Humanized monoclonal antibody and vaccines against measles virus

Abstract

A chimeric monoclonal antibody (mAb 77.1) neutralizes measles fusion protein and its cryo-EM structure in complex with MeV F in the prefusion conformation. Vaccine compositions using MeV F protein or its ectodomain are mutated to have stabilized pre-fusion conformation. Vaccine compositions may be varied, targeting other paramyxoviruses; they are mutated forms of paramyxovirus F protein with mutations at specific positions on the protein. Thermostable full-length MeV fusion proteins (FFLs) induce protective immunity and neutralizing antibodies in vivo. Vaccination with the FFL induces neutralization titer higher than with live vaccine, providing robust humoral response, likely protective also for individuals with impaired cellular immunity. The 8 specific mAbs, alone or combined with mAb 77, neutralize post fusion transition of MeV fusion protein, interrupting the F refolding process and preventing conformational changes required for membrane fusion and viral entry: mAbs G3, F3, E11, 9E1D3, H8, 10H2G3, D9, and Y10F.

Description

HUMANIZED MONOCLONAL ANTIBODY AND VACCINES AGAINST MEASLES VIRUSRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application 63 / 592,145, filed October 20, 2023, to The Trustees of Columbia University, titled “HUMANIZED MONOCLONAL ANTIBODY AGAINST MEASLES VIRUS,” U.S. provisional patent application 63 / 592,147, filed October 20, 2023, to The Trustees of Columbia University, titled “VACCINES AGAINST MEASLES VIRUS,” and U.S. provisional patent application 63 / 663,690, filed June 24, 2024, titled “DEVELOPMENT AND USE OF MONOCLONAL ANTIBODIES AGAINST F-PROTEIN OF MEASLES VIRUS,” the entirety of the disclosures of which are hereby incorporated by this reference.INCORPORA TION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 25711 byte XML file named “219WO-PCT_seq_listing.xml” created on October 21, 2024.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under AI176833, AI159085, NS 105699, and NS091263 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0004] The subject matter disclosed herein is generally directed to stabilized paramyxovirus F protein and antibodies against F protein.BACKGROUND

[0005] Measles virus (MeV) is a highly transmissible (Ro of 12-18) respiratory, enveloped, single-stranded negative-sense RNA virus. MeV belongs to the Paramyxovtridae family and causes significant morbidity and mortality among young children worldwide, despite the availability of safe and effective MeV vaccines. Measles accounts for more childhood deaths than any other vaccine-preventable disease. MeV was presumed to be eliminated in the U.S. in2000 (i.e. ≥12 months without transmission). However, reintroduction of the virus has resulted in a resurgence in recent years, causing repeated outbreaks. Outbreaks in the U.S. and other countries with high vaccination coverage are often attributed to unvaccinated individuals. However, a significant number of cases also occur in previously vaccinated people, since up to 5% of vaccine recipients do not develop adequate protective antibodies even after the recommended two-dose vaccination regimen, and because vaccine-elicited immunity likely wanes over time. In addition, the influence of anti-vaccination proponents in social media led to vaccine hesitancy among parents and pockets of unvaccinated children.

[0006] With no effective antiviral regimen currently available for MeV, insufficient vaccine coverage (which is defined as < 95% vaccination rate by WHO) can lead to a rise in MeV disease among vulnerable populations. Indeed, MeV has already undergone a global resurgence; estimated global MeV deaths rose from 89,780 in 2016 to 207,500 in 2019. The situation may worsen since the SARS-CoV-2 pandemic reduced routine childhood vaccination coverage and may expose more vulnerable individuals to infection. There have been over 122,000 cases in the first half of 2023 alone. Without global eradication, a significant increase in the number of MeV cases can be unavoidable. Even mild cases of measles infection can result in immune “amnesia”, and loss of preexisting antibodies against other infections, leaving measles convalescent individuals more vulnerable to future diseases.

[0007] Of particular concern during this resurgence in MeV cases are the rising number of individuals with compromised immune systems, such as cancer patients, transplant recipients, persons with autoimmune disorders on immunosuppressive therapies, and human immunodeficiency virus (HIV)-infected individuals, in addition to pregnant women. In such immunocompromised people, vaccination with current vaccines containing live-attenuated virus is often risky and inefficient; antibody responses are impaired, and even pre-existing immunity wanes. Measles resurgence in other parts of the world, combined with increased vaccine hesitancy, increases the risk of future measles outbreaks and the risk to vulnerable patient populations. Notably, despite the foreseeable rise in measles cases and measles deaths, there are still no effective antivirals approved for human use. Neutralizing monoclonal antibodies offer lifesaving treatments against a number of viruses, but surprisingly, there are none yet available for prevention or treatment of measles virus. Even more surprisingly, there are not yet structures of any antibodies against the measles virus by which Applicants might interpret epitopes or activities that prevent or treat infection.

[0008] The current measles virus (MeV) vaccine, developed in the early 1960s, is a live- attenuated virus created by repeatedly passaging MeV in cell culture. Although this vaccine is safe and effective for most healthy people, the MMR vaccine is contraindicated in immunocompromised individuals because the live vaccine itself can cause fatal illness in those with severely weakened immune systems1. These groups must instead rely on herd immunity for protection which is a risky proposition. Even about 5% of healthy people do not develop sufficient immune responses to the vaccine. A greater issue, which causes frequent surges in MeV cases in the United States, is insufficient vaccine coverage at home and abroad coupled with the extraordinary transmissibility of MeV (RO of 12-18 people per each infected person, with infection by the respiratory route). Even among healthy individuals, immune responses to measles vaccine can fail or wane2-6. Coupled with the extraordinary transmissibility of MeV (R° of 12-18 people per each infected person, with infection by the respiratory route), insufficient vaccine coverage presents a substantial hurdle to eradication and high likelihood that outbreaks will continue causing 10-20 million measles cases each year. In 2019, approximately 207,500 people died from MeV infection worldwide7,8, and yet vaccination rates dropped to 81%. From 20212to 2022 the death toll from measles increased by 43%9. In 2019, 1,282 measles cases were reported in the U.S. alone (the highest number since 1992), with 128 hospitalizations and 61 cases with complications (e.g., pneumonia and encephalitis), including a vertical transmission (mother to fetus) at Columbia University Irving Medical Center. The SARS-CoV-2 pandemic reduced routine childhood vaccination coverage and may expose more vulnerable individuals to infection10. Without global eradication, a significant increase in MeV cases may be unavoidable5,11-13. Apart from acute measles with all its sequelae, MeV infection causes immune amnesia, resulting in loss of specific immune responses against unrelated infectious agents and thus increasing susceptibility to other infectious diseases14.

[0009] The MeV envelope glycoproteins hemagglutinin (H, the receptor binding protein) and fusion (F) protein play critical roles in viral cell entry. F is synthesized as a precursor (Fo). After its synthesis, Fo is cleaved by furin within the virus-producing cell to yield the pre-fusion F complex, consisting of three C-terminal F1 subunits associated covalently via disulfide bonds between three N-terminal F2subunits. Even after this cleavage, F is kinetically trapped in a metastable conformation and is primed for fusion activation upon engagement of H with a cellular receptor (either CD 150 or nectin-4). After H engages the receptor, the pre-fusion F undergoes a conformational transition, starting with the refolding of N-terminal heptad repeat (HRN) exposing the fusion peptide (FP) into the target cells, and irreversibly folding into apost-fusion conformation by formation of 6-helix bundles between HRN and C-terminal heptad repeat (HRC) as it drives virus and host membrane fusion.

[0010] The H and F proteins (i.e., the fusion complex) are the main targets of neutralizing antibodies. The current vaccine elicits mostly antibodies against hemagglutinin (H): 80% of the human sera antibodies are specific for H, and only 20% for F, and the anti-F antibodies elicited by the current vaccine are poorly neutralizing15-17. The primary technical challenge for vaccine design has been obtaining the F protein in its pre-fiision state, representing an ideal target to elicit protective immunity. Several strategies have been proposed for stabilizing the pre-fusion state of the F proteins, which involve the introduction of several residues that may also alter the natural viral epitopes18-24.

[0011] A vaccine strategy based on recombinant subunit forms of the viral envelope fusion glycoprotein is needed in order to provide a safe vaccine for the growing number of people who cannot be vaccinated, including young children, pregnant women, and people with weakened immune systems.

[0012] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0013] In one aspect, the present invention provides for a thermostable full-length paramyxovirus fusion protein (FFL), wherein the FFLcomprises at least one mutation that stabilizes the pre-fusion conformation, wherein the at least one mutation comprises at least one substitution mutation at a position corresponding to residue 170, residue 442, residue 455, residue 459, or residue 506 of measles virus (MeV) F protein. In certain embodiments, the FFLfurther comprises at least one mutation that removes or inactivates the furin cleavage site corresponding to the furin cleavage site of MeV F protein. In certain embodiments, the FFLcomprises the following mutations corresponding to MeV F protein: E455G; or E170GZE455G; or E170GZE455G / G506E; or E170G / E455G / V459I / G506E; or E170G / Y442HZE455G / G506E; or E170G / Y442H / E455G / V459I / G506E. In certain embodiments, the paramyxovirus is measles virus (MeV).

[0014] In another aspect, the present invention provides for a paramyxovirus vaccine comprising the FFLof any embodiment herein.

[0015] In another aspect, the present invention provides for a method of inducing protective immunity and neutralizing antibodies, the method comprising administration of atherapeutically effective dose of the paramyxovirus vaccine of any embodiment herein to a subject in need thereof.

[0016] In another aspect, the present invention provides for a paramyxovirus vaccine composition comprising paramyxovirus F protein or its ectodomain, wherein the paramyxovirus F protein or its ectodomain comprises at least one mutation that stabilizes the pre-fusion conformation, wherein the at least one mutation comprises at least one substitution mutation at a position corresponding to residue 170, residue 442, residue 455, residue 459, or residue 506 of measles virus (MeV) F protein. In certain embodiments, the at least one mutation corresponds to E170G, Y442H, E455G, V459I, or G506E in MeV F protein. In certain embodiments, the paramyxovirus F protein or its ectodomain comprises the following mutations corresponding to MeV F protein: E455G; or E170GZE455G; orE170G / E455G / G506E; or E170G / E455G / V459I / G506E; orE170G / Y442H / E455G / G506E; or E170G / Y442H / E455G / V459I / G506E. In certain embodiments, the paramyxovirus F protein or its ectodomain further comprises at least one mutation that removes or inactivates the furin cleavage site corresponding to the furin cleavage site of MeV F protein. In certain embodiments, the vaccine composition is a measles virus (MeV) vaccine composition.

[0017] In another aspect, the present invention provides for a method of vaccinating against a paramyxovirus comprising administering to the subject the paramyxovirus vaccine composition corresponding to any embodiment herein, wherein the paramyxovirus vaccine composition comprises the F protein or its ectodomain of the paramyxovirus vaccinating against.

[0018] In another aspect, the present invention provides for a method producing neutralizing antibodies against a paramyxovirus, the method comprising: administering to a subject the paramyxovirus vaccine composition of any embodiment herein; confirming the subject produces neutralizing antibodies from the paramyxovirus vaccine composition; isolating B cells from the subject; culturing the B cells to form hybridomas; and isolating the hybridoma that produces neutralizing antibodies against the paramyxovirus. In certain embodiments, the method further comprises isolating neutralizing antibodies against the paramyxovirus from the isolated the hybridoma that produces neutralizing antibodies against the paramyxovirus. In certain embodiments, the paramyxovirus is measles virus (MeV).

