or cellulose, or a sugar alcohol such as xylitol, sorbitol, or mannitol. In some embodiments, these excipients are solid at room temperature. Some nonlimiting examples of sugar alcohols include erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotritol, maltotetraitol, or a polyglycitol.

[0111] In some embodiments, the amount of the sugar in the pharmaceutical composition is from about 25% w / w to about 98% w / w, 40% w / w to about 95% w / w, 50% w / w to about 90% w / w, 50% w / w to about 70% w / w, or from about 80% w / w to about 90% w / w. In some embodiments, the amount of the sugar in the pharmaceutical composition is from about 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w, 52.5% w / w, 55% w / w, 57.5% w / w, 60% w / w, 62.5% w / w, 65% w / w, 67.5% w / w, 70% w / w, 75% w / w, 80% w / w, 82.5% w / w, 85% w / w, 87.5% w / w, 90% w / w, to about 95% w / w, or any range derivable therein.

[0112] In some embodiments, the pharmaceutically acceptable polymer is a copolymer. The pharmaceutically acceptable polymer may further comprise one, two, three, four, five, or six subunits of discrete different types of polymer subunits. These polymer subunits may include polyoxypropylene, polyoxyethylene, or a similar subunit. In particular, the pharmaceutically acceptable polymer may comprise at least one hydrophobic subunit and at least one hydrophilic subunit. In particular, the copolymer may have hydrophilic subunits on each side of a hydrophobic unit. The copolymer may have a hydrophilic subunit that is polyoxyethylene and a hydrophobic subunit that is polyoxypropylene.

[0113] In some embodiments, expression cassettes are employed to express an engineered CCHFV glycoprotein ectodomain, either for subsequent purification and delivery to a cell / subject, or for use directly in a viral-based delivery approach. Provided herein are expression vectors which contain one or more nucleic acids encoding an engineered CCHFV glycoprotein ectodomain.

[0114] Expression requires that appropriate signals be provided in the vectors and include various regulatory elements such as enhancers / promoters from both viral and mammalian sources that drive expression of the engineered CCHFV glycoprotein ectodomain in cells. Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, / .<?., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.

[0115] The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0116] At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0117] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0118] In certain embodiments, viral promotes such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.

[0119] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

[0120] Below is a list of promoters / enhancers and inducible promoters / enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter / enhancer combination (as per the Eukaryotic PromoterData Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0121] The promoter and / or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and / or DQ p, [3-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, p- Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, a-fetoprotein, t-globin, P-globin, c-fos, c-HA-m insulin, neural cell adhesion molecule (NCAM), ai-antitrypain, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus.

[0122] Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0123] There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor- mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals.

[0124] One method for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containingadenovirus sequences sufficient to (a) support packaging of the construct and (b) to express engineered CCHFV glycoprotein ectodomain that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

[0125] The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

[0126] Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5 ’-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation. In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.

[0127] Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which wastransformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete.

[0128] Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g. , Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

[0129] The adenoviruses of the disclosure are replication defective, or at least conditionally replication defective. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is one exemplary starting material that may be used to obtain the conditional replication-defective adenovirus vector for use in the present disclosure.

[0130] Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus, adeno- associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.

[0131] In embodiments, particular embodiments, the vector is an AAV vector. AAV is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease. The virus causes a very mild immune response, lending further support to its apparent lack of pathogenicity. In many cases, AAV vectors integrate into the host cell genome, which can be important for certain applications, but can also have unwanted consequences. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in anextrachromosomal state without integrating into the genome of the host cell, although in the native virus some integration of virally carried genes into the host genome does occur. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. Recent human clinical trials using AAV for gene therapy in the retina have shown promise. AAV belongs to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae . The virus is a small (20 nm) replicationdefective, nonenveloped virus.

[0132] Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of features. Chief amongst these is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19. This feature makes it somewhat more predictable than retroviruses, which present the threat of a random insertion and of mutagenesis, which is sometimes followed by development of a cancer. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. Development of AAVs as gene therapy vectors, however, has eliminated this integrative capacity by removal of the rep and cap from the DNA of the vector. The desired gene together with a promoter to drive transcription of the gene is inserted between the inverted terminal repeats (ITR) that aid in concatemer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division, since the episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at very low frequency. AAVs also present very low immunogenicity, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly defined cytotoxic response. This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for human gene therapy.

[0133] The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteinsrequired for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.

[0134] The Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. The feature of these sequences that gives them this property is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles.

[0135] With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural cap) and packaging rep) proteins can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep-dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis.

[0136] In some aspects, the present disclosure provides pharmaceutical compositions that contain one or more salts. The salts may be an inorganic potassium or sodium salt such as potassium chloride, sodium chloride, potassium phosphate dibasic, potassium phosphate monobasic, sodium phosphate dibasic, or sodium phosphate monobasic. The pharmaceutical composition may comprise one or more phosphate salts, so as to generate a phosphate buffer solution. The phosphate buffer solution may comprise each of the phosphates to buffer a solution to a pH from about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or any range derivable therein.

[0137] In some aspects, the present disclosure comprises one or more excipients formulated into pharmaceutical compositions. An “excipient” refers to pharmaceutically acceptable carriers that are relatively inert substances used to facilitate administration or delivery of an API into a subject or used to facilitate processing of an API into drugformulations that can be used pharmaceutically for delivery to the site of action in a subject. Furthermore, these compounds may be used as diluents in order to obtain a dosage that can be readily measured or administered to a patient. Non-limiting examples of excipients include polymers, stabilizing agents, surfactants, surface modifiers, solubility enhancers, buffers, encapsulating agents, antioxidants, preservatives, nonionic wetting or clarifying agents, viscosity increasing agents, and absorption-enhancing agents.

[0138] In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and can preferably include an adjuvant. Water is a particular carrier when the pharmaceutical composition is administered by injections, such an intramuscular injection. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0139] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington’s Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

[0140] Engineered proteins or nucleic acids encoding engineered proteins of the present disclosure, as described herein, can be formulated for parenteral administration, e.g. ,formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, intra- tumoral or even intraperitoneal routes. The formulation could alternatively be administered by a topical route directly to the mucosa, for example by nasal drops, inhalation, or by nebulizer. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0141] Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0142] The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0143] Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes. Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

[0144] The compositions disclosed herein may be used to treat both children and adults. Thus a human subject may be less than 1 year old, 1-5 years old, 5-16 years old, 16-55 years old, 55-65 years old, or at least 65 years old.

[0145] Preferred routes of administration include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, and intraoccular injection. Particularly preferred routes of administration include intramuscular, intradermal and subcutaneous injection.IV. Immunogenic Compositions

[0146] Immunogenic compositions comprising an engineered CCHFV glycoprotein ectodomain and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to a subject by a variety of modes, for example, by an intranasal route. Standard methods for preparing administrable immunogenic compositions are described, for example, in such publications as Remington’s Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995.

[0147] The immunogenic compositions can be formulated for administration to a subject by a variety of administration modes, including mucosal administration modes such as by oral, rectal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to other surfaces, and non-mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes.

[0148] Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing.

[0149] The immunogenic composition can contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually 1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

[0150] The immunogenic composition can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0151] The immunogenic composition may optionally include an adjuvant to enhance the immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, A1PO4, alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the recombinant virus, and cytokines, nonionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many other suitable adjuvants well known in the art, may be used as an adjuvant (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product.

[0152] In some embodiments, the immunogenic composition can be provided in unit dosage form for use to induce an immune response in a subject, for example, to prevent or inhibit CCHFV infection in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and / or a metering mechanism for administering the unit dose or multiples thereof.

[0153] The immunogenic composition typically contains an effective amount of engineered CCHFV glycoprotein ectodomain, and can be prepared by conventional techniques. Preparation of immunogenic compositions, including those for administration to human subjects, is generally described in Pharmaceutical Biotechnology, Vol. 61 Vaccine Design — the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., U.S.A. 1978. Typically, the amount of antigen in each dose of the immunogenic composition is selected as an amount which induces an immune response without significant adverse side effects.

[0154] The amount of the engineered CCHFV glycoprotein ectodomain included in the therapeutic composition can vary depending upon the specific antigen employed, the route and protocol of administration, and the target population, for example. For protein therapeutics, typically, each human dose will comprise 1-1000 pg of protein, such as from about 1 pg to about 100 pg, for example, from about 1 pg to about 50 pg, such as about 1 pg, about 2 pg, about 5 pg, about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 30 pg, about 40 pg, or about 50 pg. The amount utilized in an immunogenic composition is selected based on the subject population (e.g., infant or elderly). An optimal amount for a particular composition can be ascertained by standard studies involving observation of antibody titers and other responses in subjects. It is understood that a therapeutically effective amount of an engineered CCHFV glycoprotein ectodomain comprises an amount that is ineffective at eliciting an immune response by administration of a single dose, but that is effective upon administration of multiple dosages, for example in a prime-boost administration protocol.V. Methods of Eliciting an Immune Response

[0155] The engineered CCHFV glycoprotein ectodomain can be administered to a subject to elicit an immune response. Upon immunization, the subject responds by producing antibodies specific for the CCHFV glycoprotein ectodomain. In addition, innate and cell- mediated immune responses are induced, which can provide antiviral effectors as well as regulating the immune response. The immune response can be a protective immune response, for example a response that prevents or reduces subsequent infection with a virus including the trimeric antigen. The immune response can be a therapeutic immune response, for example a response that treats or inhibits current infection with a virus including the trimeric antigen and illnesses associated therewith.

[0156] Typical subjects intended for treatment with the compositions and methods of the present disclosure include humans, as well as non-human primates and other animals. To identify subjects for prophylaxis or treatment according to the methods of the disclosure, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease of condition, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods. These and other routine methodsallow the clinician to select patients in need of therapy using the methods and immunogenic compositions of the disclosure. In accordance with these methods and principles, an engineered CCHFV glycoprotein ectodomain and / or other biologically active agent can be administered according to the teachings herein as an independent prophylaxis or treatment program, or as a follow-up, adjunct or coordinate treatment regimen to other treatments.