[0019] In another aspect, the present invention provides for an antibody against measles virus F protein, comprising: one or more light chain variable region complementarity determining regions (CDRs) selected from the group consisting of: a first CDR comprising thesequence RASKSISKYLA (SEQ ID NO: 1), a second CDR comprising the sequence SGSTLQS (SEQ ID NO: 2), and a third CDR comprising the sequence CQQHNEYTLT (SEQ ID NO: 3); or one or more heavy chain variable region CDRs selected from the group consisting of: a first CDR comprising the sequence DYAWN (SEQ ID NO: 4), a second CDR comprising the sequence ISYTLTTGYNPSLKSRI (SEQ ID NO: 5), and a third CDR comprising the sequence RSGWLLPYWYFDV (SEQ ID NO: 6). In certain embodiments, the one or more light chain variable region CDRs are grafted onto a human antibody. In certain embodiments, the one or more heavy chain variable region CDRs are grafted onto a human antibody. In certain embodiments, the light chain variable region comprises a first CDR comprising the sequence RASKSISKYLA (SEQ ID NO: 1), a second CDR comprising the sequence SGSTLQS (SEQ ID NO: 2), and a third CDR comprising the sequence CQQHNEYTLT (SEQ ID NO: 3). In certain embodiments, the heavy chain variable region comprises a first CDR comprising the sequence DYAWN (SEQ ID NO: 4), a second CDR comprising the sequence ISYTLTTGYNPSLKSRI (SEQ ID NO: 5), and a third CDR comprising the sequence RSGWLLPYWYFDV (SEQ ID NO: 6). In certain embodiments, the light chain variable region comprises a first CDR comprising the sequence RASKSISKYLA (SEQ ID NO: 1), a second CDR comprising the sequence SGSTLQS (SEQ ID NO: 2), and a third CDR comprising the sequence CQQHNEYTLT (SEQ ID NO: 3); and the heavy chain variable region comprises a first CDR comprising the sequence DYAWN (SEQ ID NO: 4), a second CDR comprising the sequence ISYTLTTGYNPSLKSRI (SEQ ID NO: 5), and a third CDR comprising the sequence RSGWLLPYWYFDV (SEQ ID NO: 6). In certain embodiments, the sequence of the light chain variable region is:DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNELLIYSGSTLQSGIPS RFRGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYTLTFGGGTKLELKR (SEQ ID NO: 7). In certain embodiments, the sequence of the heavy chain variable region is: DVQLQESGPGLVKPSQSLSLTCTVSGYSITSDYAWNWIRQFPGNKLEWMGYISYTLT TGYNPSLKSRISITRDSSKNQFFLQLNSVTTEDTATYYCARSGWLLPYWYFDVWGAG TTVTVSS (SEQ ID NO: 8). In certain embodiments, the antibody is encoded by GS66077-1 pTRIOZ-hIgGl_HRV3C_77.1.

[0020] In another aspect, the present invention provides for an antibody against measles virus F protein, comprising: one or more light chain variable region complementarity determining regions (CDRs) selected from the group consisting of: a first CDR comprising the sequence RATSSVVYMH (SEQ ID NO: 9), a second CDR comprising the sequenceATSNLAS (SEQ ID NO: 10), and a third CDR comprising the sequence QQWNSNPYT (SEQ ID NO: 11); or one or more heavy chain variable region CDRs selected from the group consisting of: a first CDR comprising the sequence NYWIE (SEQ ID NO: 12), a second CDR comprising the sequence EILPGNSYTNYNEKFRGKA (SEQ ID NO: 13), and a third CDR comprising the sequence PLSGNY (SEQ ID NO: 14). In certain embodiments, the one or more light chain variable region CDRs are grafted onto a human antibody. In certain embodiments, the one or more heavy chain variable region CDRs are grafted onto a human antibody. In certain embodiments, the light chain variable region comprises a first CDR comprising the sequence RATSSVVYMH, a second CDR comprising the sequence ATSNLAS, and a third CDR comprising the sequence QQWNSNPYT. In certain embodiments, the heavy chain variable region comprises a first CDR comprising the sequence NYWIE, a second CDR comprising the sequence EILPGNSYTNYNEKFRGKA, and a third CDR comprising the sequence PLSGNY. In certain embodiments, the light chain variable region comprises a first CDR comprising the sequence RATSSVVYMH, a second CDR comprising the sequence ATSNLAS, and a third CDR comprising the sequence QQWNSNPYT; and the heavy chain variable region comprises a first CDR comprising the sequence NYWIE, a second CDR comprising the sequence EILPGNSYTNYNEKFRGKA, and a third CDR comprising the sequence PLSGNY. In certain embodiments, the sequence of the light chain sequence is:certain embodiments, the sequence of the heavy chain sequence is:

[0021] In another aspect, the present invention provides for a humanized antibody comprising one or more CDRs from a single antibody selected from the group consisting of: C6, 9E1D3, H8, D9, and 10H2G3.

[0022] In another aspect, the present invention provides for a method of neutralizing postfusion transition of measles virus (MeV) fusion protein in a subject, the method comprising administration of an effective amount of a first monoclonal antibody (mAb) selected from the group consisting of: G3, C6, F3, E11, 9E1D3, G4, H8, 10H2G3, D9, and Y10F, wherein the antibody is administered alone or in combination with mAb 77 to a subject in need thereof.

[0023] In certain embodiments, the administration allows initial activation and refolding of the MeV F ectodomain and interrupts the refolding process of the F to prevent conformational changes required for membrane fusion and viral entry. In certain embodiments, the first mAb, alone or in combination with mAb 77, forms a complex with F.

[0024] In another aspect, the present invention provides for a method of inhibiting measles virus spread through cells, the method comprising administering to the cells the humanized monoclonal antibody of any embodiment herein.

[0025] In another aspect, the present invention provides for a method of treating measles virus infection in a subject, the method comprising administering to the subject the humanized monoclonal antibody of any embodiment herein.

[0026] In certain embodiments, the humanized monoclonal antibody administered within 24 hours of exposure to measles virus.

[0027] In another aspect, the present invention provides for a method of prophylactically treating measles, the method comprising administering to the subject the humanized monoclonal antibody of any embodiment herein. In certain embodiments, the humanized monoclonal antibody is administered no more than 12 hours before anticipated exposure to measles virus.

[0028] In certain embodiments, the humanized monoclonal antibody is administered onto a mucosal surface. In certain embodiments, the humanized monoclonal antibody is administered intranasally.

[0029] The foregoing and other aspects, features, and advantages will be apparent from theDESCRIPTION and DRAWINGS, and from the CLAIMS if any are included.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0031] An understanding of the features and advantages of the present invention will hereinafter be described in conjunction with the appended and / or included DRAWINGS, where like designations denote like elements, and:

[0032] FIG. 1 - mAb 77.1 displays robust antiviral activity in vitro and in vivo. A) Antiviral potency of mAb 77.1 (violet) in a viral entry assay to measure inhibition of infection in Vero cells expressing SLAM / CD150. The activity was compared to the established MeV inhibitors, 3G (green) and HRC4 (blue). Values for IC50and Hill slope were estimated using logistic regression, and values were: 3G: IC50=15O.l±16.2 nM, Hills slope=0.37±0.01, HRC4: IC50=1.17±0.27 nM, Hills slope=0.62±0.07, mAb 77.1: IC50=0.034±0.005 nM, Hills slope=0.52±0.04. B) In vivo passive immunization experiment to assess in vivo efficacy of mAb 77.1. Cotton rats received intraperitoneal (IP) injection of 0.1 mg / kg or 1 mg / kg mAb 77.1 12 hours before challenge with MeV. Animals in the control group received PBS before challenge. Values correspond to MeV titers in lung tissue collected 4 days post-infection. Significance showed using the Mann-Whitney-Wilcoxon test. P-value annotation legend: ns (non-significant): p <= 1.00, *: 0.01 < p <= 0.05, **: 0.001 < p <= 0.01, ***: 0.0001 < p <= 0.001.

[0033] FIG. 2 - mAb 77.1 mirrors the action of the HRC4 inhibitor and stabilizes the extended state of F protein. A) Schematic representation of the red blood cell (RBC) assay conducted in HEK293T cells expressing a chimeric HN / H receptor with MeV F protein. Without fusion inhibitors, the HN / H receptor binds RBCs. The MeV H stalk region subsequently activates the F protein, facilitating its conformational refolding to a fusion intermediate that anchors fusion peptide in the RBC membrane, culminating in the fusion of RBC and H293T membranes. B) In the presence of zanamivir, a small-molecule inhibitor that competes with sialic acids present at RBCs on HN, reversible binding of RBCs to HN occurs. The addition of 3G locks the F protein in its pre-fusion conformation, resulting in only reversible binding of most RBCs to the HN / H. C) The subsequent fusion checkpoint is evaluated through the combined administration of zanamivir and ACK buffer. Zanamivir acts as described in panel B, whereas ACK buffer induces lysis of RBCs. At this checkpoint, fusion intermediates with anchored fusion peptides within RBC membranes resist zanamivir-induceddissociation but are lysed in the presence of ACK buffer. D) At the final fusion checkpoint, complete fusion of RBC and HEK293T membranes is achieved. Here, ACK buffer addition has no impact on the fused RBCs. E) A bar chart summarizing the results of a mechanistic assay comparing various known inhibitors. For each condition, cells were categorized based on their interaction with RBCs: reversibly bound (dark blue) where the RBCs were only adhered by chimeric H and could be easily removed by treatment with zanamivir; irreversibly bound (pink) where RBCs were initially attached by chimeric H, but subsequent F protein refolding anchored the fusion peptide in the RBC membrane, making the RBCs attachment zanamivir-insensitive and retaining RBC on the HEK293T cell surface; and fused (light blue) where RBCs underwent membrane fusion with HEK293T cells expressing the fusion complex.

[0034] FIG. 3 - FECTOstructure shows that the 77.1 Fab fragment engages FECTOat the base of the head domain, as elucidated by cryo-EM. A) Primary structure annotation of FECTO. Functional domains are delineated as color-coded rectangular regions, with the following key: SP for Signal Peptide, FP for Fusion Peptide, HRN for N-terminal Heptad Repeat, DIII for Domain III, DI for Domain I, DII for Domain n, and HRC for C-terminal Heptad Repeat. The presence of Twin Strep-Tag II is indicated by '2xST'. White hexagonal symbols denote glycosylation sites within the F2 region. A black loop represents natural disulfide bonds. The furin cleavage site, which divides the precursor F0 into F2 and F1 subunits, is marked with black scissors icons. The locations of two stabilizing mutations are indicated by arrows. B) A ribbon representation of the 2.3 Å structure of FECTOin the presence of FIP. C) Domain description of FECTO, colored from N-terminus to C-terminus (red-purple), as in A). D) A ribbon representation of FECTOin complex with three 77.1 Fab fragments. The Fabs interact symmetrically with the lower section of the head domain in a cleft between domains I and II. The FECTOF1 chain is represented in green, the F2 chain in gray, the 77.1 Fab heavy variable domain (VH) in violet, and the light variable domain (VL) in pale pink. The cryo-EM map delineated as a silhouette, also encompasses the masked-out region, including the constant light and heavy segments (CL and CH) of the Fab 77.1. E) CDR loops Hl and H2 establish hydrogen bonds with three distinct FECTOpolypeptide chains: F2B (light gray), F1B(dark green), and F1A(light green). The interacting residues on FECTOand Fab 77.1 are shown in the respective colors of their chains. F) The majority of the Fab 77.1 interactions are executed by the VH part of the antibody, with all three heavy chain complementarity-determining regions (CDRs H1, H2, and H3) engaging with the head domain, and only a single CDR L2 from the VL domain contributing to the interaction. Black rectangles show regions enlarged inE) and G). G) The CDR loops H-3 and L-2 only interact with a single FECTOchain, F1. Like in panel E, the interacting residues are highlighted in the color of the respective chains.

[0035] FIG. 4 - The post-fusion conformation of FECTOexhibits rearrangement of key hinge regions and domain repositioning in projection of heptad repeats and fusion machinery. A) The cryo-EM structure of FECTOin its post-fusion conformation reveals a transformation of the HRN and HRC, forming a canonical post-fusion 6-helix bundle. The F1 chains are illustrated in shades of blue, and the F2 chains are in shades of pink. The cryo-EM map is displayed as a silhouette, with two notable glycosylation sites indicated and visualized only at the map level. B) Domain-based coloring of FECTO in post-fusion conformation as in Fig 2B. C) A global alignment comparison between FECTOin pre-fusion (depicted in white with silhouette) and post-fusion (in domain color scheme) conformations is shown. Optimal global alignment per chain was attained by aligning FECTOon the domain I (highlighted in light green). Regions that undergo major conformational changes are surrounded in boxes and shown in panels D and E. D) Domain III undergoes the most dramatic shift, moving its center of mass over 11 Å and rotating by 52°. Most of the fold remains unchanged when local alignment is performed on the sequence region corresponding to the domain region, with the most notable changes seen in the HRN, which releases the fusion peptide and refolds it into an extended helix. The furin-containing loop is depicted as a dashed line, and yellow scissors symbolize the furin cleavage site. E) Domain II undergoes a twisting motion during the refolding step. However, only slight differences are observed in the fold after local alignment for the residue range corresponding to the domain. The most significant change is noted in the C-terminal part of the domain, comprised of the HRC, which alters direction almost 180° around residue G433. HRC in color indicates the post-fusion structure, while HRC in black denotes the pre-fiision structure. D) Mapping of residues comprising the mAb 77.1 epitope on both the pre- and postfusion states of FECTOreveals the displacement of the epitope in the post-fusion state, thereby elucidating the absence of 77.1 binding to FECTOin its post-fusion conformation.

[0036] FIG. 5 - Structural analysis of MeV F sequence conservation and viral evolution assay reveals consistent conservation across the protein and a lack of viral variants that are resistant to mAb 77.1. A) A conservation map of the MeV F full-length (FL) primary sequence was derived from over 830 F sequences sourced from the NCBI protein database. The sequences were aligned using Clustal Omega multiple sequence alignment (MSA), and the AL2CO algorithm was employed to calculate the conservation score. MeV F demonstrates uniform sequence conservation, with only two regions indicating lowerconservation: the signal peptide and the cytoplasmic tail depicted with black lines on top. B) When mapping the AL2C0 score onto the tertiary structure of FECTOin both its pre- and postfusion states, no conservation hotspots are evident. In general, residues less exposed show higher conservation compared to solvent-exposed residues. C) Although most residues forming the 77.1 epitope demonstrate high sequence conservation (light gray), two residues, G37 and R437 (shown in shades of yellow), have been identified with natural variants. D) Results from the cell-based fusion inhibition assay elucidate the impact of G37R and R436K mutations on the binding affinity of 77.1. The R436K mutation, although frequent, exhibited minimal influence on antibody-mediated fusion inhibition. Conversely, the G37R mutation demonstrated a capability to evade inhibition by 77.1. E) Viral evolution assay results from control (mock), as well as passages in the presence of HRC4 and passages 4 and 5 in the presence of mAb 77.1, were evaluated for mutations in the F gene. Only mutations with an allele frequency greater than 5% and a minimum read depth of 20 were included in the analysis. Notably, passage 5 under mAb 77.1 conditions did not yield any mutations that met these filtering criteria.

[0037] FIG. 6 - Elucidation of mAb 77.1 mechanism of action through NS EM and cryo-EM analyses. A) 2D class averages obtained from NS EM of the FECTO-Fab 77.1 complex subjected to a heating experiment at 50°C for 5 minutes. Upon thermal activation, FECTO initiates its refolding process. The presence of Fab 77.1 restricts FECTO from following the canonical folding pathway. A stepwise separation sequence is below the NS EM 2D class averages, detailing the retention and subsequent FECTO separation. B) 2D cryo-EM class averages of Fab 77.1 stabilizing a truncated fragment of FECTO. The accompanying schematic is presented on the right for clarification. C) Approximately 7 Å resolution density map of the Fab 77.1-FECTO fragment superimposed on the trimeric FECTO-Fab 77.1 complex. A zoomed-in view of the corresponding density associated with only a stabilized portion of FECTO is shown on the right. The interaction between Fab 77.1 and FECTO maintains the previously characterized epitope in a stabilized form, inhibiting further refolding. Domain coloring follows the scheme presented in Figure 3. Post-triggered antibody recognition in the presence or absence of mAb 77.1. In the presence of mAb 77.1, more post-trigger epitope of F is present on cells expressing F after incubation at 55°C. D) Canonical model outlining the typical refolding pathway of F protein. E) Proposed mechanism of action for mAb 77.1, supported by NS EM 2D class averages, depicting the observed populations of FECTO-Fab 77.1 complexes throughout the experimental conditions.

[0038] FIG. 7 - Detailed plot from Fig 2 showing red blood cell assay after 48h of transfection incubated at 37°C for 60 min, including controls for inhibitors and mAb 77.1 concentrations.

[0039] FIG. 8 - Purification assessment, NS EM of FECTO, binding affinity of FFab 77.1 to FECTO, complex formation, and assessment of FECTOstability using DSF, focusing on the effects of pH and salt concentration. A) The purity of FECTOand Fab 77.1 is verified using SDS-PAGE. FECTOexhibits a single band corresponding to approximately 54 kDa (F1+F2) in non-reducing conditions (non-red) and about 37 kDa in reducing conditions (red). Due to its high glycosylation, F2 travels as a smear at around 20 kDa. B) Negative stain electron microscopy (NS EM) 2D classes of pre-fusion confirmation of FECTOafter purification. C) BioLayer interferometry binding curves of FECTOand Fab 77.1 reveal high association (3.48-105M-1s-1) and slow dissociation kinetics (1.25·10-4s-1) of Fab 77.1, with a calculated KDof 3.61 -10-10. D) The size exclusion profile of the FECTOand Fab 77.1 complex. The purple line signifies the 1 : 1 mixture of FECTO:Fab 77.1, with a fully formed complex peak at approximately 11.7 ml and a Fab 77.1 peak at 16.5 ml. The green line represents the FECTOelution profile. E) Screening of buffer pH to analyze its influence on FECTO'S apparent melting temperature (Tma). Five different pH values were examined, with the resulting FECTOTma values measured and depicted in the corresponding graphs. The trend across the pH levels is shown in the last chart. F) Analysis of FECTOstability across a range of salt concentrations. The study explored six salt concentrations (60 - 1460 mM) and their impact on FECTOstability expressed as Tma. The trend demonstrating the relationship between salt concentration and FECTOstability is illustrated in the graph below.

[0040] FIG. 9 - Representative micrographs for each dataset with particle picks from the final particle stack (left) and selected 2D classes (right). A) FECTO -Fab 77.1, B) FECTO in post-fusion conformation, C) FECTO-[FIP-HRC]2-PEG11.

[0041] FIG. 10 - Final map quality, local resolution, FSC, and particle angular distribution for each recorded dataset. A) FECTO-Fab 77.1, B) FECTO in post-fusion conformation, C) FECTO-[FIP-HRC]2-PEG11.

[0042] FIG. 11 - Cryo-EM data processing for each recorded dataset as described in the methods section. A) FECTO-Fab 77.1, B) FECTOin post-fusion conformation, C) FECTO-[FIP- HRC]2-PEG11.

[0043] FIG. 12 - Structure analysis and comparison of FECTO. A) Application of local scaling to the FECTO-[FIP-HRC]2-PEG11map using OccuPy. On the left, the relative occupancy of the unsharpened map is shown, and on the right, an equalized map that enhances regions with low occupancy or resolution, illustrating glycans (N29, N61, and N67), the furin- containing loop, and the density from [FIP-HRC]2-PEG11. B) The equalized map is presented at both low (on the left) and high (on the right) contour levels. The locations of FIP and HRC are indicated with arrows. The density corresponding to FIP was clearly visible in the unequalized map, whereas the density associated with HRC only became noticeable at the high contour level in the equalized map. C)Differences between disulfide-stabilized FECTO(X-ray), and native FECTO (cryo-EM). The graph shows the Cα root mean square deviation (RMSD) between the X-ray structures (FECTOAPO: 5YXW, FECTO FIP: 5YZD, FECTO AS-48: 5YZC), and the cryo-EM FECTO-[FIP-HRC]2-PEG11. Each model was globally aligned based on a single monomer (F1+F2), after which the RMSD was calculated between residues present in both structures. Regions with the highest RMSD include the loop 248-265 and the stalk region highlighted in D and E. D) The ribbon diagram presents the global alignment of the X- ray and cryo-EM models, as in C. The areas exhibiting high RMSD are indicated with arrows. E) Close-up view of the areas marked in D), with the starting and ending residues indicated. The color coding matches that in D).

[0044] FIG. 13 - Quantitative analysis of expression, cell-surface staining and fusion inhibition of F protein variants by 77.1 antibody. A) Western blot analysis using anti-HRC antibodies to detect F protein variants after 24h and 48h in cell-surface biotinylation assays conducted at 32°C and 37°C. B) Bar graph representing the relative integrated band intensities derived from C). The data are normalized to the WT F protein band intensity at 24h 37°C. C) Cell surface staining of F protein variants after 48h at 32°C in the presence of 77.1 antibody, adjusted for average expression levels as determined from panel D. Dotted lines connect discrete data points for clarity. D) Fusion inhibition assay conducted at 32°C comparing 77.1 antibody with various F protein variants. Data are normalized to the highest signal per variant. Solid lines represent nonlinear regression logistic curves fit to the data, whereas dotted lines merely serve to connect discrete data points for ease of interpretation. Fit values were obtained from global fits to all data points with shared IC50and Hill slope and were: WT: IC50=1.2±0.3 nM, slope=0.73±0.11, R436K: IC50=0.58±0.27 nM, slope=0.48±0.08, R436G: IC50=65.7±41.7 nM, slope=0.12±0.02. E) Same as in panel D but presenting unnormalized data to elucidate the relative magnitude of fusion activity among different F protein variants. F) Computationalanalysis of the impact of point mutations on the electrostatic surface potential maps of F2MECT0. The color scale represents electrostatic potential in volts, with red indicating -10, white indicating 0, and blue indicating +10. Arrows pinpoint the locations of mutations under investigation.

[0045] FIG. 14 - Allele frequency distribution of nonsynonymous and synonymous mutations in viral evolution assay. MeV was cultured in the presence of HRC4 (dimeric lipopeptide), mAb 77, or without any inhibitors for 4 to 5 passages. Nucleotide polymorphisms and corresponding amino acid changes relative to the wild-type strain are depicted. A) Comprehensive mutational profile for all samples. B) Refined mutational profile, filtered for allele frequencies ≥ 5% and sequencing depth ≥ 20. Points are color-coded to match the sequence depth, as indicated by the color bar at the bottom.

[0046] FIG. 15 - Comparative analysis of the shape and dimensions between FECTOin its pre- and post-fusion conformations. A) The final models derived for both FECTOconformations are presented, with their respective dimensions indicated. B) Visual representation of the domains, elucidating the changes in domain conformations and overall shape transitioning from the pre- to post-fusion state. The domains are color-coded consistently across both models for ease of comparison.