[0157] In some embodiments, a subject is selected for treatment that has, or is at risk for developing, a CCHFV infection, for example because of exposure or the possibility of exposure to CCHFV. Such a subject can then be administered an effective amount of an engineered CCHFV glycoprotein ectodomain to induce an immune response to the CCHFV glycoprotein ectodomain. The immune response can neutralize autologous virus (e.g., the strain of the engineered CCHFV glycoprotein ectodomain) or heterologous virus (e.g., strains other than the strain of the engineered CCHFV glycoprotein ectodomain). In some such embodiments, the immune response inhibits subsequent CCHFV infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by CCHFV) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (prevention of detectable CCHFV infection), as compared to a suitable control.

[0158] The immunogenic composition may be administered by any suitable method, including but not limited to, via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application.

[0159] An effective amount of the engineered CCHFV glycoprotein ectodomain and / or other biologically active agent 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.

[0160] The engineered CCHFV glycoprotein ectodomain can be administered to the subject in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The effective amount of the engineered CCHFV glycoprotein ectodomain can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yieldclinically significant results to alleviate one or more symptoms or detectable conditions associated with a targeted disease or condition as set forth herein.

[0161] The administration of an effective amount of a engineered CCHFV glycoprotein ectodomain of the disclosure can be for either prophylactic or therapeutic purpose. When provided prophylactically, the engineered CCHFV glycoprotein ectodomain is provided in advance of any symptom, for example in advance of infection, such as in the form of a yearly flu shot. The prophylactic administration of the engineered CCHFV glycoprotein ectodomain serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the engineered CCHFV glycoprotein ectodomain is provided at (or shortly after) the onset of a symptom of disease or infection. Thus, when used to prevent or treat a viral infection (such as CCHFV infection), the engineered CCHFV glycoprotein ectodomain of the disclosure can be provided prior to the anticipated exposure to virus so as 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.

[0162] Determination of effective dosages is typically based on 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. Suitable models in this regard include, for example, murine, rat, porcine, feline, ferret, nonhuman primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the engineered CCHFV glycoprotein ectodomain (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease).

[0163] The actual dosage of the engineered CCHFV glycoprotein ectodomain will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the vaccine for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. As described above in the forgoing listing ofterms, a therapeutically effective amount is also one in which any toxic or detrimental side effects of the engineered CCHFV glycoprotein ectodomain and / or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of an engineered CCHFV glycoprotein ectodomain and / or other biologically active agent within the methods and formulations of the disclosure is about 0.01 mg / kg body weight to about 10 mg / kg body weight, such as about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, or about 10 mg / kg, for example 0.01 mg / kg to about 1 mg / kg body weight, about 0.05 mg / kg to about 5 mg / kg body weight, about 0.2 mg / kg to about 2 mg / kg body weight, or about 1.0 mg / kg to about 10 mg / kg body weight. In some embodiments, the dosage includes a set amount of an engineered CCHFV glycoprotein ectodomain, such as from about 1-300 .g, for example, a dosage of about 10-300 pig, about 60 pg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 pg. As used herein with reference to a concentration or amount, “about” refers to +1-5%. Therefore, “about 100 pg” refers to 95-105 pg.

[0164] Upon administration of an effective amount of an engineered CCHFV glycoprotein ectodomain (for example, via injection, aerosol, oral, topical or other route), the immune system of the subject typically responds to the engineered CCHFV glycoprotein ectodomain by producing antibodies specific for the CCHFV glycoprotein ectodomain. Such a response signifies that an effective amount of the engineered CCHFV glycoprotein ectodomain was delivered. An effective amount can be achieved by single or multiple administrations (including, for example, multiple administrations per day), daily, or weekly administrations. For each particular subject, specific dosage regimens can be evaluated and adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the vaccine. In some embodiments, the antibody response of a subject administered the compositions of the disclosure will be determined in the context of evaluating effective dosages / immunization protocols. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from the subject. Decisions as to whether to administer booster inoculations and / or to change the amount of the composition administered to the individual can be at least partially based on the antibody titer level. Theantibody titer level can be based on, for example, an immunobinding assay which measures the concentration of antibodies in the serum which bind to a specific antigen, for example, CCHFV glycoprotein ectodomain.

[0165] An immunogenic composition including one or more of the engineered CCHFV glycoprotein ectodomains can be used in coordinate (or prime-boost) vaccination protocols or combinatorial formulations. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate engineered CCHFV glycoprotein ectodomain formulations, each directed toward eliciting an anti-viral immune response, such as an immune response to CCHFV glycoprotein ectodomain. Separate immunogenic compositions that elicit the anti-viral immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate (or prime-boost) immunization protocol.

[0166] There can be several boosts, and each boost can be a different disclosed immunogen. In some examples that the boost may be the same immunogen as another boost, or the prime. The prime and boost can be administered as a single dose or multiple doses, for example two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. Multiple boosts can also be given, such one to five (e.g., 1 , 2, 3, 4 or 5 boosts), or more. Different dosages can be used in a series of sequential immunizations. For example a relatively large dose in a primary immunization and then a boost with relatively smaller doses.

[0167] In some embodiments, the boost can be administered about two, about three to eight, or about four, weeks following the prime, or about several months after the prime. In some embodiments, the boost can be administered about 5, about 6, about 7, about 8, about 10, about 12, about 18, about 24, months after the prime, or more or less time after the prime. Periodic additional boosts can also be used at appropriate time points to enhance the subject's “immune memory.”

[0168] The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers and / or neutralizing activity during the course of the immunization program. To assess neutralization activity, following immunization of a subject, serum can becollected from the subject at appropriate time points, frozen, and stored for neutralization testing. Methods to assay for neutralization activity are known and are further described herein, and include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry based assays, single-cycle infection assays. In some embodiments, the serum neutralization activity can be assayed using a panel of pseudo viruses. In addition, the clinical condition of the subject can be monitored for the desired effect. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, the dose of the disclosed immunogen can be increased or the route of administration can be changed.VI. Immunodetection Methods

[0169] In still further embodiments, the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting CCHFV glycoprotein ectodomain. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e. , long term stability) of antigens. Alternatively, the methods may be used to screen various antibodies for appropriate / desired reactivity profiles.

[0170] Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of CCHFV glycoprotein ectodomain also is provided. The steps of various useful immunodetection methods have been described in the scientific literature. In general, the immunobinding methods include obtaining a sample suspected of containing CCHFV glycoprotein ectodomain, and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[0171] These methods include methods for detecting or purifying CCHFV glycoprotein ectodomain from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the CCHFV glycoprotein ectodomain will be applied to the immobilized antibody. The unwantedcomponents will be washed from the column, leaving the CCHFV glycoprotein ectodomainexpressing cells immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column.

[0172] The immunobinding methods also include methods for detecting and quantifying the amount of CCHFV glycoprotein ectodomain or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing CCHFV glycoprotein ectodomain and contact the sample with an antibody that binds CCHFV glycoprotein ectodomain or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing CCHFV glycoprotein ectodomain, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid (e.g., a nasal swab), including blood and serum, or a secretion, such as feces or urine.

[0173] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e. , to bind to CCHFV glycoprotein ectodomain. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0174] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art.

[0175] The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0176] Further methods include the detection of primary immune complexes by a two- step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[0177] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence ofthe antibody / antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0178] Another known method of immunodetection takes advantage of the immuno- PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.A. ELISAs

[0179] Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.

[0180] In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the CCHFV glycoprotein ectodomain is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-CCHFV glycoprotein ectodomain antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection may also be achieved by the addition of a second anti-CCHFV glycoprotein ectodomain antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0181] In another exemplary ELISA, the samples suspected of containing the CCHFV glycoprotein ectodomain (e.g. , potentially infected cells) are immobilized onto the well surface and then contacted with the anti-CCHFV glycoprotein ectodomain antibodies of the disclosure. After binding and washing to remove non-specifically bound immune complexes, the boundanti-CCHFV glycoprotein ectodomain antibodies are detected. Where the initial anti-CCHFV glycoprotein ectodomain antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-CCHFV glycoprotein ectodomain antibody, with the second antibody being linked to a detectable label.

[0182] Irrespective of the format employed, ELIS As have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.L0183] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0184] In ELIS As, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

[0185] “Under conditions effective to allow immune complex (antigen / antibody) formation” means that the conditions preferably include diluting the antigens and / or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS) / Tween. These added agents also tend to assist in the reduction of nonspecific background.

[0186] The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically fromabout 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25 °C to 27°C, or may be overnight at about 4°C or so.

[0187] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

[0188] To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS -Tween).

[0189] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g. , by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2’-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g. , using a visible spectra spectrophotometer.B. Western Blot

[0190] The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[0191] Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes),using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.

[0192] The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pl), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

[0193] In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties ( / '.«, binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies.C. Immunohistochemistry

[0194] The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art.

[0195] Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and / or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and / or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and / or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings.

[0196] Permanent- sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing / pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted.D. Immunodetection Kits

[0197] In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect CCHFV glycoprotein ectodomain, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to a CCHFV glycoprotein ectodomain, and optionally an immunodetection reagent.

[0198] In certain embodiments, the antibody may be pre-bound to a solid support, such as a column matrix and / or well of a microtitre plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to asecondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0199] Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

[0200] The kits may further comprise a suitably aliquoted composition of CCHFV glycoprotein ectodomain, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

[0201] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.E. Flow Cytometry and FACS

[0202] The antibodies of the present disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser- or impedance-based technology employed in many detection assays, including cell counting, cell sorting, biomarker detection and protein engineering. The technology suspends cells in a stream of fluid and passing them through an electronic detection apparatus, which allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of disorders, and has many applications in basic research, clinical practice and clinical trials.

[0203] Fluorescence-activated cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting a heterogenous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescent characteristics of each cell. In general, the technology involves a cell suspension entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescence of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based immediately prior to fluorescence intensity being measured, and the opposite charge is trapped on the droplet as it breaks form the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge.