[0047] FIG. 16 - Structural and biophysical investigation of the effects of 77.1 on FECTO. A) Per residue RMSD analysis between FEcro-Fab 77.1 and [FECTO-FIP-HRC]2-PEG11structure. Two RMSD statistics per residue were calculated: based on all atoms in the residue (blue) and only between Ca (orange). Black dots positioned on top of the lines indicate the locations of the residues involved in the 77.1 epitope. The dashed lines indicate the mean RMSD across the entire residue range. B) The influence of Fab 77.1 binding is demonstrated through structural mapping of the Cα RMSD between two pre-fusion FECTOstructures in complex with either Fab 77.1 or [FIP-HRC]2-PEG11. The intensifying red gradient indicates an increase in Cα RMSD, with arrows pinpointing the antibody-binding epitope. C) Differential scanning fluorimetry (DSF) experiments were conducted with FECTOand complexes of FECTOwith [FIP-HRC]2-PEG11and Fab 77.1. The thermal stability of FECTO, stabilized by two point mutations, was with a Tmappof 47.6°C. The addition of [FIP-HRC]2-PEG11enhanced the thermal stability to 56.7°C, whereas the Fab 77.1 only slightly stabilized FECTO, raising the temperature to 51.6°C.

[0048] FIG. 17 - Analysis of Time-Resolved Heating Experiment with FECTO-Fab 77.1.A, B) 5-minute, 50°C heat-treated controls for Fab 77.1 and FECTO2M. C) Unheated control ofthe FECTO-Fab 77.1 complex. D, E, F, G) Time-resolved thermal analysis of FECTO-Fab 77.1. The most representative or the sole class obtained during the 2D classification round are shown. All datasets were processed using identical parameters. Obtained states are color-coded: violet: Fab, green: FECTO / FECTO-Fab 77.1 in pre-fusion conformation, slate blue: FECTOin post-fusion conformation, orange: relaxed / open state of FECTO-Fab 77.1, red: Fab-stabilized FECTOfragment.

[0049] FIG. 18 - Comparison of Paramyxovirus-specific, pre-fusion F targeted antibodies. Three main epitopes can be distinguished among all 12 characterized antibodies, including mAb 77.1. A) Apex binding antibodies (Fab PIA174: 6MJZ, Fab 12B2: 7KI4, Fab66: 6T3F, Fab 4H3: 7UOP (unpublished), Fab 3x1 : 8DG8, Fab 2D3: 7UP9 (unpublished) recognize top part of domain III and F2. B) Face binding antibodies (Fab 1 A9: 7UPK (unpublished), Fab 5B3: 6TYS, Fab 1F5: 7KI6, Fab 1H8: 7UPA (unpublished)) mainly focus on domain I and lower part of domain III C) Base binding antibodies (Fab 2B12: 7UPD (unpublished), Fab 1H1: 7UPB (unpublished), Fab 77.1: this study) bind primarily domain n, bottom of domain I and the linker region between domain II and HRC. Interestingly, Fab 1H1 also recognizes post-fusion conformation of Nipah F. D) Angle of approach of antibodies included in A-C. Axis was determined based on the long dimension of a whole Fab structure fitting into available Fab fragment. Axes are colored based on the antibody colors from A-C.

[0050] FIG. 19 - Grafting 77.1 mAb. The 77.1 mAb, with variable domains (VL and VH) derived from 77.4 (mouse) was cloned in pTRIOZ-hlgG1 vector.

[0051] FIG. 20 - Inhibition of viral spread. Effect of humanized monoclonal antibody (77.1 mAb) on the areas of plaques formed by the indicated recombinant MeV viruses with eGFP in infected Vero SLAM (A) or Vero cells (B). The mAb were added after the 120-min adsorption period. Representative images of Vero SLAM cells after 72 h post-infection were taken using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek) (bars, 200 μm). Areas of infection were measured using BioTek Gen5 software on images randomly acquired from one experiment in duplicate. Statistical analysis was performed using the Unpaired t test with Welch’s correction (**, p< 0.05; *** p< 0.005; ****, p < 0.0001).

[0052] FIG. 21 - 77.1 mAb inhibits MeV infection in vivo. Cotton rats were infected intranasally with 105TCID50 / ml MeV B3-eGFP wild type. Animals received either the indicated amount of the mAb 77.1 or vehicle subcutaneously 12 hours prior infection. Animals were euthanized 5 days after infection. The results are presented as TCID50 / g of lung tissue.Statistical analysis was performed using the one-way ANOVA with Dunnett’s multiple comparison test (** p = 0.001, **** p< 0.0001).

[0053] FIG. 22 - Vaccination with full-length stabilized measles F is protective in vivo. A) 2D class averages derived from cryo-EM of the full-length E170G / E455G F construct embedded in either a lipid nanodisc (upper panel) or amphipol (lower panel). The high degree of flexibility in the membrane-proximal region impedes the reconstruction of the transmembrane segment. B) Cryo-EM maps of the F protein constructs, resolved at resolutions of 2.5 Å (within nanodisc) and 2.7 Å (within amphipol), demonstrating the high quality of obtained protein constructs. The lower contour map, depicted as a silhouette, shows the unresolved transmembrane (TM) region. Q Immunization with the stabilized, full-length F in amphipol yields complete lung protection (red). Compared to this, immunization with purified MeV antigen (gradient purified, UV-inactivated MeV (Pueschel, K.; Tietz, A.; Carsillo, M.; Steward, M.; Niewiesk, S., Measles virus-specific CD4 T-cell activity does not correlate with protection against lung infection or viral clearance. Journal of virology 2007, 81 (16), 8571- 8.1951373; and Reich, A.; Erlwein, O.; Niewiesk, S.; ter Meulen, V.; Liebert, U. G, CD4+ T cells control measles virus infection of the central nervous system. Immunology 1992, 76 (2), 185-91)) led to lower, -tenfold reduction in viral load (blue). Statistical significance was determined using the Mann- Whitney-Wilcoxon test. P-value annotations: ns denotes p <= 1, * denotes 0.01 < p <= 0.05, ** denotes 0.001 < p <= 0.01, and *** denotes 0.0001 < p <= 0.001.

[0054] FIG. 23 - Ectodomain of MeV F bearing E170G and E455G is highly thermostable. A) Molecular representation of the 2.6 Å-resolution structure of the F ectodomain (residues 1-495) in complex with mAb77.1 Fab fragments overlayed on the lower contour cryo-EM map. The antibody interacts with domain II of F, stabilizing the lower section of the head domain through its simultaneous interaction with two F1 and one F2 chain. B) In vivo efficacy of mAb77.1 demonstrated through cotton rat passive immunization experiments. mAb77.1, administered prior to MeV infection at doses of 0.1 and 1 mg / kg, resulted in lower viral titer at both concentrations compared to the virus-only control group. Significance showed using the Mann-Whitney- Wilcoxon test. P-value annotation legend: ns: p <= 1.00e+00, *: 1.00e-02 < p <= 5.00e-02, **: 1.00e-03 < p <= 1.00e-02, ***: 1.00e-04 < p <= 1.00e-03. C) Differential scanning fluorimetry shows the high thermostability of the F ectodomain and the influence exerted by the mAb77.1 Fab fragments and [FIP-HRC-PEG4]2-chol fusion inhibitors (Bovier, F. T.; Rybkina, K.; Biswas, S.; Harder, O.; Marcink, T. C.; Niewiesk, S.; Moscona,A.; Alabi, C. A.; Porotto, M., Inhibition of Measles Viral Fusion Is Enhanced by Targeting Multiple Domains of the Fusion Protein. ACS Nano 2021).

[0055] FIG. 24 - Vaccine-enhanced disease / lung histology. For induction of vaccine- enhanced disease, cotton rats (5 animals / group) were immunized with formalin-inactivated (FA) 105pfu MeV twice s.c. in a three-week interval and challenged with wildtype virus after 52 days. Lung pathology was scored on a scale from 0 (nothing) to 100 (severe).

[0056] FIG. 25 - Structural Characterization of F Protein Constructs. A) Primary structure annotation of FFL: Functional domains are delineated by color-coded rectangular regions with the following designations: SP (Signal Peptide), FP (Fusion Peptide), HRN (N- terminal Heptad Repeat), DIll (Domain III), DI (Domain I), DII (Domain II), HRC (C-terminal Heptad Repeat), TM (Transmembrane Region), and CT (Cytoplasmic Tail). White hexagons indicate glycosylation sites within the F2 region. Natural disulfide bonds and the furin cleavage site (which divides the precursor F0 into F2 and F1 subunits) are represented by a black loop and black scissors, respectively. B) SDS-PAGE analysis of purified FFLconstructs: Constructs of wild-type (wt), 2M, and 3M. The introduction of stabilizing mutations is shown to enhance protein yield. C) Structural representation of FFL: ribbon diagrams depict FFLin pre-fusion (left) and post-fusion (right) conformations, highlighting point mutations E170G and E455G (2M), 2M+Afur (3M), and 3M+Y442H+V459I+G506E (6M). The cell membrane is illustrated as a gray rectangle, with unmodelled portions of F depicted schematically. The color scheme corresponds to that in A). D) Negative stain electron microscopy 2D classes depict the enhanced stability of the pre-fusion conformation across all FFLconstructs. While F wt was purified only in the post-fusion conformation, the 6M construct lacked detectable post-fusion conformation. Particles come from -100 micrographs for each construct. E) Cryo-EM 2D classes of FFL2M visualized in nanodisc (upper) and amphipol (lower) environments. The transmembrane region is noted for lacking detailed structural information, possibly due to high flexibility or alignment procedure focusing on the head region. F) Cryo-EM reconstruction of FFLconstructs of FFL2M in nanodisc and amphipol and FFL3M in amphipol. High resolution is observed in the head region, but the transmembrane domain is consistently absent in the reconstructions. A low-resolution silhouette in black shows the Gaussian blurred map at 1.5 standard deviations, highlighting the membrane region. G) Model of FFL2M head region that contains F1 and F2 fragments (in green and gray, respectively), with three glycosylation sites per protomer emphasized in magenta. Each unique glycosylation site is annotated alongside apie chart representing the glycosylation heterogeneity at that site, as determined by mass spectrometry.

[0057] FIG. 26 - Immunization of cotton rats with stabilized F protein variants demonstrates protection against viral challenge and elevated antibody responses to prefusion epitopes. A) Cotton rat (CR) immunization with stabilized F proteins. Animals were immunized either with 2 doses of 5 μg of the indicated protein in alum at four-week intervals or with a single dose of 104TCID50of the Edmonston-Zagreb MeV vaccine. Viral titers are reported, with a detection limit of 2 logio / g. The line within each box plot indicates the median viral titer. Significance was assessed by Mann-Whitney-Wilcoxon test, with p-value categorized as non-significant (ns): p < 1.00, *: 0.01 < p < 0.05, **: 0.001 < p < 0.01, ***: 0.0001 < p < 0.001. B) Neutralizing antibody titers were obtained from CR sera four weeks after the last immunization. FFL3M and FFL6M induced complete protection against viral challenge and induced higher neutralizing antibody titers than immunization with live MeV vaccine. Individual animal titers are represented by points, with the mean depicted by bars and the 95% confidence interval indicated by error bars. C) Conformation-specific sera binding assay. Conformation-specific antibody binding to the F protein (using cells expressing wt F) was assessed using sera from immunized rats. The presence of antibodies targeting both pre- and post-triggered epitopes of the F protein is noted. Post-triggered epitopes were induced by incubation of cells expressing F at 55 °C for 30 minutes. Proteins with FFL2M which are more likely to shift to the post-fusion conformation, show a higher recognition of this conformation than those with FFL3M and 6M. The graphical representation uses bars for means and error bars for the 95% confidence interval, consistent with B). D) Neutralization activity directly correlates with F protein pre-fusion recognition. Data from B and C, excluding PBS and MeV controls, are represented in a correlation plot. Immunizations are shown in columns (1 left, 2 right) and epitope specificity as rows (pre-fusion top, post-fusion bottom). A linear regression is shown as a solid (R2>0.5) and dashed (R2< 0.5) lines and a 95% confidence interval is shown in gray.

[0058] FIG. 27 - Time-resolved stability of F variants on the cell surface exposed to 55°C and 65°C. A) Comparison between the wild-type (wt) strain F and the vaccine strain of MeV F. Lines represent exponential decay function. B) Stability increase resulting from the addition of point mutations identified in central nervous system (CNS) MeV strains. Solid lines indicate the exponential decay function fit, while dashed lines represent data connectivity. C) Accumulation of stabilizing point mutations enhances the stability of pre-trigger F on the cellsurface exposed to 65°C. All data were fitted using a single exponential decay function. Data were obtained from a single biological replicate consisting of three technical replicates. D) Stability of variants shown in panel C, presented as unfolding half-lives derived from the exponential decay fit. Error bars indicate the standard error of the half-lives.

[0059] FIG. 28 - Reducing SDS-PAGE gel analysis of purified MeV F constructs. The reducing SDS-PAGE analysis of purified MeV F constructs demonstrates distinct band patterns for different constructs. The FECTO2M construct shows as two bands corresponding to F1 and F2. The FFL2M construct, similar to FECTO2M (Science in press), shows a prominent F1 band at approximately 45 kDa, with the F2 band not well visible. In contrast, the FFL3M and FFL6M constructs display only the F0 band, indicating the absence of proteolytic cleavage.

[0060] FIG. 29 - Biophysical Characterization of Investigated F Constructs via Nanotemper Prometheus Panta. Top Panel: The first derivative of the nano-differential scanning fluorimetry (nano-DSF) signal at 330 nm indicates the apparent melting temperatures of FECTO2M, FFL2M, FFL3M, and FFL6M. The incorporation of stabilizing mutations results in increased melting temperatures of the purified F constructs. Bottom Panel: As determined from dynamic light scattering experiments, the cumulant radius reveals that the full-length (FL) constructs exhibit significantly less aggregation than the ectodomain. Additionally, constructs with abolished Furin cleavage sites show markedly lower aggregation following unfolding.

[0061] FIG.30 - Cryo-EM data processing flowchart. Selected classes are shown in green. A) FFL2M in nanodisc. B) FFL2M + [FIP-HRC]2-PEG11in nanodisc. C) FFL2M + [FIP-HRC]2- PEG11in amphipol. D) FFL3M in amphipol. E) FFL6M in amphipol.

[0062] FIG. 31 - Cryo-EM statistics of recorded datasets. The first row corresponds to local resolution mapped on the final volume, the second shows the Fourier shell correlation, the third shows local resolution histogram derived from the data in row 1, the fourth row shows the angular distribution, and the fifth and sixth rows show representative micrograph and particle locations present in the final particle stack. A) FFL2M in nanodisc. B) FFL2M + [FIP- HRC]2-PEG11in nanodisc. C) FFL2M + [FIP-HRC]2-PEG11in amphipol. D) FFL3M in amphipol. E) FFL6M in amphipol.

[0063] FIG. 32 - Cryo-EM Data on the MeV Transmembrane Region. A) Cryo-EM 2D classes of FFL2M in nanodisc. Particles were aligned on either the ectodomain (left) or nanodisc (right). Despite alignment on the nanodisc region, no internal structure (transmembrane region) was observed. The cartoon to the right of the 2D classes illustrates the effect of the alignment on the observed image. In the ectodomain-aligned particles, thenanodisc density is blurred and features are missing. In nanodisc-aligned particles, features inside the nanodisc (lipid bilayer) are visible, but the head domain is blurred and heterogeneously positioned. B) Maximum projection of the final volume (left), raw map (center), and amplified locally scaled map in Occupy (right) of FFL2M in nanodisc (top), FFL2M + [FIP-HRC]2-PEG11in amphipol (center), and FFL3M in amphipol (bottom) show the absence of structural features within the transmembrane region. The amplified locally scaled map, however, reveals clear density for other flexible parts of the structure, such as glycans N29, N61, and N67, as well as partial density for the Afiir loop. Local-scale values are shown at the bottom and apply to all maps. C) Computational investigation of the C-terminal part of the F protein. For the F stalk region (residues 460-487), linker (residues 488-492), transmembrane region (TM, residues 495-515), and cytoplasmic tail (CT, residues 516-550), secondary structure was predicted using AlphaFold 2 (AF2) and the protein large language model ProtT5. Both models indicate that the linker region lacks defined secondary structure, similar to the CT. Secondary structure for ProtT5 is depicted as follows: ●: helix, ►: beta sheet, — : loop. D) Hypothetical hybrid model of FFLincorporating the cryo-EM structure of FFL2M in amphipol (residues 24-485) and AF2 prediction (residues 487-528). The linker was modeled based on partial density present in the cryo-EM map, and the TM and CT regions were based on AF2 prediction. The conformation of the linker may result in the separation of the TM helices and increase the flexibility of the head region. Additionally, the predicted TM helix spans ~40 Å, matching the width of the lipid bilayer. E) Multiple sequence alignment (MSA) of representative members of paramyxoviruses. The stalk and TM regions are mapped based on the MeV F sequence. A similar pattern of residues is observed in the linker region across other family members. F) AF2 predictions of the stalk-linker-TM region based on the MSA of MeV F. Out of 19 species, more than half show a flexible linker depicted as a kink in the secondary structure. Only one representative structure is shown out of five predicted; some structures displayed mixed straight-bent predictions, but only the bent form is presented.

[0064] FIG. 33 - Glycan shielding of F. A) Glycan envelope of F shown with conformer library of determined glycans. Two glycans, N61 (purple) and N67 (dark pink) create a dense barrier on the F apex. N29 (pink) is placed at the bottom of the protein. Possibly due to the placement and inaccessibility to downstream glycosylation enzyme, this glycan is high mannose. The minimal glycosylation leaves majority of the F face exposed to the solvent. B) Glycan shielding effect calculated based on A). The highest shielding effect is present close to the glycosylation site and apex of the protein shows higher shielding than the bottom.

[0065] FIG. 34 - FFLconstructs immunization does not induce vaccine-enhanced respiratory disease in cotton rats. F protein constructs (5 μg), vehicle control (1 mL 1:1 PBS:Adjuphos), or measles Edmonston-Zagreb strain (104TCID50) was administered subcutaneously to cotton rats. At day 28 post-primary immunization, F protein constructs or vehicle control were administered again. At day 56 post-primary immunization, cotton rats were infected with measles virus (wt B3). Cotton rats were euthanized at day 4 post-infection and the right lung lobes were prepared for histopathological evaluation. A-F) Representative regions of peribronchiolar inflammatory infiltrates for each treatment or control group. A) The vehicle control group exhibits minimal peribronchiolar infiltrates. B) Edmonston-Zagreb vaccinated animals exhibit more pronounced peribronchiolar inflammatory infiltrates, occasionally forming complete cuffs (*) around affected airways, with a higher proportion of lymphocytes and plasma cells. C-F) Animals vaccinated and boosted with F protein constructs demonstrate similar or lower degree of peribronchiolar infiltrates compared to Edmonston- Zagreb vaccinated animals (C = FFL2M; D = FFL3M; E = FFL6M). Substantial interstitial and perivascular inflammatory infiltrates are absent.

[0066] FIG. 35 - Neutralization of Live Virus and Screen for hybridomas producing neutralizing antibodies. Hybridomas from a mouse vaccinated with purified and stabilized MeV F protein were screened for neutralization of Measles B3 virus. Vero SLAM cells (Vero cells stably expressing Measles receptor CD 150) were treated with serial dilutions of supernatant from cultured hybridomas prior to infections with 1 ,000 PFU / well of MeV B3 with mCherry. Fluorescent plaques were counted two days following infection and percent inhibition of plaque formation was calculated by normalizing to the no treatment condition for each plate. Hybridoma supernatants that neutralized >80% (dotted line) at the top concentration were selected. Mouse hybridoma producing previously characterized mouse anti measles F antibody 77.4 was used as a positive control. In addition, a purified humanized version of 77.4, 77.1, was used as a technical control within each plate.

[0067] FIG. 36 - Neutralization of Live Virus dose response. Antibodies from neutralizing hybridoma supernatant were purified using protein G conjugated magnetic beads and neutralization of live virus was tested using 10 nM antibody with 1:2 serial dilutions.

[0068] FIG. 37 - Recognition of pre vs post triggered MeV F conformational state. Screening for conformation specific MeV F antibodies. HEK-293T cells transiently transfected with Measles B3 strain F were incubated at 4 degrees Celsius or 55 degrees Celsius for 30 minutes to maintain the pre-triggered state of F (4°C) or induce the post triggered state of F(55° C). Hybridoma supernatants were added to the cells at 4°C and a fluorescent secondary anti-protein G antibody. Transition to the post-triggered state was confirmed by disappearance of previously characterized 77.4.

[0069] FIG. 38 - Sequences of antibodies. (SEQ ID NOS: 17-26)

[0070] FIG. 39 - Mechanism of Action. A) Red blood cell (RBC) fusion assay was conducted in HEK293T cells expressing a chimeric H / HN receptor with MeV F protein, (i) Without fusion inhibitors, the H / HN receptor binds RBCs, and the MeV H stalk region activates the F protein, facilitating its refolding to a fusion intermediate that anchors the fusion peptide in the RBC membrane, culminating in the fusion of RBC and H293T membranes, (ii) In the presence of Zanamivir, a small-molecule inhibitor that competitively binds the HN sialic acid receptor binding site, reversible binding of RBCs to HN is observed. Adding 3G locks the F protein in its pre-fusion conformation, resulting in reversible binding of most RBCs to the H / HN. (iii) The subsequent fusion checkpoint is evaluated through combined administration of Zanamivir and ACK buffer. ACK buffer induces lysis of RBCs. At this checkpoint, fusion intermediates with anchored fusion peptides within RBC membranes resist zanamivir-induced dissociation but are lysed in the presence of ACK buffer, (iv) At the final fusion checkpoint, complete fusion of RBC and HEK293T membranes is achieved. Here, ACK buffer addition has no impact on the fused RBCs. B) Bar chart summarizing the results of a mechanistic assay comparing known inhibitors and neutralizing hybridoma supernatant fluid. For each condition, cells were categorized based on their interaction with red blood cells (RBCs): reversibly bound (dark blue) in which RBCs were adhered only by chimeric H and could be removed by treatment with Zanamivir; irreversibly bound (pink) in which RBCs were initially attached by chimeric H, but subsequent F protein refolding anchored the fusion peptide in the RBC membrane, making RBC attachment insensitive to Zanamivir with RBC retained on the HEK293T cell surface; and fused (light blue) in which RBCs underwent membrane fusion with HEK293T cells expressing the fusion complex.

[0071] FIG. 40 - Illustrates that current vaccines elicit mostly antibodies against hemagglutinin: 80% of human sera antibodies are against H, and only 20% against F.

[0072] FIG. 41 - Plaque reduction assay. Vero SLAM cells were infected with 1000 PFU / well of MeV B3 WT in the presence of serial dilutions of monoclonal antibodies Y10F or 77.1. After 2 days, the plate was imaged, and total fluorescence was calculated by Cytation 5. The percentage of inhibition was calculated relative to no treatment.