[0204] In certain embodiments, to be used in flow cytometry or FACS, the antibodies of the present disclosure are labeled with fluorophores and then allowed to bind to the cells of interest, which are analyzed in a flow cytometer or sorted by a FACS machine.VII. Examples

[0205] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Prediction and design of a stabilized GP38-Gn construct

[0206] To express the GP38-Gn heterodimer, the native furin cleavage site (RSKR) between the mucin-like domain (MLD) and GP38 was initially kept, but an R516S mutation was designed to abrogate the SKI-I cleavage site between GP38 and Gn, resulting in an uncleaved linker (SRLL, FIG. 10A). This single amino acid substitution boosted expression (FIG. 10B). However, even when co-expressed with furin protease, SDS-PAGE analysisrevealed bands from the purified protein that were consistent with incomplete cleavage of the MLD from the GP38-Gn heterodimer. To address this, the furin cleavage site was altered to six consecutive arginine residues (6xR), which resulted in near complete cleavage of the MLD from the GP38-Gn heterodimer.

[0207] AlphaFold2 (Tunyasuvunakool et al., 2021) was used to predict a model of the full coding sequence of the CCHFV IbArl0200 M segment to facilitate further optimization of the GP38-Gn construct (FIG. 1). The predicted GP38-Gn heterodimer displayed a conserved domain architecture observed among the Gn and E2 proteins that accompany class II fusion proteins, as described by Guardado-Calvo & Rey (2021), and was structurally similar to the Gn protein of Andes virus, the prototype hantavirus (FIG. 1A). Based on the domain architecture of CCHFV Gn in the AlphaFold2 prediction and the strategies used to crystallize structures of hantavirus Gn head and base (Serris et al., 2020), two truncations of the ectodomain construct were designed: one lacking the MPER (residues 520-663, termed GP38- GnH+c) and one lacking both the MPER and Gn domain C (residues 520-587, termed GP38- GnH, FIG. 2A). Although both truncations increased the expression of the GP38-Gn ectodomain, GP38-GnHexpressed substantially better than either GP38-Gn ectodomain or GP38-GnH+c(FIG. 2B).

[0208] To fortify the GP38-Gn heterodimer, three disulfide bonds were designed between the C-terminal P-strand of GP38 and the N-terminal -strand of Gn domain B and these disulfide bonds were tested in the ectodomain construct. Only Q496C / V562C improved expression (FIG. 2C), with a modest 2-fold increase. The shortest truncation (GP38-GnH) was combined with the disulfide substitution (Q496C / V562C) to generate the best-expressing construct, GP38-GnH DS, that expressed at a final yield of 7 mg / L in FreeStyle 293-F cells (FIGS. 3A, 3B).

[0209] To evaluate if the Q496C / V562C mutation in GP38-GnH DSformed the intended disulfide bond, GP38-GnHand GP38-GnH DSvariants that replaced the SRLL linker with a 6xR cleavage site were co-expressed with furin to cleave GP38-Gn at the 6xR junction. Introducing the 6xR cleavage site between GP38 and GnH(GP38-6xR-GnH) eliminated detectable purified protein. However, the incorporation of Q496C / V562C minimized the deleterious impact of the 6xR cleavage site, as expression of GP38-6xR-GnH DSwas only slightly decreased relative to GP38-GnH DS. In agreement with the result in the ectodomain construct, the expression of GP38-GnH DSwas increased relative to GP38-GnH. As expected, both reducing and non-reducing SDS-PAGE showed a single band for GP38-GnHand GP38-GnH DSat the estimated molecular weight. In contrast, the reducing SDS-PAGE for GP38-6xR-GnH DSshowed bands at 38 kDa and 17 kDa corresponding to GP38 and Gn11, respectively, whereas the non-reducing gel showed a single band at the expected molecular weight for the GP38-GnH DSheterodimer. Together, these data confirm that Q496C / V562C results in the successful formation of a disulfide bond between GP38 and Gn.

[0210] To evaluate the effect of Q496C / V562C on thermostability, differential scanning fluorimetry (DSF) was performed on GP38-GnH, GP38-GnH DS, and GP38-6xR-GnH"DS. GP38-GnHhad a melting temperature (Tm) of 47°C whereas GP38-GnH DShad a Tm of 60°C, indicating that the addition of the disulfide bond increased the Tm by 13°C. Interestingly, replacing the SRLL linker between GP38 and Gn with a 6xR cleavage site decreased Tm by 3°C, demonstrating that the SRLL linker not only boosted expression, but also increased thermostability of GP38-GnH DS(FIG. 3C). Collectively, these data demonstrate that both the R516S and Q496C / V562C mutations contribute to increased thermostability and enable the production of a GP38-Gn heterodimeric complex.Example 2 - A 2.5 A crystal structure of the GP38-Gn heterodimer reveals a hydrophobically packed interface

[0211] Initial attempts to structurally characterize GP38-GnH DSvia X-ray crystallography resulted in crystals that diffracted poorly. To aid growth of well-diffracting crystals, a construct that minimized the flexible loop between GP38 and Gn (residues 509-532) was created by replacing it with an 8 amino-acid Gly-Ser linker (FIG. 4A). This construct successfully yielded crystals in space group P2]2]2i that diffracted X-rays to 2.5 A resolution (FIG. 4B, FIG. 11). After iterative rounds of model building and refinement, the final structure had an Rwork / Rfree of 21.3% / 25.5%.

[0212] In the crystal structure, GnHcontains a two-strand domain B fold (residues 550- 572) and an incompletely resolved -ribbon domain, in which only one of the two strands was observed (residues 573-583 resolved, 533-549 unresolved). Within domain B, an N-linked GlcNAc was observed at Asn557, which is a glycosylation site with 100% conservation among 8 nairovirus sequences analyzed (FIG. 4C, FIG. 12). In comparison to the previously published crystal structure of the GP38 monomer (Mishra et al., 2020), the conformation of GP38 in the GP38-Gn heterodimer is nearly identical, with a few notable exceptions. In contrast with themonomeric GP38 structure, an O-linked GlcNAc on Thr341 is resolved and a conformational difference in GP38’s C-terminal P-strands (residues 488-499) was observed. These C-terminal -strands are shifted in the heterodimer to constitute an interface between GP38 and Gn domain B that is further stabilized by the Q496C / V562C disulfide bond. The interface between GP38 and Gn is predominantly hydrophobic and features only a small network of hydrogen bonds, consisting of mostly mainchain-mainchain hydrogen bonds in addition to a contact between the sidechains of GP38 Gln422 and Gn Tyr573. Hydrophobicity analysis highlighted four He residues (positions 541 , 553, 555, 575) and one Tyr (Tyr573) of GnHthat compose a major hydrophobic packing interface (FIG. 4D). This hydrophobic patch packs neatly against hydrophobic residues within the interfacing pocket of the GP38 core.Example 3 - Immunization with GP38-Gn heterodimers does not elicit neutralizing antibodies

[0213] To investigate the immunogenicity of GP38-Gn constructs and their ability to elicit a neutralizing antibody response, C57BL / 6J mice were immunized at 0 and 3 weeks with 10 pg of GP38, GP38-GnH DSor GP38-GnH DS+c, adjuvanted with 50 pg poly(I:C). Mock- immunized mice received PBS, and sera were harvested from all animals at 10 weeks postimmunization (FIG. 5A). The elicited antibody binding titers to GP38, GP38-GnH DSand GP38- GnH-Ds+cwerequantifiecjviaELISA and the area under the curve (AUC) was calculated (FIGS. 5B, 13). As expected, the mock-immunized sera bound poorly to the coating antigens (median logioAUC values of -1.1 for GP38, -1.6 for GP38-GnH DS, and -1.2 for GP38-GnH DS+c). Sera from GP38-immunized mice bound to the GP38 coating antigen with a median logioAUC of - 0.11 but bound to GP38-GnH DSand GP38-GnH DS+cless, with values of -0.8 and -0.5, respectively. The sera from GP38-GnH DS-immunized mice bound to both heterodimeric coating antigens better than the GP38 coating antigen (median logioAUC values of -0.8 for GP38, -0.2 for GP38-GnH DS, and 0.2 for GP38-GnH DS+c), indicating that immunization with GP38-GnH DSelicited Gn-specific antibodies. Interestingly, sera from mice immunized with GP38-GnH DS+cbound to all three coating antigens better than sera from mice immunized GP38 or GP38-GnH DS(median logioAUC values of 0.6 for GP38, 0.8 for GP38-GnH DS, and 0.8 for GP38-GnH DS+c). These data demonstrate that GP38-Gn heterodimers are immunogenic and elicit Gn-specific antibodies, and that inclusion of Gn domain C increases immunogenicity in comparison to Gn11alone.

[0214] Next, the ability of the sera to neutralize infection of CCHFV transcription- and entry-competent virus-like particles (tecVLPs) was assessed (Zivcec et al., 2015) (FIG. 5C). As expected, anti-Gc neutralizing mAh ADI-36145 neutralized infection of Vero cells, whereas anti-GP38 non-neutralizing mAh ADI-46152, the mock-immunized sera, and GP38- immunized sera did not neutralize (Fels et al., 2021; Shin et al., 2024). Additionally, neither GP38-GnH DS- nor GP38-GnH DS+c-immunized sera neutralized infection. In contrast, the Gn protein of hantaviruses has been shown to contain multiple neutralizing epitopes. Therefore, these data highlight a distinct functional difference between the accompanying proteins of hantaviruses (Gn) and CCHFV (GP38-Gn) by demonstrating that GP38-Gn heterodimers do not contain neutralizing epitopes and therefore are not likely involved in receptor binding.Example 4 - A 3.4 A Cryo-EM structure of GP38-GnH DS-Gc reveals a stable heterotrimeric complex in the pre- fusion conformation

[0215] To gain a complete structural understanding of the glycoprotein complex, a construct containing GP38-GnH DSin association with the fusion glycoprotein Gc was engineered. It was hypothesized that, similar to hantaviruses, the Gn ‘head’ subdomain (GP38- Gnn-DsorCCHFV) would make a stable complex with Gc if expressed with a flexible tether between them (Serris et al., 2020). Therefore, the construct contained the native CCHFV signal sequence and MLD followed by GP38- GnH DSjoined to Gc (residues 1061-1545) by a Gly-Ser linker. This heterotrimeric construct was named GP38-GnH DS-Gc (FIG. 5A). The protein expressed with a yield of 0.8 mg / L in FreeStyle 293-F cells, was monodisperse as judged by size-exclusion chromatography, and had a Tm of 65°C, which was higher than GP38 (45°C) or Gc (48°C) (FIG. 14).