[0073] FIG. 42 - Structural characterization of mAbs Y10F and 77.1. A) Cryo-EM structure (2.6 Å) of the FECTOin complex with the Y10F Fab fragment. Y10F specifically recognizes and binds the apex region of the F protein, engaging asymmetrically with two F1 protomers and one F2 chain. B) Cryo-EM structure (3.1 Å) of a mAb77.1 and Y10F antibodies binding a single FECTO.

[0074] FIG. 43 - 77.1 and Y10F activity on MeV F stability. MeV B3 F expressing cells were incubated with varying concentrations of Y10F or 77.1 human monoclonal antibodies for 30’, afterward the cells were transferred to 55 °C for 0, 15, or 30 minutes and subsequently incubated with 10 nM of C6 (mouse mAb that recognized the MeV F in the post-triggered state) at 4°C. Secondary antibodies (anti-mouse Alexa fluor 488 and anti-human Alexa Fluor 594). Cells were imaged on Cytation 5 Imager, and fluorescent cells were assessed. A) Illustrates MeV F arrangements from pre- to post-fusion state. B) Y10F maintains signal after incubation at 55°C while 77.1 does not. C) C6 is a mouse mAb that recognizes MeV F protein in its post-triggered state. C6 signal post-incubation at 55°C in the presence of Y10F is lower compared to 77.1 or mock treated.

[0075] FIG. 44 - 77.1 and Y10F block fusion via different mechanisms. A) Illustrates a RBC fusion assay. Cells expressing MeV and MeV H / HPIV3 HNChimeric receptor binding protein (to enable sialic acid binding) were allowed to bind to red bloods cells (RBCs) at 4° C for 30 min. Unbound RBCs were washed, and cells were treated with monoclonal antibodies in A) 77.1, in B) Y10F, for 30 min at 4°C. The cells were then brought to 37°C for 60 min to enable F triggering and fusion with RBCs. RBCs either: 1) were released by loss of receptor engagement or viral neuraminidase activity during incubation at 37°C; 2) remain bound by HZHN chimera but could be dislodged by treatment with small molecule zanamivir and were therefore reversibly bound: 3) remain bound by insertion of F’s fusion peptide into RBC membrane but not fused with glycoprotein bearing cell (irreversibly bound): 4) fused with glycoprotein bearing cells. B) mAb77.1 blocks fusion at the irreversibly bound stage, indicating that it allows F activation and insertion into the target cells, but it blocks the subsequent refolding required for membrane merger. C) mAb Y10F blocks fusion at the reversibly bound stage, indicating that it prevents F activation.

[0076] FIG. 45 - Sequences of Y10F kappa light chain and Y10F heavy chain (SEQ IDNOS: 15-16).

[0077] FIG. 46 - Illustrates the measles virus (MeV).

[0078] FIG. 47 - Illustrates the measles virus (MeV) entry.

[0079] FIG. 48 - Illustrates MeV architecture and structure.

[0080] FIG. 49 - Illustrates single particle cryogenic electron microscopy.

[0081] FIG. 50 - Illustrates structural studies of MeV glycoproteins.

[0082] FIG. 51 - Illustrates MeV F refolding.

[0083] FIG. 52 - Illustrates MeV F refolding.

[0084] FIG. 53 - Viral entry inhibition assay comparing mAb77 to previously described inhibitors.

[0085] FIG. 54 - Shows the structure of F with Fab77.

[0086] FIG. 55 - Shows the structure of F with Fab77.

[0087] FIG. 56 - Shows the structure of F with Fab77.

[0088] FIG. 57 - Shows the structure of F with Fab77.

[0089] FIG. 58 - Red blood cell fusion assay using 3G, HRC4, and mAb77.1.

[0090] FIG. 59 - Differential scanning fluorimetry of Fab77 and F.

[0091] FIG. 60 - Temperature-resolved dynamic light scattering of F and Fab77.

[0092] FIG. 61 - Negative stain electron microscopy (NS EM) images of F.

[0093] FIG. 62 - Negative stain electron microscopy (NS EM) images of F and Fab77.

[0094] FIG. 63 - Cryo-electron microscopy (cryo-EM) images of F and Fab77.

[0095] FIG. 64 - Structural images showing that Fab stabilizes the epitope.

[0096] FIG. 65 - Illustration showing the neutralization mechanism of a novel neutralizing antibody against MeV that halts the refolding before the membrane merging.

[0097] FIG. 66 - Shows that the mAb77 epitope is highly conserved.

[0098] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0099] Detailed aspects and applications of the disclosure are described below in the following drawings and detailed description of the technology. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

[0100] In the following description, and for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various aspects of the disclosure. It will be understood, however, by those skilled in the relevant arts, that embodiments of the technology disclosed herein may be practiced without these specificdetails. It should be noted that there are many different and alternative configurations, devices and technologies to which the disclosed technologies may be applied. The full scope of the technology disclosed herein is not limited to the examples that are described below.

[0101] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a step” includes reference to one or more of such steps.

[0102] The words “exemplary,” “example,” or various forms thereof are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” or as an “example” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Furthermore, examples are provided solely for purposes of clarity and understanding and are not meant to limit or restrict the disclosed subject matter or relevant portions of this disclosure in any manner. It is to be appreciated that a myriad of additional or alternate examples of varying scope could have been presented, but have been omitted for purposes of brevity.

[0103] When a range of values is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0104] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -!% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0105] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to”, and are not intended to (and do not) exclude other components.

[0106] As required, detailed embodiments of the present disclosure are included herein. It is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. Therefore, specific structural and functional detailsdisclosed herein are not to be interpreted as limits, but merely as a basis for teaching one skilled in the art to employ the present invention. The specific examples below will enable the disclosure to be better understood. However, they are given merely by way of guidance and do not imply any limitation.

[0107] The present disclosure may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific materials, devices, methods, applications, conditions, or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed inventions. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about," it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0108] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (MJ. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bardet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0109] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0110] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breastmilk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0111] The terms “subj ect,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0112] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiments). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily - but may be - all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within thescope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0113] Reference is made to Zyla DS, Della Marca R, Niemeyer G, et al. A neutralizing antibody prevents postfusion transition of measles virus fusion protein. Science. 2024;384(6703):eadm8693.

[0114] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.OVERVIEW

[0115] Embodiments disclosed herein provide for mAb 77.4 and its humanized mAb (77.1), for which the structure and activities are uncovered here, and is one of the few neutralizing monoclonal antibodies (mAbs) yet described against the MeV fusion protein.

[0116] Measles virus (MeV) is a highly transmissible (Ro of 12-18) respiratory, enveloped, single-stranded negative-sense RNA virus. MeV belongs to the Paramyxoviridae family and causes significant morbidity and mortality among young children worldwide, despite the availability of safe and effective MeV vaccines1. Measles accounts for more childhood deaths than any other vaccine-preventable disease. MeV was presumed to be eliminated in the U.S. in 2000 (i.e. ≥12 months without transmission)2. However, reintroduction of the virus has resulted in a resurgence in recent years, causing repeated outbreaks3-7. Outbreaks in the U.S. and other countries with high vaccination coverage are often attributed to unvaccinated individuals. However, a significant number of cases also occur in previously vaccinated people7"9, since up to 5% of vaccine recipients do not develop adequate protective antibodies even after the recommended two-dose vaccination regimen, and because vaccine-elicited immunity likely wanes over time10”14. In addition, the influence of anti-vaccination proponents in social media led to vaccine hesitancy among parents and pockets of unvaccinated children15.

[0117] With no effective antiviral regimen currently available for MeV, insufficient vaccine coverage (which is defined as < 95% vaccination rate by WHO) can lead to a rise in MeV disease among vulnerable populations. Indeed, MeV has already undergone a global resurgence; estimated global MeV deaths rose from 89,780 in 2016 to 207,500 in 2019. The situation may worsen since the SARS-CoV-2 pandemic reduced routine childhood vaccination coverage and may expose more vulnerable individuals to infection16. There have been over 122,000 cases in the first half of 2023 alone17. Without global eradication, a significant increasein the number of MeV cases can be unavoidable18-21. Even mild cases of measles infection can result in immune “amnesia”22,23, and loss of preexisting antibodies against other infections, leaving measles convalescent individuals more vulnerable to future diseases.

[0118] Of particular concern during this resurgence in MeV cases are the rising number of individuals with compromised immune systems, such as cancer patients, transplant recipients, persons with autoimmune disorders on immunosuppressive therapies, and human immunodeficiency virus (HlV)-infected individuals, in addition to pregnant women. In such immunocompromised people, vaccination with current vaccines containing live-attenuated virus is often risky and inefficient24 29; antibody responses are impaired, and even pre-existing immunity wanes30-37. Measles resurgence in other parts of the world, combined with increased vaccine hesitancy, increases the risk of future measles outbreaks and the risk to vulnerable patient populations (see CDC reports36,38-45). Notably, despite the foreseeable rise in measles cases and measles deaths, there are still no effective antivirals approved for human use. Neutralizing monoclonal antibodies offer lifesaving treatments against a number of viruses, but surprisingly, there are none yet available for prevention or treatment of measles virus. Even more surprisingly, there are not yet structures of any antibodies against the measles virus by which Applicants might interpret epitopes or activities that prevent or treat infection. Mab 77.446, for which structure and activities are uncovered here, is one of the few neutralizing mAbs yet described against the MeV fusion protein.

[0119] MeV infects via coordinated action of the hemagglutinin (H, the receptor binding protein) and fusion (F) envelope glycoproteins, to mediate virus attachment and entry into target lymphocyte cells. Vaccination results in neutralizing antibodies against these two proteins, although earlier work suggests that more neutralizing antibodies are directed against hemagglutinin (H), and that anti-F antibodies elicited by the current vaccine are poorly neutralizing47-49. Better understanding of the structures of F, conformational changes that drive fusion, and how antibodies may inhibit structural changes to neutralize MeV are needed.

[0120] F is synthesized as a precursor (Fo) that is cleaved in the producer cell to yield N- terminal F2and C-terminal F1subunits that remain linked by a disulfide bond. The pre-fusion F complex on the viral surface is a trimer comprising three F1and three F2subunits. This trimeric F structure is kinetically trapped in a metastable conformation, primed for fusion activation upon engagement of the H glycoprotein by the cell-surface receptor50-55. After receptor engagement by H, the pre-fusion F undergoes a structural transition, extending and inserting the hydrophobic fusion peptide into the target cell. During entry, F refolds into theclassical 6-helix bundle, post-fusion structure, which brings together the N-terminal heptad repeat (HRN) and the C-terminal heptad repeat (HRC), triggering subsequent fusion of viral and cell membranes56-63. Before refolding to the low-energy, post-fusion state, however, the F protein adopts a series of intermediate conformations that require domain conformational rearrangement and refolding.

[0121] Targeting the intermediate state of F is a key antiviral approach. Applicants previously demonstrated that peptides derived from a consensus sequence of the MeV HRC domain can inhibit viral entry, fusion, and spread in vitro and can prevent infection in vivo64-67'. HRC-derived peptides (e.g. HRC, residues 450-485) mimic the interactions between the native HRC domains and the HRN domains of MeV F proteins, thus inhibiting the transition of F into its post-fusion state and, consequently, viral-host membrane fusion65. The MeVHRC- peptides have been shown to block fusion after F activation and before refolding, likely targeting an extended intermediate of the F protein68. Applicants have recently modified the MeV HRC peptide, by adding a carbobenzoxy-d-Phe-l-Phe-Gly sequence at the N-terminal domain of the previously described HRC peptide68. This new peptide (called FIP-HRC) is superior in potency to the original HRC, can interact with the F before its activation, and stabilize the pre-fusion state of the protein68. Applicants have also recently described a mouse monoclonal antibody (mAb 77.4) that, like HRC-peptides, blocks MeV viral infection through fusion inhibition46. The structural basis for the activity of a humanized version of mAb 77.4, termed mAb 77.1, and FIP-HRC molecules is not yet known. Here, Applicants describe three cryo-EM structures: prefusion MeV F in complex with mAb 77.1, prefusion MeV F in complex with FIP-HRC fusion inhibiting peptide, and F alone in the post-fusion conformation.

[0122] The present study features the first structure of MeV F in complex with any antibody; the structure is in the prefusion conformation, and this is the only neutralizing antibody yet described for F. The mAb is potent in vitro and in vivo; suggesting that other anti- F antibodies yet to be uncovered can have complementary efficacy. This work also provides the first structure of MeV F in the post-fusion conformation, providing the first pre- and postfusion sequence matched pair for the paramyxovirus family. These structures and biochemical characterization enable Applicants to model the conformational changes occurring during the transition from pre-fusion to post-fusion states. The findings indicate that the antibody anchors to a tripartite epitope specific to the pre-fusion form of MeV F and achieves its neutralizing effect by arresting F at an intermediate stage, blocking the protein from completing its final refolding step to achieve the post-fusion conformation and membrane fusion.

[0123] Embodiments disclosed herein also provide for a thermostable (e.g., stabilized) recombinant subunit vaccine that can fill this unmet need in developed countries and is so stable even at high temperatures that it needs no cold chain. This vaccine would eliminate the cold chain requirement of the current live attenuated vaccine and thereby enable distribution in developing nations and facilitate global measles eradication.

[0124] The MeV transmembrane glycoproteins hemagglutinin (H) and fusion protein (F) play critical roles in viral cell entry. The F is kinetically trapped in a metastable conformation, primed for fusion activation upon engagement of H with a cellular receptor (either CD 150 or nectin-4). After H engages the receptor, the pre-fusion F undergoes a conformational transition, irreversibly refolding into a post-fusion conformation as it drives fusion of virus and host membranes. The H and F proteins (i.e., the fusion complex) are the main targets of neutralizing antibodies. The technical challenge for vaccine design has been stabilizing F for vaccine use. Applicants have identified specific mutations in the measles F protein that stabilize the prefusion conformation. Using these substitutions, Applicants have purified both full-length and ectodomain forms of the pre-fusion state of the measles F protein, and Applicants found that they stably maintain the pre-fusion state at high temperature and induce a protective immune response in vivo. Importantly, these are natural mutations combined from selected strains.

[0125] Here, Applicants described MeV specific pre-fusion F stabilizing mutations occurring during viral evolution. MeV field isolates from the central nervous system (CNS) demonstrate that the MeV fusion complex — and specifically mutations in the MeV F protein — can facilitate viral spread in the CNS, even in the absence of canonical receptors25-27. Acquired mutations in the F protein derived from the CNS of infected individuals demonstrate a balance between mutations that decrease thermostability and those that partially compensate for decreases in stability. In previous work, Applicants reported that introducing these compensatory mutations alone into the wild-type fusion protein increases its stability26,27. Leveraging the findings, Applicants recently obtained the structure of a stabilized F ectodomain bearing two stabilizing mutations (E170G and E455G)26,27. The full-length (FL) structure of MeV F has not yet been resolved. To obtain a stable purified MeV FFL, Applicants introduced initial mutations (E170G and E455G) and explored additional stabilizing substitutions. Applicants found that the stabilizing mutations are additive, and the combination of 5 mutations (E170G, Y442H, E455G, V459I, and G506E) produced the most stable F protein. In addition, Applicants have mutated the furin cleavage site to further enhance protein stability. When expressed on the surface of cells, the F bearing E170G, Y442H, E455G, V459L, and G506Emaintains the pre-fusion state (assessed by recognition of pre-fusion antibody27,28) even after exposure to 55°C, 60°C, and 65°C for a prolonged period. Applicants hypothesized that these stabilized pre-fusion FFLproteins will elicit in vivo protection. Applicants purified FFLforms of the pre-fusion state of the MeV F protein bearing these stabilizing mutations, and Applicants have found that they indeed induce a protective immune response in vivo and elicit neutralizing antibodies.Terminology and Definitions

[0126] Paramyxoviridae (from Greek para- “by the side of" and myxa “mucus”) is a family of negative-strand RNA viruses in the order Mononegavirales. Vertebrates serve as natural hosts. Diseases associated with this family include measles, mumps, and respiratory tract infections. The family has four subfamilies, 17 genera, three of which are unassigned to a subfamily, and 78 species. Measles is caused by the measles virus of the genus Morbillivirus within the family Paramyxoviridae. Virions are enveloped and can be spherical or pleomorphic and capable of producing filamentous virions. The diameter is around 150 nm. Genomes are linear, around 15kb in length. Fusion proteins and attachment proteins appear as spikes on the virion surface. Matrix proteins inside the envelope stabilize virus structure. The nucleocapsid core is composed of the genomic RNA, nucleocapsid proteins, phosphoproteins and polymerase proteins. The genome is nonsegmented, negative-sense RNA, 15-19 kilobases in length, and contains six to 10 genes. Viruses in the Paramyxoviridae family are also antigenically stable, meaning that the glycoproteins on the viruses are consistent between different strains of the same type.

[0127] The gene sequence is: Nucleocapsid - phosphoprotein - matrix - fusion - attachment - large (polymerase). The fusion protein F projects from the envelope surface as a timer, and mediates cell entry by inducing fusion between the viral envelope and the cell membrane by class I fusion. The cell attachment proteins, H / HN / G, span the viral envelope and project from the surface as spikes. They bind to proteins on the surface of target cells to facilitate cell entry. Proteins are designated H (hemagglutinin) for morbilliviruses as they possess hemagglutination activity, observed as an ability to cause red blood cells to clump in laboratory tests. HN (Hemagglutinin-neuraminidase) attachment proteins occur in respiroviruses, rubulaviruses and avulaviruses. These possess both hemagglutination and neuraminidase activity, which cleaves sialic acid on the cell surface, preventing viral particles from reattaching to previously infected cells. Attachment proteins with neitherhemagglutination nor neuraminidase activity are designated G (glycoprotein). These occur in henipaviruses.

[0128] The nucleotide and protein, polypeptide and peptide sequences corresponding to various paramyxovirus fusion protein genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information 's Genbank and GenPept databases located at the National Institutes of Health website.

[0129] As used herein “thermostable” refers to a protein that maintains its conformation at a temperature for greater than 1, 3, 5, 7, 9, 12, 30, or greater than 60 minutes, such as greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 30 days. The temperature that a protein is thermostable at can include 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, or 75°C.

[0130] The terms “therapeutic agent”, “therapeutic capable agent” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.

[0131] As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested. As used herein “treating” includes ameliorating, curing, preventing it from becoming worse, slowing the rate of progression, or preventing the disorder from re-occurring (i.e., to prevent a relapse).

[0132] The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will provide an image for detection by any oneof the imaging methods described herein. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

[0133] A “pharmaceutical composition” refers to a composition that usually contains an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to cells or to a subject.NEUTRALIZING ANTIBODIES

[0134] In example embodiments, neutralizing antibodies are disclosed that recognize the F protein of a paramyxovirus (e.g., measles virus). In example embodiments, the antibodies recognize the F protein while it is in its pre-fusion state. In example embodiments, the neutralizing antibody recognize the F protein of MeV. In example embodiments, the antibodies include CDRs specific for binding the F protein. In example embodiments, the CDRs may be included in any antibody backbone disclosed herein. In example embodiments, the antibodies are humanized. In example embodiments, the antibodies are monoclonal antibodies.

[0135] In example embodiments, the antibodies comprise the following CDRs: one or more light chain variable region complementarity determining regions (CDRs) selected from a first CDR comprising the sequence RASKSISKYLA, a second CDR comprising the sequence SGSTLQS, and a third CDR comprising the sequence CQQHNEYTLT; or one or more heavy chain variable region CDRs selected from a first CDR comprising the sequence DYAWN, a second CDR comprising the sequence ISYTLTTGYNPSLKSRI, and a third CDR comprising the sequence RSGWLLPYWYFDV.

[0136] In example embodiments, the antibodies comprise the following CDRs: one or more light chain variable region complementarity determining regions (CDRs) selected from a first CDR comprising the sequence RATSSVVYMH, a second CDR comprising the sequence ATSNLAS, and a third CDR comprising the sequence QQWNSNPYT; or one or more heavy chain variable region CDRs selected from a first CDR comprising the sequence NYWIE, a second CDR comprising the sequence EILPGNSYTNYNEKFRGKA, and a third CDR comprising the sequence PLSGNY.

[0137] The term “antibody” is used interchangeably with the term “immunoglobulin” herein, and includes intact antibodies, fragments of antibodies, e.g., Fab, F(ab')2 fragments, scFva, and intact antibodies and fragments that have been mutated either in their constant and / or variable region (e.g., mutations to produce chimeric, partially humanized, or fullyhumanized antibodies, as well as to produce antibodies with a desired trait, e.g., enhanced binding and / or reduced FcR binding). The term “fragment” refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained via chemical or enzymatic’ treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. Exemplaiy fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, VHHand scF and / or Fv fragments. As used herein, a preparation of antibody protein “having less than about 50% of non-antibody protein (also referred to herein as a “contaminating protein”), or of chemical precursors, is considered to be “substantially free.” 40%, 30%, 20%, 10% and more preferably 5% (by dry weight) of non-antibody protein, or of chemical precursors is considered to be substantially free. When the antibody protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume or mass of the protein preparation.

[0138] In an embodiment, the antibody is a fragment or portion thereof. In an example embodiment, the antibody is an epitope binding protein or portion thereof. The term “binding portion" of an antibody (or “antibody portion”) includes one or more complete domains, e.g., a pair of complete domains, as well as fragments of an antibody that retain the ability to specifically bind to a target molecule. It has been shown that the binding function of an antibody can be performed by fragments of a full-length antibody. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, Fv, single chains, single-chain antibodies, e.g., scFv, and single domain antibodies.

[0139] In an embodiment, the antibody is an antibody fragment or portion. Examples of portions of antibodies encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VHand CHI domains; (ii) the Fab' fragment, which’ is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VHand CHI domains; (iv) the Fd' fragment having VHand CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VLand VHdomains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., 341 Nature 544 (1989)) which consists of a VHdomain or a VLdomain that binds antigen; (vii)’isolated CDR regions or isolated CDR regions presented in a functional framework; (viii) F(ab')2 fragmentswhich are bivalent fragments including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., 242 Science 423 (1988); and Huston et al., 85 PNAS 5879 (1988)); (x) “diabodies” with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; Hollinger et al., 90 PNAS 6444 (1993)); (xi) “linear antibodies” comprising a pair of tandem Fd segments (VH-Ch1-VH-Ch1) which, together with complementary light chain oligopeptides, form a pair of “antigen binding regions” (Zapata et al., Protein Eng. 8(10): 1057-62 (1995); and U.S. Patent No. 5,641,870).

[0140] The term “antigen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding). As such these antibodies or fragments thereof are included in the scope of the disclosure, provided that the antibody or fragment binds specifically to a target molecule.