[0216] Cryo-EM grids of the GP38-GnH DS-Gc protein in complex with two antibodies were prepared and imaged on a Titan Krios equipped with a K3 detector. Two antibodies — an antigenic site II anti-Gc antibody ADI-36125 and an antigenic site V anti-GP38 antibody ADI- 46152 — were complexed with GP38-GnH DS-Gc to increase mass of the complex and aid particle alignment (Fels et al., 2021; Shin et al., 2024). After curation, a total of 10,739 micrographs were accepted, from which a final stack of 728,031 particles allowed us to obtain a 3.6 A resolution reconstruction of GP38- GnH DS-Gc in complex with the two antibodies. The cryo-EM map revealed an ordered [3-ribbon domain of GP38-GnH DSand a stable complex between GP38-GnH DSand Gc. To improve resolution in the areas of interest, local refinementwas performed using a mask encompassing GP38-GnH DS, domain II of Gc, and the variable regions of the antibodies, which yielded a 3.4 A resolution reconstruction (FIGS. 5B, 15, 16).

[0217] In the GP38-GnH DS-Gc model, the same hydrogen bonding interface was observed between GP38 and Gn as seen in the crystal structure of GP38-GnH DS. However, four additional hydrogen bonds and one salt bridge were resolved between GP38 and the [3-ribbon domain of GP38-GnH DS(FIG. 5F). GP38 residues Gln373, Glu415, Asn417, and His437 form hydrogen bonds with Gn residues Ile541, Thr536, Glu537, and Glu540 respectively, and GP38 Lys488 forms a salt bridge with Gn Asp563. The newly resolved contacts amend the incomplete characterization of the Gn [3-ribbon domain as well as constitute a substantial interface between GP38 and Gn. In total, 1189 A2of surface area on Gn11is buried by GP38. Surprisingly, GP38 not only shares an interface with Gn, but also shares an interface with Gc in the form of a pi-cation stacking interaction between GP38 Arg493 and Gc Trpl 197 — a key residue in the Gc fusion loops that is indispensable for syncytia formation (FIG. 5C; Mishra et al., 2022).

[0218] In agreement with the crystal structure of GP38-GnH DS, the cryo-EM map supported an N-linked glycan attached to Asn557 of Gn. However, in contrast with the single GlcNAc observed in the crystal structure due to the digestion of the branched glycan via EndoH to aid in crystallization, the cryo-EM map resolved a branched GlcNAcsMan (FIG. 5C). The GP38-GnH DS-Gc model contextualizes the significance of this glycan by revealing that the branched glycan shields the cd fusion loop of Gc, the epitope of a neutralizing antibody ADI- 37801 (Mishra et al., 2022). Although CCHFV Gn structurally lacks the capping loop seen in hantaviruses, this glycan, in combination with GP38 Arg493, leaves Trpl 197 and Trpl 199 relatively inaccessible to solvent. In addition to shielding the Gc fusion loops, Gn makes two polar contacts in the domain II tip: a sidechain-mainchain interaction between Gn Glu569 and Gc Cysl 165, and a sidechain-sidechain interaction between Gn Asp544 and Gc Thrl 164 (FIG. 5D). Outside of the domain II tip, two residues of Gn make hydrogen bonds in the domain II base of Gc (FIG. 5E). The sidechain of Gn Arg535 makes hydrogen bonds with the sidechain of Gc Aspl214 and the mainchain of Gc Phel216, whereas mainchain and sidechain atoms of Gn Thr531 contact mainchain atoms of Gc Leu 1292 and Asp 1291, respectively. Together, these data demonstrate that Gn has extensive interfaces with both GP38 and Gc, and GP38- GnII DS-Gc forms a heterotrimeric complex fortified by many polar interactions.

[0219] Hydrophobicity analysis of Gc in the complex revealed two prominent hydrophobic patches: one at the domain II base and one at the domain II tip that is composed of the Trp residues of the fusion loops. While the hydrophobic patch composed of four He residues (positions 541, 553, 555, 575) and Tyr573 of Gn was seen in both the crystal structure of GP38-GnH DSand the cryo-EM model of GP38-GnH DS-Gc, two additional hydrophobic patches were discovered on Gn that pack tightly with Gc. In conjunction with the N-linked glycan on Asn557, a hydrophobic patch at the apex of Gn sandwiches the Gc fusion loops, and a hydrophobic patch of residues at the base of GnHpack against the patch of hydrophobic residues at the base of Gc domain II (FIG. 17).

[0220] The Gc analysis also revealed that the fusion loops adopt the pre-fusion conformation, presumably restrained by the presence of GP38-GnH DS. This is in contrast to the fusion loop conformations that have been previously observed in the structure of monomeric CCHFV Gc in complex with two neutralizing antibodies, as well as structures of the CCHFV Gc post-fusion trimer (Ei et al., 2022; Mishra et al., 2022). Comparison of the pre-fusion heterotrimeric GP38-GnH DS-Gc cryo-EM structure with the published post-fusion homotrimeric Gc crystal structure showed a canonical conformational change in the fusion loops that is comparable to what has been observed in hantavirus Gc structures (FIG. 5G). Similar to the hantaviruses, the biggest conformational change occurs in the cd and ij loops, whereas the be loop remains relatively unchanged between the pre- and post-fusion conformations. As expected, from the pre-fusion to post-fusion conformation, Trpl l91 , Trpl 197, and Trpl 199 rotate from buried to exposed positions. In contrast to the Trp residues, Asnl l94 and Argl l89 transition from exposed to buried positions and form hydrogen bonds with each other. This structural analysis indicates that the presence of Gn, and possibly GP38 as well, is required to restrain the hydrophobic fusion loops of Gc in the prefusion conformation.

[0221] Additional disulfide substitutions and point substitutions were tested in the GP38-Gn-Gc construct, and were found to boost expression and thermostability (FIGS. 8A- 8D). Yet further disulfide substitutions and combination of disulfide substitutions boosted expression, thermostability, and prefusion stability in the GPC ectodomain (FIGS. 9A-9G).Example 5 - Immunization with GP38-GnH DS-Gc confers 40% protection against lethal IbAr 10200 challenge in mice and elicits neutralizing antibodies

[0222] Several GP38- and Gc-targeting antibodies are protective in mice, and Gc is the only known target of neutralizing antibodies, highlighting the relevance of both GP38 and Gc in the development of a vaccine antigen (Fels et al., 2021 ; Durie et al., 2022; Suschak et al., 2021 ; Shin et al., 2024). Because GP38-GnH DS-Gc contains the glycoproteins relevant in the immune response against CCHFV and is relatively thermostable in comparison to the monomeric glycoproteins, it was hypothesized that immunization with GP38-GnH DS-Gc would elicit neutralizing antibodies and protect mice from lethal challenge with IbArl0200. C57BL / 6J mice were immunized at 0 and 3 weeks with 10 pg of GP38, Gc, GP38 + Gc (5 pg each), or GP38-GnH DS-Gc, adjuvanted with a 1 : 1 dilution of Addavax. Mock-immunized mice received PBS. On week 6, sera were harvested for analysis. Mice were challenged with 100 plaque forming units (PFU) of IbArl0200 on week 7, and one day post-challenge, mice were transiently immunosuppressed via treatment with 1.5 mg / mouse of IFN-blocking antibody mAb-5A3 (FIG. 7A). Weight and survival were monitored for 28 days post-challenge. As expected, by the sixth day post-challenge, all mock-immunized mice succumbed to infection. Interestingly, 40% of mice immunized with GP38, GP38 + Gc, or GP38-GnH DS-Gc were protected for 28 days after lethal challenge, whereas only 20% of Gc -immunized mice were protected (FIG. 7B). Notably, mice immunized with GP38 + Gc had improved weight loss recovery and a lower clinical score than all other groups (FIG. 18). These results indicate that, with respect to the surface glycoproteins of CCHFV, GP38 is a key immunogen necessary for the protection against lethal infection, providing 40% protection, and vaccination regimens that include Gc or Gn-Gc did not improve survival.

[0223] The ability of serially diluted sera from vaccinated animals to neutralize IbArl0200 infection of VeroE6 cells was tested and the AUC quantified (FIG. 7C). As expected, sera of mock-immunized and GP38 -immunized mice did not neutralize infection, whereas sera from Gc-immunized mice neutralized infection, with a logi AUC value of 52. GP38 + Gc-immunized mice sera also neutralized infection, with a logwAUC value of 18, but did not exhibit a statistically significant difference from the sera of mock-immunized mice. The lower neutralizing capacity is expected as only 5 pg of Gc was immunized in the GP38 + Gc group. Sera from mice immunized with GP38-GnII DS-Gc neutralized infection, with a logioAUC value of 37, which was statistically different from the mock-immunized control.These results indicate that immunization with GP38-GnH DS-Gc elicits neutralizing antibodies and the incorporation of GP38, Gn, and Gc in a single chain construct affords an advantage of eliciting higher neutralizing antibody titers than a GP38 + Gc subunit vaccination strategy.

[0224] The ability of the sera to bind whole irradiated virus, GP38, Gn, Gc, and GP38- GnH DS-Gc was evaluated by measuring binding titers via ELISA and quantifying the AUC. All sera from mice immunized with the CCHFV immunogens bound to whole irradiated virus, and sera from mock-immunized mice did not, confirming that all immunogens elicited CCHFV- specific antibodies (FIG. 7D). Next, the ability of the sera to bind the monomeric glycoproteins was evaluated. Interestingly, sera from mice immunized with GP38-GnH DS-Gc bound to GP38 better than sera from GP38-immunized mice by roughly two logs (FIG. 7E). None of the sera bound to monomeric Gn, including the sera from mice immunized with GP38-GnH DS-Gc (FIG. 7F), indicating no Gn-specific antibodies were detected. Sera from mice immunized with Gc, GP38 + Gc, or GP38-GnH DS-Gc bound Gc to varying degrees, with sera from Gc-immunized mice showing the strongest binding (FIG. 7G). The ability of the sera to bind GP38-GnH DS-Gc was also tested. Surprisingly, sera from GP38-immunized mice did not bind the heterotrimer. Sera from GP38 + Gc-immunized mice bound the heterotrimer but to a lesser extent than the sera from Gc- or GP38-GnH DS-Gc-immunized mice (logioAUC values of 0.4, 5.0, and 4.9, respectively, FIG. 7H). Together, these data indicate that immunization with GP38-GnH DS-Gc elicits neutralizing antibodies, generates a strong GP38-specific antibody response, and produces Gc-specific antibodies with the ability to neutralize infection.

[0225] Mice had disparate humoral immune responses after vaccination (FIGS. 7C- 7H). GP38-specific antibodies were undetectable for three mice in the GP38-immunized and GP38-GnH DS-Gc-immunized groups and seven mice in the GP38 + Gc-immunized group (FIG. 7E). For the GP38-immunized and GP38-GnH DS-Gc-immunized groups, five mice in each group had a strong GP38-specific response (AUC > 1). Of these mice, four survived in each group. Additionally, most surviving mice from the GP38-GnH DS-Gc-immunized group had high neutralizing antibody titers, high Gc-specific antibody titers, and high GP38-GnH DS-Gc- specific antibody titers. Together, these data suggest a correlation between a robust humoral immune response and survival outcomes and indicate that elicitation of GP38-specific antibodies may be required for protection against lethal challenge.

[0226] Since CCHFV IbAr 10200 is a laboratory-passaged virus, binding of the sera to GP38 derived from the CCHFV isolates Afg09, Oman, M18-China, Turkey 2004, and Kosova-Hoti was tested via ELISA (FIGS. 19A-19F). Sera from mice immunized with GP38 or GP38- GnH DS-Gc bound all GP38 proteins tested. Sera of mice immunized with GP38-GnH-DS-Gc bound to GP38 of Afg09, Oman, and M18-China similarly to IbArl0200 (AUC values of 3.3, 4.4, 2.0, and 3.7, respectively) and bound Kosova-Hoti and Turkey 2004 to a lesser extent (AUC values of 0.7 and 0.6, respectively). Notably, mice immunized with GP38-GnH DS-Gc had higher levels of cross-reactive GP38-specific antibodies than mice immunized with GP38 alone for all strains of GP38 tested. These data demonstrate that GP38-GnH DS-Gc elicits highly cross- reactive GP38-specific antibodies that bind GP38 derived from clinically relevant isolates.Example 7 - Material and Methods

[0227] Protein expression and purification ofGP38-Gn variants. All variants included the CCHFV M segment (Isolate IbArl0200, GenBank AF467768.2) native signal sequence followed by the MLD, a furin cleavage site (RRRRRR; SEQ ID NO: 18), and GP38-Gn. Codon-optimized (GenScript) variants were cloned into the mammalian expression vector paH upstream of a human rhinovirus 3C (HRV3C) protease cleavage site, an 8X His tag, and a Twin-Strep tag. GP38-Gn plasmids were transiently co-transfected (4: 1 ratio) with a plasmid encoding for a human furin ectodomain (residues 1-794) into 40 mLs of FreeStyle293F cells (ThermoFisher). Cells were transfected at a density of 1 million cells / mL using 25 kDa linear polyethylenimine (PEI). Cultures were grown for 6 days in a shaking incubator at 37°C supplied with 8% CO2 and 80% humidity. After 6 days, culture supernatant was separated via centrifugation and passed through a 0.22 pm filter. To evaluate differences in expression, all variants were purified from supernatant using 1 mL of StrepTactin resin following the manufacturer’s instructions (IBA). The elution fractions were concentrated to 100 pL. Purified protein was flash-frozen in liquid nitrogen and stored at -80°C. Expression experiments of GP38-Gn variants in this study were performed at least twice.

[0228] Protein expression and purification of GP38-GnH DS-Gc. The GP38-GnH DS-Gc ectodomain was a single-chain construct that included the native CCHFV M segment signal sequence followed by the MLD, a furin cleavage site (RRRRRR; SEQ ID NO: 18), GP38 (248- 515, Q496C), an R516S mutation in the SKI-I cleavage site, resulting in a linker (SRLL, mutation underlined), Gn (520-587, V562C), a TEV-cleavable Gly-Ser linker with the sequence ENLYFQGGGGSGGGSGGGSENLYFQG (SEQ ID NO: 19; TEV cleavage sites in bold) followed by Gc (1061-1545) upstream of an HRV3C protease cleavage site, an 8X His tag, and a Twin-Strep tag. A codon-optimized (GenScript) gene was cloned into themammalian expression vector paH. Expression and purification of GP38-GnH DS-Gc were performed as described for the GP38-Gn variants. For structural studies, GP38-GnH DS-Gc was further purified by size-exclusion chromatography (SEC) using a Superdex 200 Increase 10 / 300 GL column (Cytiva) in a buffer composed of 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% NaNs- Purified protein was concentrated, flash frozen in liquid nitrogen, and stored at - 80°C.

[0229] Protein expression and purification of CCHFV immunogens. The GP38 construct encoded the native signal sequence followed by the MLD, a furin cleavage site (RRRRRR; SEQ ID NO: 18), and GP38. The Gc construct encoded the native signal sequence followed by the MLD, a furin cleavage site (RRRRRR; SEQ ID NO: 18), GP38, another furin cleavage site (RRRRRR; SEQ ID NO: 18), followed by Gc (1061-1545). The GP38-Gn and GP38-GnH DS-Gc constructs were as described. Codon-optimized (GenScript) genes were cloned into the mammalian expression vector paH upstream of a human rhinovirus 3C (HRV3C) protease cleavage site, an 8X His tag, and a Twin-Strep tag. Expression and purification of immunogens were performed as described for the GP38-Gn variants. Immunogens were further purified by size-exclusion chromatography (SEC) using a Superdex 200 Increase 10 / 300 GL column (Cytiva) in phosphate buffered saline (PBS). GP38, GP38- GnH DS, GP38-GnH DS+c, and GP38-GnH DS-Gc were used in subsequent ELISA experiments.

[0230] X-ray crystallography. To crystallize GP38-GnH DS, a construct was expressed that minimized a flexible linker region between GP38 and Gn11. This GP38-Gn variant included the signal sequence, MLD, and furin cleavage site as previously described for the other GP38- Gn variants but residues 509-532 (spanning both GP38 and Gn) were replaced with a Gly-Ser linker (GGGSGGGS; SEQ ID NO: 20). To trim the N-linked glycans, the purified protein was brought to a final concentration of 1 mg / mL in a buffer composed of 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN3 and was treated with His-tagged EndoH for 3 hours at a 10:1 ratio of protein to EndoH (wt / wt) at room temperature. During the last hour of EndoH digestion, His-Tagged HRV3C was added at a 50:1 ratio of protein to HRV3C (wt / wt) to remove the affinity tags. Digested protein was passed over Ni-NTA resin to remove cleaved tags, EndoH, and HRV3C. The flow-through was collected, which contained untagged GP38-GnH DS. The protein was further purified by SEC using a Superdex 200 Increase 10 / 300 GL column (Cytiva) in a buffer composed of 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaNs. After concentrating to 7.1 mg / mL, the protein (0. 1 u L) was mixed with 0.05 u L mother liquor (8% v / v Tascimate™pH 8.0, 20% wt / vol polyethylene glycol 3,350) using an NT8 (Formulatrix), and a 0.15 pL drop was spotted onto an MRC2 crystallization tray and sealed to allow for vapor diffusion. The crystal was soaked in mother liquor supplemented with glycerol to a final concentration of 30% (vol / vol), looped, and flash-frozen in liquid nitrogen. Remote data collection was performed at NYX beamline 19-ID at NSLS-II. Diffraction data were indexed and integrated in iMOSFLM (Battye el al., 2011) before being merged and scaled to 2.5 A using Aimless (Evans et al., 2013). Molecular replacement was performed in Phaser (McCoy et al., 2007) using GP38 (PDB ID: 6VKF) as a starting model. The model was built and refined using Coot (Emsley et al., 2010) and Phenix (Liebschner et al., 2019).

[0231] Cryo-EM. 0.5 mg / mL GP38-GnII DS-Gc was incubated with 1.2-fold molar excess of ADI-36125 and ADI-46152 Fab in 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN3, and 0.0035% amphipol (wt / vol, Anatrace). The sample was incubated at room temperature for 30 minutes and then deposited onto a glow-discharged Protochips C-Flat 400 mesh 1.2 pm / 1.3 pm grid. After 5 s of wait time, the grid was blotted for 4 s with a force of 1 using a Vitrobot Mark IV (ThermoFisher) and plunge-frozen into liquid ethane. A total of 13,208 micrographs were collected — 8,061 micrographs at 0° tilt and 5,147 micrographs at 30° tilt — from a single grid on a FEI Titan Krios (ThermoFisher) equipped with a K3 direct electron detector (Gatan) at the end of a Gatan Biocontinuum Imaging Filter (Gatan) operated with a 20 eV slit width. The microscope was operated with an accelerating voltage of 300 kV and a total electron flux of 69 e 7 A2. Data were collected at a magnification of 105,000X, corresponding to a calibrated pixel size of 0.83 A / pix. CryoSPARC v4.0.1 was used for data processing, which includes patch-based motion correction, patch-based CTF correction, particle-picking, and then curation of the particles via iterative rounds of 2D classification. 3D volumes were generated using ab initio reconstruction, and data were further processed via heterogeneous refinement, homogenous refinement, and subsequently non-uniform homogeneous refinement of final classes. The processing workflow can be found in (FIG. 15) Models were docked into the experimental EM map using ChimeraX (Pettersen et al., 2021) and further refined by Phenix and Coot. The starting models for Gc and ADI-46152 were from PDB ID 7L7R and 8VWW. For GP38 and Gn, the starting model was the crystal structure from this manuscript (PDB ID). For ADI-36125, an initial homology model was generated using the SAbPred server (Abanades et al., 2022).

[0232] Differential scanning fluorimetry. All variants were prepared at a concentration of 3 pM with a final concentration of 10X SYPRO Orange Protein Gel Stain (Sigma- Aldrich) in a white, opaque 96-well plate. 15 fluorescence measurements ( ex = 465 nm, Zem = 580 nm) were taken per °C using a Roche LightCycler 480 II, with a temperature ramp rate of 0.04°C / second, and a temperature range of 22°C to 95°C. Data were plotted as the derivative of the melting curve. Each curve is an average of three technical replicates.

[0233] Cell culture. Vero and VeroE6 cells, immortalized epithelial cell lines isolated from the kidney of an adult female African grivet monkey (RRID:CVCL-0059 and RRID: CVCL-0574, respectively), were obtained from the American Type Culture Collection (ATCC). SW13 cells, a cell line isolated from the adrenal gland and cortex of a 55-year old female patient with carcinoma (RRIDD:CCL-105), were obtained from ATCC. BSR-T7 cells (RRID: CVCL_RW96), generated by stable T7 RNA polymerase expression in BHK-21 cells, were a kind gift from K.-K. Conzelmann. The parent cell line (RRID: CVCL_1915) was isolated from the kidney of a 1 -day-old male golden hamster. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose; Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 1% penicillinstreptomycin (P / S; Thermo Fisher Scientific), and 1% GlutaMAX (Thermo Fisher Scientific). All cell lines were maintained in a humidified 37°C incubator supplied with 5% CO2.

[0234] In vivo immunization with GP38-Gn constructs. For immunization with GP38- Gn constructs, 6-8 week old female C57BL / 6J mice were purchased from the Jackson Laboratory (JAX: 000664). Mice (n =6) were immunized with 10 pg of GP38, GP38-GnH DS, GP38-GnH DS+c, or an equal volume of phosphate-buffered saline (PBS) adjuvanted with 50 pg of poly(EC) (High Molecular Weight) VacciGrade (InvivoGen) and boosted three weeks after in the same fashion. Constructs and adjuvant were each diluted in endotoxin-free PBS (Millipore) to a total volume of 100 pL each before they were mixed to obtain a total of 200 pL per mouse. 200 pL of immunogen + adjuvant was delivered by intraperitoneal (IP) injection for each mouse. Mice were bled two days before prime (day -2) and ten weeks post-boost (day 70). Sera were isolated from whole blood by allowing the blood to coagulate for 1 hour at room temperature and separated by microcentrifugation (10000 g, 10 min). Sera were aliquoted and stored at -80°C for subsequent use in enzyme-linked immunosorbent assay (ELISA) and tecVLP neutralization assays.

[0235] ELlSAs for GP38-Gn immunization. ELISAs for each serum sample were run in duplicate. Flat bottom, high binding, half-area 96-well plates (Coming) were coated with 75 ng of each immunogen in PBS at 4°C overnight. The initial coating was decanted, and the plate was then blocked with 5% nonfat dry milk (BioRad) in PBS for 3 hours at 37°C. Serial 3-fold dilutions were made for each mouse serum sample starting at a 1 :100 dilution in 3% bovine serum albumin (Fisher) in PBS and incubated with the coated immunogens for 1.5 hours at 37°C. Sera / immunogen samples were incubated with an anti-mouse IgG-HRP secondary (Jackson Immunolabs; Cat. 1 15-035-003) in 3% nonfat dry milk in PBS for 1 hour at 37°C. Finally, the ELISA was developed for 8 min at room temperature with 1-Step TMB Ultra Substrate (Thermo) before being neutralized with 0.5 M H2SO4. Binding was quantified using Cytation 5 cell imaging multimode reader (BioTek) to measure absorbance at 450 nm. After background subtraction, area under the curve (AUC) values were computed for each ELISA curve (Baseline Y =0, Ignore peaks <10% of the distance from min to max Y). AUCs were log-transformed to fulfill normality and homoscedasticity requirements, and log-transformed AUCs for each serum and immunogen group were compared by 2-way ANOVA with Tukey corrections for multiple comparisons. Statistical tests were performed in GraphPad Prism 10.0.3.

[0236] Generation of tecVLPs bearing CCHFV IbAr 10200 glycoproteins. The amino acid sequence for the IbAr 10200 was derived from GenBank M segment sequences with an accession number NC_005300. Transcription- and entry-competent virus-like particles (tecVLPs) bearing CCHFV glycoproteins were generated as previously described (Zivcec et al., 2015; Fels et al., 2021). Briefly, BSR-T7 cells were transfected with plasmids encoding the CCHFV nucleoprotein (NP), glycoprotein complex (GPC), polymerase (L), as well as T7 polymerase, and a minigenome encoding Nano-Gio Luciferase. 15 hours post-transfection, transfection media was replaced on cells with fresh DMEM growth media. 48 hours posttransfection, tecVLP -containing supernatants were collected, clarified by low-speed centrifugation, and finally pelleted by ultracentrifugation at 25,000 rpm for 2.5 hours. Pelleted tecVLPs were resuspended in plain DMEM overnight before storage at -80°C prior to use in any infection experiments.

[0237] tecVLP neutralization assay. Neutralization assays were performed at select dilutions. Vero cells were seeded in 96-well cell culture plates (Corning) at 18,000 cells per well 24 hours before infection. The number of tecVLPs to add was empirically determinedsuch that the maximum luminescence signal of infected cells was ~500x that of background. Mouse sera were incubated with tecVLPs at either a 1:20 or 1 :100 dilution in DMEM supplemented with 2% FBS, 1% P / S, and 1% GlutaMAX for 1 hour at 4°C. 90 pL of media was removed from the target cells, and 50 pL virus / sera mixture was in duplicate. The infection was allowed to proceed 14-16 hours at 37°C and 5% CO2. The infection media was then decanted, cells were washed once with PBS, and the luciferase signal was developed using the Nano-Gio luciferase assay system (Promega) per the manufacturer’s instructions. Infectivity was quantified by luminescence signal using Cytation 5 cell imaging multimode reader (Biotek) with the following run parameters: 200 gain, 6 mm height, 10 sec integration time. Percent infectivity values were calculated for each serum sample relative to sera-free, infected wells. Sera group infectivity values were assessed for normality and were compared by 2-way ANOVA against the control group (mock-immunized) with Turkey corrections for multiple comparisons (GraphPad Prism 10.0.3).

[0238] Ethics statement for lbArl0200 in vivo challenge study. Murine challenge studies were conducted under Institutional Animal Care and Use Committee (IACUC)- approved protocols in compliance with the Animal Welfare Act, PHS Policy, and other applicable federal statutes and regulations. The facility where these studies were conducted (USAMRIID) are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and adhere to the principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2013. Humane endpoints were utilized during these studies and mice that were moribund, according to an endpoint score sheet and in line with IACUC approved criteria, were humanely euthanized.

[0239] IbAr 10200 in vivo challenge study. 4- week old male and female C57BL / 6J mice (strain #000664; The Jackson Laboratory) were vaccinated two times at 3 week intervals with 10 pg of recombinant protein or PBS control diluted at a 1:1 ratio with Addavax (InvivoGen) via the IP route. Constructs were diluted in endotoxin-free PBS (ThermoFisher Scientific) to a total volume of 200 |iL each before they were mixed with 200 pL Addavax to obtain a total of 400 pL per mouse. Whole blood was collected prior to vaccination on days 0, 21, and 42 by submandibular bleed and sera were isolated from whole blood as described above. Mice were challenged on day 53. For the challenge, all mice were challenged with 100 plaque forming units (PFU) of CCHFV-IbAr 10200 by the IP route. 24 hours post-challenge, mice were transiently immunosuppressed by treatment with 1.5 mg / mouse of mAb-5A3 (LeincoTechnologies Inc.) via the IP route. Mice were monitored daily for weight changes, clinical score, and survival. Mice were scored on a 4-point grading scale; 1 defined by decreased grooming and ruffled fur, 2 defined by subdued behavior when un-stimulated, 3 defined by lethargy, hunched posture, and subdued behavior even when stimulated, and 4 defined by bleeding, unresponsiveness, severe weakness, or inability to walk. All mice scoring a 4 were considered moribund and were euthanized based on lACUC-approved criteria. Daily observations were increased to a minimum of twice daily while mice were exhibiting a clinical score of 3.

[0240] Viruses. The authentic CCHFV isolate CCHFV-IbArl0200 was used in this study.

[0241] Neutralization assays against authentic CCHFV. Neutralization assays were conducted as described previously (Fels et al., 2021). In brief, heat inactivated serum was diluted 1 :5 and then serial 3-fold dilutions were generated. CCHFV-Ib Ar 10200 was incubated with dilutions for 1 hour at 37°C. The serum-virus mixture was added to monolayers of VeroE6 cells in a 96-well plate at a final multiplicity of infection (MOI) of 0.06 and incubated for 1 hour at 37 °C. Infection medium was then removed, and fresh cell culture medium without sample was added. 48 hours post-infection, culture medium was removed, and plates were fully submerged in 10% buffered formalin and fixed for at least 4 hours at room temperature. Plates were removed from formalin and permeabilized with 0.2% Triton-X for 10 minutes at room temperature and treated with blocking buffer (Cell Stain Buffer; ThermoFisher). Infected cells were detected by consecutive incubation with CCHFV-specific antibody 9D5 (3 pg / ml; BEI NR-40270) and secondary detection antibody (goat anti-mouse) conjugated to AlexaFluor 488 (1 :2000 dilution; Invitrogen). Percent infection was determined using the Cytation5 high- content imaging instrument and data analysis was performed using the Gen5.11 software (BioTek). Percent infectivity values were calculated for each serum sample relative to sera- free, infected wells and normalized to naive control samples. AUC values were computed for each neutralization curve (Baseline Y=0, Ignore peaks <10% of the distance from min to max Y) and log transformed. AUCs were compared by 2-way ANOVA with Turkey corrections for multiple comparisons (GraphPad Prism 10.0.3).

[0242] Preparation of irradiated CCHFV whole antigen. SW13 cells were infected with CCHFV-IbAr 10200 at an MOI of 0.01 and incubated for 1 hour at 37°C. After incubation, infection medium was removed, and fresh cell culture medium was added. 72 hours post-infection, supernatants from CCHFV-IbArl0200 infected SW13 cells were harvested and cleared from cell debris through load-speed centrifugation at 2,000 rpm for 10 minutes at 4°C. Clarified supernatants were centrifuged at 12,000 rpm for 4 hours at 4°C to pellet virus and pelleted virus resuspended in cold lx Tris-NaCl-EDTA (TNE) buffer (Quality Biologies). Virus was loaded onto a 20-60% sucrose gradient and spun at 40,000 rpm (SW-41 rotor) for 16 hours at 4°C with no brake. Virus band was collected and treated by gamma-irradiation (8xl06rads).

[0243] Enzyme-linked immunosorbent assay (ELISA). High bind half-area ELISA plates (Corning) were coated overnight (~18 hours) at 4°C with either 125 ng CCHFV rGc, Sheep Fc-Tag (Native Antigen), 125 ng rGn, His-Tag (Native Antigen), 75 ng GP38 protein, 75 ng GP38-GnH DS-Gc, or 125 ng irradiated CCHFV whole antigen diluted in PBS. The following day, plates were blocked with 5% skim milk (BD Biosciences) diluted in PBS containing 0.05% Tween-20 (PBST) for 2 hours at 37°C. Sera samples were diluted 1 : 10 with subsequent 3 -fold dilutions (dilution range 1: 10 to 1:7290) in blocking buffer and plates were loaded with dilutions in duplicate. Plates were incubated at ambient temperature for 2 hours, washed three times with PBST, and then incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse (1:2000 dilution; Jackson ImmunoResearch) diluted in blocking buffer for 1 hour at ambient temperature. After incubation, plates were washed three times with PBST and then developed with TMB substrate (ThermoFisher Scientific). Reaction was stopped using 0.16 M sulfuric acid and absorbance was read at 450 nm wavelength, detected using a SpectraMax (Molecular Biosciences) microplate reader. Naive sera collected prior to vaccination was used as an internal control for each group. A cutoff value was determined based on the average absorbance of the naive control starting dilution plus 3 standard deviations. Only sample dilutions whose average were above this cut-off were registered as a positive signal. AUC values were computed for each ELISA curve (Baseline Y=0, Ignore peaks <10% of the distance from min to max Y). AUCs were log-transformed and compared for each serum and immunogen group by 2-way ANOVA with Tukey corrections for multiple comparisons. Statistical tests were performed in GraphPad Prism 10.0.3.

[0244] Quantification and statistical analysis. Statistical details, including the number of replicates (n), measures of precision, and the statistical test used for each experiment can be found in the corresponding figure legends and in the results section. All area under the curvevalues (ELISA and microneutralization assays) were log transformed prior to statistical analysis. All statistical analyses were conducted in GraphPad Prism 10.0.3.* * *

[0245] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claimsREFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.Abanades B, Georges G, Bujotzek A, Deane CM. AB looper: fast accurate antibody CDR loop structure prediction with accuracy estimation. Bioinformatics. 2022;38(7): 1877-80.Ahata B, Akcapinar GB. CCHFV vaccine development, current challenges, limitations, and future directions. Front Immunol. 2023; 14: 1238882.Altamura LA, Bertolotti-Ciarlet A, Teigler J, Paragas J, Schmaljohn CS, Doms RW. Identification of a novel C-terminal cleavage of Crimean-Congo hemorrhagic fever virus PreGN that leads to generation of an NSM protein. J Virol. 2007;81(12):6632-42.Appelberg S, John L, Pardi N, Vegvari A, Bereczky S, Ahlen G, et al. Nucleoside-Modified mRNA Vaccines Protect IFNAR(- / -) Mice against Crimean-Congo Hemorrhagic Fever Virus Infection. J Virol. 2022 ;96(3):e0156821.Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr. 2011 ;67(Pt 4):271-81.Bereczky S, Lindegren G, Karlberg H, Akerstrom S, Klingstrom J, Mirazimi A. Crimean- Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptorknockout mice. J Gen Virol. 2010;91(Pt 6): 1473-7.Bertolotti-Ciarlet A, Smith J, Strecker K, Paragas J, Altamura LA, McFalls JM, et al. Cellular localization and antigenic characterization of crimean-congo hemorrhagic fever virus glycoproteins. J Virol. 2005 ;79(10):6152-61.Centers for Disease Control and Prevention, Crimean-Congo Hemorrhagic Fever (CCHF)2013. Available on the world wide web at cdc.gov / vhf / crimean- congo / index.html#:~:text=Crimean%2DCongo%20hemorrhagic%20fever%20is,East %2C%20and%20the%20Indian%20subcontinent.&text=lxodid%20(hard)%20ticks% 20are%20both,for%20the%20CCHF%20virus.Dowall SD, Graham VA, Rayner E, Hunter L, Watson R, Taylor I, et al. Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses. PLoS One. 2016;l l(6):e0156637.Durie IA, Tehrani ZR, Karaaslan E, Sorvillo TE, McGuire J, Golden JW, et al. Structural characterization of protective non-neutralizing antibodies targeting Crimean-Congo hemorrhagic fever virus. Nat Commun. 2022;13(l):7298.Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):486-501.Ergonul O, Celikbas A, Baykam N, Eren S, Dokuzoguz B. Analysis of risk-factors among patients with Crimean-Congo haemorrhagic fever virus infection: severity criteria revisited. Clin Microbiol Infect. 2006; 12(6):551 -4.Ergonul O. Crimean-Congo haemorrhagic fever. Lancet Infect Dis. 2006;6(4):203-14.European Centre for Disease Prevention and Control, The spatial distribution of Crimean- Congo haemorrhagic fever in Europe and neighbouring areas. 2023.Evans PR, Murshudov GN. How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr. 2013;69(Pt 7): 1204- 14.Fels JM, Maurer DP, Herbert AS, Wirchnianski AS, Vergnolle O, Cross RW, et al. Protective neutralizing antibodies from human survivors of Crimean-Congo hemorrhagic fever. Cell. 2021 ;184(13):3486-501 e21.Ganaie SS, Schwarz MM, McMillen CM, Price DA, Feng AX, Albe JR, et al. Lrpl is a host entry factor for Rift Valley fever virus. Cell. 2021 ;184(20):5163-78 e24.Garrison AR, Alkhovsky SV, Avsic-Zupanc T, Bente DA, Bergeron E, Burt F, et al. ICTV Virus Taxonomy Profile: Nairoviridae. J Gen Virol. 2020;101(8):798-9.Garrison AR, Shoemaker CJ, Golden JW, Fitzpatrick CJ, Suschak JJ, Richards MJ, et al. A DNA vaccine for Crimean-Congo hemorrhagic fever protects against disease and death in two lethal mouse models. PLoS Negl Trop Dis. 2017;l l(9):e0005908.Garrison AR, Smith DR, Golden JW. Animal Models for Crimean-Congo Hemorrhagic Fever Human Disease. Viruses. 2019;l 1(7).Golden JW, Fitzpatrick CJ, Suschak JJ, Clements TL, Ricks KM, Sanchez-Lockhart M, Garrison AR. Induced protection from a CCHFV-M DNA vaccine requires CD8(+) T cells. Virus Res. 2023;334: 199173.Golden JW, Shoemaker CJ, Lindquist ME, Zeng X, Daye SP, Williams JA, et al. GP38- targeting monoclonal antibodies protect adult mice against lethal Crimean-Congo hemorrhagic fever virus infection. Sci Adv. 2019;5(7):eaaw9535.Guardado-Calvo P, Rey FA. The surface glycoproteins of hantaviruses. Curr Opin Virol.2021;50:87-94.Guardado-Calvo P, Rey FA. The Viral Class II Membrane Fusion Machinery: Divergent Evolution from an Ancestral Heterodimer. Viruses. 2021 ; 13( 12).Hawman DW, Feldmann H. Crimean-Congo haemorrhagic fever virus. Nat Rev Microbiol. 2023;21(7):463-77.Hawman DW, Meade- White K, Leventhal S, Appelberg S, Ahlen G, Nikouyan N, et al. Accelerated DNA vaccine regimen provides protection against Crimean-Congo hemorrhagic fever virus challenge in a macaque model. Mol Ther. 2023 ;31(2):387-97.Hinkula J, Devignot S, Akerstrom S, Karlherg H, Wattrang E, Bereczky S, et al. Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and / or Virus-Like Particles Induces Protection and Survival in Challenged Mice. J Virol. 2017;91(10). langra RK, Herbert AS, Li R, Jae LT, Kleinfelter LM, Slough MM, et al. Protocadherin-1 is essential for cell entry by New World hantaviruses. Nature. 2018;563(7732):559-63.Kortekaas J, Vloet RP, McAuley AJ, Shen X, Bosch BJ, de Vries L, et al. Crimean-Congo Hemorrhagic Fever Virus Subunit Vaccines Induce High Levels of Neutralizing Antibodies But No Protection in STAT1 Knockout Mice. Vector Borne Zoonotic Dis. 2015;15(12):759-64.Li N, Rao G, Li Z, Yin J, Chong T, Tian K, et al. Cryo-EM structure of glycoprotein C from Crimean-Congo hemorrhagic fever virus. Virol Sin. 2022;37(l):127-37.Liebschner D, Afonine PV, Baker ML, Bunkoczi G, Chen VB, Croll TI, et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol. 2019;75(Pt 10):861-77.McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40(Pt 4):658-74.Mishra AK, Hellert J, Freitas N, Guardado-Calvo P, Haouz A, Fels JM, et al. Structural basis of synergistic neutralization of Crimean-Congo hemorrhagic fever virus by human antibodies. Science. 2022;375(6576): 104-9.Mishra AK, Moyer CL, Abelson DM, Deer DJ, El Omari K, Duman R, et al. Structure and Characterization of Crimean-Congo Hemorrhagic Fever Virus GP38. J Virol. 2020;94(8).Mittler E, Serris A, Esterman ES, Florez C, Polanco LC, O'Brien CM, et al. Structural and mechanistic basis of neutralization by a pan-hantavirus protective antibody. Sci Transl Med. 2023 ;15(700) :eadg 1855.Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 2021;30(l):70-82.Punch EK, Hover S, Blest HTW, Fuller J, Hewson R, Fontana J, et al. Potassium is a trigger for conformational change in the fusion spike of an enveloped RNA virus. J Biol Chem. 2018;293(26):9937-44.Rissanen I, Krumm SA, Stass R, Whitaker A, Voss JE, Bruce EA, et al. Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen. mBio. 2021 ;12(4):e0253120.Rodriguez SE, Cross RW, Fenton KA, Bente DA, Mire CE, Geisbert TW. Vesicular Stomatitis Virus-Based Vaccine Protects Mice against Crimean-Congo Hemorrhagic Fever. Sci Rep. 2019;9(1):7755.Sanchez AJ, Vincent MJ, Erickson BR, Nichol ST. Crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by Furin- like and SKI-1 proteases to generate a novel 38-kilodalton glycoprotein. J Virol. 2006;80(l):514-25.Scher G, Bente DA, Mears MC, Cajimat MNB, Schnell MJ. GP38 as a vaccine target for Crimean-Congo hemorrhagic fever virus. NPJ Vaccines. 2023;8(l):73.Serris A, Stass R, Bignon EA, Muena NA, Manuguerra JC, Jangra RK, et al. The Hantavirus Surface Glycoprotein Lattice and Its Fusion Control Mechanism. Cell. 2020;183(2):442-56 el6.Shepherd AJ, Swanepoel R, Leman PA. Antibody response in Crimean-Congo hemorrhagic fever. Rev Infect Dis. 1989; 11 Suppl 4:S801-6.Shin et al., Crimean-Congo Hemorrhagic Fever Survivors Elicit Protective Non-Neutralizing Antibodies that Target 11 Overlapping Regions on Viral Glycoprotein GP38. bioRxiv, doi: 10.1101 / 2024.03.02.583110, March 6, 2024.Stass R, Engdahl TB, Chapman NS, Wolters RM, Handal LS, Diaz SM, et al. Mechanistic basis for potent neutralization of Sin Nombre hantavirus by a human monoclonal antibody. Nat Microbiol. 2023;8(7): 1293-303.Suschak JJ, Golden JW, Fitzpatrick CJ, Shoemaker CJ, Badger CV, Schmaljohn CS, Garrison AR. A CCHFV DNA vaccine protects against heterologous challenge and establishes GP38 as immunorelevant in mice. NPJ Vaccines. 2021 ;6( 1):31.Tunyasuvunakool K, Adler J, Wu Z, Green T, Zielinski M, Zidek A, et al. Highly accurate protein structure prediction for the human proteome. Nature. 2021;596(7873):590-6.Vincent MJ, Sanchez AJ, Erickson BR, Basak A, Chretien M, Seidah NG, Nichol ST. Crimean- Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI- 1. J Virol. 2003;77(16):8640-9.World Health Organization, Annual review of diseases prioritized under the Research and Development Blueprint 2018. Available on the world wide web at who.int / news- room / events / detail / 2018 / 02 / 06 / default-calendar / 2018-annual-review-of-diseases- prioritized-under-the-research-anddevelopment-blueprint.World Health Organization, Crimean-Congo haemorrhagic fever 2020. Available on the world wide web at who.int / health-topics / crimean-congo-haemorrhagic-fever.Xu ZS, Du WT, Wang SY, Wang MY, Yang YN, Li YH, et al. LDLR is an entry receptor for Crimean-Congo hemorrhagic fever virus. Cell Res. 2024.Zivcec M, Metcalfe MG, Albarino CG, Guerrero LW, Pegan SD, Spiropoulou CF, Bergeron E. Assessment of Inhibitors of Pathogenic Crimean-Congo Hemorrhagic Fever Virus Strains Using Virus-Like Particles. PLoS Negl Trop Dis. 2015;9(12):e0004259.Zivcec M, Safronetz D, Scott D, Robertson S, Ebihara H, Feldmann H. Lethal Crimean-Congo hemorrhagic fever virus infection in interferon alpha / beta receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy. J Infect Dis. 2013;207(12):1909-21.

Claims

CLAIMS1 . An engineered protein comprising an engineered CCHFV glycoprotein ectodomain that comprises GP38 and GnHand Gc, wherein the GP38 and Gn11portion comprises a sequence at least 90% identical to the sequence of positions 248-587 of SEQ ID NO: 1, and wherein the Gc portion comprising a sequence at least 90% identical to the sequence of positions 1061- 1544 of SEQ ID NO: 1, and wherein the engineered ectodomain comprises at least one substitution or substitution set selected from the group consisting of: Q496C / V562C; G371C / Y546C; S370C / Y546C; K404D; G527C / K1162C; R565C / P1161C; R579C / D1214C; and C1354S, with the positions being relative to the sequence of SEQ ID NO: 1.

2. The engineered protein of claim 1, wherein the engineered ectodomain comprises at least one substitution set selected from the group consisting of:(a) Q496C / V562C and R565C / P1161C;(b) Q496C / V562C and R579C / D1214C;(c) Q496C / V562C and G371C / Y546C;(d) G371C / Y546C and R565C / P1161C;(e) G371C / Y546C and R579C / D1214C; and(f) R565C / P1161C and R579C / D1214C, with the positions being relative to the sequence of SEQ ID NO: 1.

3. The engineered protein of claim 1 or 2, wherein the Gc region is fused to the C-terminus of the GnHdomain by a flexible linker.

4. The engineered protein of claim 3, wherein the flexible linker is a glycine-serine linker.

5. The engineered protein of any one of claims 1-4, wherein the engineered CCHFV glycoprotein ectodomain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of positions 250-1100 of any one of SEQ ID NOs: 10-17 and 31-36.

6. An engineered protein comprising an engineered CCHFV glycoprotein ectodomain that comprises GP38 and Gn11, wherein the engineered CCHFV glycoprotein ectodomain comprises a sequence at least 90% identical to the sequence of positions 248-587 of SEQ ID NO: 1, and wherein the engineered CCHFV glycoprotein ectodomain comprises a disulfide bond between the C-terminal P-strand of GP38 and the N-terminal -strand of the GnHdomain B.

7. The engineered protein of claim 6, wherein the disulfide bond is between positions Q496C and V562C, with the positions being relative to the sequence of SEQ ID NO: 1.

8. The engineered protein of claim 6 or 7, wherein the cleavage site between GP38 and GnHis mutated.

9. The engineered protein of any one of claims 6-8, further comprising a R516S substitution.

10. The engineered protein of any one of claims 6-9, further comprising a Gn domain C region.

11. The engineered protein of any one of claims 6-10, wherein the engineered CCHFV glycoprotein ectodomain comprises a sequence at least 90% identical to the sequence of positions 248-663 of SEQ ID NO: 1.

12. The engineered protein of any one of claims 6-11, further comprising a Gn MPER region.

13. The engineered protein of any one of claims 6-12, wherein the engineered CCHFV glycoprotein ectodomain comprises a sequence at least 90% identical to the sequence of positions 248-690 of SEQ ID NO: 1.

14. The engineered protein of any one of claims 1-13, further comprising a mucin-like domain region.

15. The engineered protein of any one of claims 1-14, wherein the engineered CCHFV glycoprotein ectodomain comprises a sequence at least 90% identical to the sequence of positions 25-587 of SEQ ID NO: 1.

16. The engineered protein of any one of claims 1-15, wherein the cleavage site between the mucin- like domain and GP38 is mutated.

17. The engineered protein of claim 16, comprising a substitution corresponding to RSKR (residues corresponding to positions 244-247 of SEQ ID NO: 1) to RRRRRR (SEQ ID NO: 18).

18. The engineered protein of any one of claims 1-17, wherein the engineered CCHFV glycoprotein ectodomain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of positions 250-589 of SEQ ID NO: 6.

19. The engineered protein of any one of claims 1-18, wherein the engineered CCHFV glycoprotein ectodomain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of positions 250-665 of SEQ ID NO: 7.

20. The engineered protein of any one of claims 1-19, wherein the engineered CCHFV glycoprotein ectodomain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of positions 250-692 of SEQ ID NO: 8.

21. The engineered protein of any one of claims 1-20, wherein the engineered CCHFV glycoprotein ectodomain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of positions 250-589 of SEQ ID NO: 9.

22. The engineered protein of any one of claims 1-21, wherein the engineered CCHFV glycoprotein ectodomain comprises the amino acid sequence of position 250-589 of SEQ ID NO: 9.

23. The engineered protein of any one of claims 6-22, wherein the engineered CCHFV glycoprotein ectodomain is fused or conjugated to a trimerization domain.

24. The engineered protein of claim 23, wherein the trimerization domain is positioned C- terminally relative to the engineered CCHFV glycoprotein ectodomain.

25. The engineered protein of any one of claims 1-24, wherein the engineered CCHFV glycoprotein ectodomain is fused or conjugated to a transmembrane domain.

26. An engineered CCHFV glycoprotein trimer comprising at least one subunit according to any one of claims 1-25.

27. A nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence of an engineered protein of any one of claims 1-25.

28. The nucleic acid of claim 27, wherein the nucleic acid comprises a DNA expression vector.

29. The nucleic acid of claim 27, wherein the nucleic acid comprises an mRNA.

30. An immunogenic composition comprising the engineered protein, engineered trimer, or nucleic acid molecule of any one of the prior claims, and a pharmaceutically acceptable carrier.

31. The composition of claim 30, further comprising an adjuvant.

32. A method of preventing a CCHFV infection or a disease associated with a CCHFV infection in a subject, comprising administering to the subject an effective amount of the immunogenic composition according to claim 30 or 31.

33. The immunogenic composition of claim 30 or 31 for use in inducing an immune response to a CCHFV glycoprotein ectodomain in a subject.

34. Use of the engineered protein, engineered trimer, or nucleic acid molecule of any one of the prior claims in the manufacture of an immunogenic composition for inducing an immune response to a CCHFV glycoprotein ectodomain.

35. A method of producing antibodies that bind to a CCHFV glycoprotein ectodomain, comprising administering to a subject an effective amount of the immunogenic composition according to claim 30 or 31.

36. A composition comprising the engineered protein of any of claims 1-25 or the engineered trimer of claim 25 bound to an antibody.

Images