[0141] In an embodiment, the antibody is a single-chain antibody (scFvs). The term “single-chain variable fragment", as used herein refers to a fusion protein containing the variable region(s) of the heavy (VH) and light (VL) of an immunoglobulin that are connected via a linker peptide. The linker peptide typically ranges from about 10 to about 25 amino acids. The linker can be flexible and can contain one or more glycine residues for flexibility. The linker can contain one or more serine or threonine residues to increase or modify solubility. The VHand light (VL) can be linked via the linker in any order. In an embodiment, N terminus of the VHand is coupled, via a linker, C terminus of the (VL). In an embodiment, C terminus of the VHand is coupled, via a linker, N terminus of the (VL). In an embodiment, the scF V is a bivalent or trivalent scFvs. In an embodiment bitrivalent or trivalent scFvs are bi or trispecific, meaning that they can target 2 or 3, respectively, different epitopes. See also e.g., Hollinger, Philipp; Prospero, T; Winter, G (July 1993). “Diabodies": small bivalent and bispecific antibody fragments”. Proceedings of the National Academy of Sciences of the United States of America. 90 (14): 6444-8; incq, S; Bosman, F; Buyse, MA; Degrieck, R; Celis, L; De Boer, M; Van Doorsselaere, V; Sablon, E (2001). “Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80 / CD86-CD28 costimulatory pathway”. Protein Expression and Purification. 22 (1): 11- 24. doi:10.1006 / prep.2001.1417; Le Gall, F.; Kipriyanov, SM; Moldenhauer, G; Little, M (1999). “Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19:effect of valency on cell binding”. FEBS Letters. 453 (1): 164-168. doi:10.1016 / S0014- 5793(99)00713-9; Huston, J. S.; Levinson, D.; Mudgett-Hunter, M.; Tai, M. S.; Novotny, J.; Margolies, M. N.; Crea, R. (1988). “Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli”. Proceedings of the National Academy of Sciences of the United States of America. 85 (16): 5879-5883; de Graaf et al., Methods Mol Biol. 2002;178:379-87. doi: 10.1385 / 1-59259-240- 6:379; Zhou, H.X., J Mol Biol. 2003 May 23;329(1):l-8. doi: 10.1016 / s0022-2836(03)00372- 3; Bird and Walker. Trends Biotechnol. 1991 Apr;9(4): 132-7. doi: 10.1016 / 0167- 7799(91)90044-1; Worn et al., J Mol Biol. 2001 Feb 2;305(5):989-1010. doi: 10.1006 / jmbi.2000.4265.

[0142] As used herein, “heavy chain antibody,” “VHH" or “single-domain antibodies” (sdAbs) refers to an antibody which is composed only of two heavy chains and lacks the two light chains usually found in antibodies (see, e.g., Henry and MacKenzie, Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs. 2018 Aug-Sep; 10(6): 815-826). VHH can refer to an antibody or VHH domain. Single-domain antibodies (sdAb) are also referred to as a “nanobody”, which is defined herein as an antibody fragment composed of a single monomeric variable antibody domain. As used herein “VHH" is used interchangeably with “nanobody.” The -12-15 kDa variable domains of these antibodies (VHHs and VNARs) can be produced recombinantly and can recognize antigen in the absence of the remainder of the antibody heavy chain. In common antibodies, the antigen binding region consists of the variable domains of the heavy and light chains (VH and VL). Heavy-chain antibodies can bind antigens despite having only VH domains. In an embodiment, the heavy chain antibody is an antibody derived from cartilaginous fishes (immunoglobulin new antigen receptor (IgNAR)) or camelid ungulates. Non-limiting examples of camelids include dromedaries, camels, llamas and alpacas.

[0143] It is intended that the term “antibody” encompass any Ig class or any Ig subclass (e.g., the IgGl, IgG2, IgG3, and IgG4 subclasses of IgG) obtained from any source (e.g., humans and non-human primates, and in rodents, lagomorphs, caprines, bovines, equines, ovines, etc.).

[0144] The term “Ig class” or “immunoglobulin class”, as used herein, refers to the five classes of immunoglobulin that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE. The term “Ig subclass” refers to the two subclasses of IgM (H and L), three subclasses of IgA (IgA1, IgA2, and secretory IgA), and four subclasses of IgG (IgGl,IgG2, IgG3, and IgG4) that have been identified in humans and higher mammals. The antibodies can exist in monomeric or polymeric form; for example, IgM antibodies exist in pentameric f-rm, and IgA antibodies exist in monomeric, dimeric or multimeric form.

[0145] The term “IgG subclass” refers to the four subclasses of immunoglobulin class IgG - IgG1, IgG2, IgG3, and IgG4 that have “been identified in humans and higher mammals by the heavy chains of the immunoglobulins, V1 - γ4, respectively. The term “single-chain immunoglobulin” or “single-chain antibody” (used interchangeably herein) refers to a protein having a two-polypeptide chain structure consisting of a heavy and a light chain, said chains being stabilized, for example, by interchain peptide linkers, which has the ability to specifically bind antigen. The term “domain” refers to a globular region of a heavy or light chain polypeptide comprising peptide loops (e.g., comprising 3 or 4 peptide loops) stabilized, for example, by β pleated sheet and / or intrachain disulfide bond. Domains are further referred to herein as “constant” or “variable”, based on the relative lack of sequence variation within the domains of various class members in the case of “constant” domain, or the significant variation within the domains of various class members in the case of a “variable” domain. Antibody or polypeptide “domains” are often referred to interchangeably in the antibody or polypeptide “regions”. The “constant” domains of an antibody light chain are referred to interchangeably as “light chain constant regions”, “light chain constant domains”, “CL” regions or “CL” domains.” The “constant” domains of an antibody heavy chain are referred to interchangeably as “heavy chain constant regions”, “heavy chain constant domains”, “CH” regions or “CH” domains.” The “variable” domains of an antibody light chain are referred to interchangeably as “light chain variable regions”, “light chain variable domains”, “VL” regions or “VL” domains. The “variable” domains of an antibody heavy chain are referred to interchangeably as “heavy chain constant regions”, “heavy chain constant domains”, “VH” regions or “VH” domains.

[0146] The term “region” can also refer to a part or portion of an antibody chain or antibody chain domain (e.g., a part or portion of a heavy or light chain or a part or portion of a constant or variable domain, as defined herein), as well as more discrete parts or portions of said chains or domains. For example, light and heavy chains or light and heavy chain variable domains include “complementarity determining regions” or “CDRs” interspersed among “framework regions” or “FRs”, as defined herein.

[0147] The term “conformation” refers to the tertiary structure of a protein or polypeptide (e.g., an antibody, antibody chain, domain or region thereof). For example, the phrase “light(or heavy) chain conformation” refers to the tertiary structure of a light (or heavy) chain variable region, and the phrase “antibody conformation" or “antibody fragment conformation” refers to the tertiary structure of an antibody or fragment thereof.

[0148] “Specific binding" of an antibody means that the antibody exhibits appreciable affinity for a particular antigen or epitope and, generally, does not exhibit significant cross reactivity. “Appreciable” binding includes binding with an affinity of at least 25 μM. Antibodies with affinities greater than 1 x 107M-1(or a dissociation coefficient of 1μM or less or a dissociation coefficient of Inm or less) typically bind with correspondingly greater specificity. Values intermediate of those set forth herein are also intended to be within the scope of the present disclosure and antibodies of the disclosure bind with a range of affinities, for example, 100nM or less, 75nM or less, 50nM or less, 25nM or less, for example 10nM or less, 5nM or less, InM or less, or in embodiments 500pM or less, 100pM or less, 50pM or less or 25pM or less. An antibody that “does not exhibit significant cross reactivity” is one that will not appreciably bind to an entity other than its target (e.g., a different epitope or a different molecule). For example, an antibody that specifically binds to a target molecule will appreciably bind the target molecule but will not significantly react with non-target molecules or peptides. An antibody specific for a particular epitope will, for example, not significantly cross react with remote epitopes on the same protein or peptide. Specific binding can be determined according to any art-recognized means for determining such binding. Preferably, specific binding is determined according to Scatchard analysis and / or competitive binding assays.

[0149] As used herein, the term “affinity” refers to the strength of the binding of a single antigen-combining site with an antigenic determinant. Affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, on the distribution of charged and hydrophobic groups, etc. Antibody affinity can be measured by equilibrium dialysis or by the kinetic BIACORE™ method. The dissociation constant, Kd, and the association constant, Ka, are quantitative measures of affinity.

[0150] As used herein, the term “monoclonal antibody” refers to an antibody derived from a clonal population of antibody-producing cells (e.g., B lymphocytes or B cells) which is homogeneous in structure and antigen specificity. The term “polyclonal antibody" refers to a plurality of antibodies originating from different clonal populations of antibody-producing cells which are heterogeneous in their structure and epitope specificity, but which recognize acommon antigen. Monoclonal and polyclonal antibodies may exist within bodily fluids, as crude preparations, or may be purified, as described herein. “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0151] The antibodies as defined for the present disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.VACCINE COMPOSITIONS

[0152] In example embodiments, a paramyxovirus fusion protein is used as an immunogen in a vaccine against a paramyxovirus. In example embodiments, the fusion protein is the MeV fusion protein. In example embodiments, the immunogen includes the full-length fusion protein. In example embodiments, the fusion protein includes the ectodomain of the fusion protein. In example embodiments, the MeV fusion protein includes one or more stabilizing mutations. In example embodiments, the stabilizing mutations make the fusion protein morethermostable. In example embodiments, orthologous mutations can be made in the fusion protein of any other paramyxovirus to generate a stabilized immunogen. As used herein, “orthologous mutations” refers to mutations that occur in orthologous genes in different strains or species of paramyxoviruses. Orthologous mutations may not occur at the same residue number, however one skilled in the art occur can identify conserved residues that occur in the homologous position or domain of the orthologous gene. For example, one skilled in the art can perform an alignment of orthologous genes.In Vitro Peptide / Polypeptide Synthesis

[0153] Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, in vitro translation, or the chemical synthesis of proteins or peptides. The nucleotide and protein, polypeptide and peptide sequences corresponding to various paramyxovirus fusion protein genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information 's Genbank and GenPept databases located at the National Institutes of Health website. The coding regions for known genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.

[0154] Peptides can be readily synthesized chemically utilizing reagents that are free of contaminating bacterial or animal substances (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963). In certain embodiments, peptides are prepared by (1) parallel solid-phase synthesis on multi-channel instruments using uniform synthesis and cleavage conditions; (2) purification over a P-HPLC column with column stripping; and re-washing, but not replacement, between peptides; followed by (3) analysis with a limited set of the most informative assays. The Good Manufacturing Practices (GMP) footprint can be defined around the set of peptides for an individual patient, thus requiring suite changeover procedures only between syntheses of peptides for different- patients.

[0155] Alternatively, a nucleic acid (e.g., a polynucleotide) encoding a peptide of the invention may be used to produce the peptide in vitro. The polynucleotide may be, e.g., DNA, cDNA, PNA, CNA, RNA, either single- and / or double-stranded, or native or stabilized formsof polynucleotides, such as e.g. polynucleotides with a phosphorothiate backbone, or combinations thereof and it may or may not contain introns so long as it codes for the peptide. In one embodiment, in vitro translation is used to produce the peptide. Many exemplary systems exist that one skilled in the art could utilize (e.g., Retie Lysate IVT Kit, Life Technologies, Waitham, MA).

[0156] An expression vector capable of expressing a polypeptide can also be prepared. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression, if necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria, insect cells, or mammalian cells), although such controls are generally available in the expression vector.

[0157] Expression vectors comprising the polynucleotides encoding the fusion protein, as well as host cells containing the expression vectors, are also contemplated. The protein may be provided in the form of RNA or cDNA molecules encoding the desired protein. One or more proteins of the invention may be encoded by a single expression vector.

[0158] The term "polynucleotide encoding a polypeptide” encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and / or non-coding sequences. Polynucleotides can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.

[0159] In embodiments, the polynucleotides may comprise the coding sequence for the protein fused in the same reading frame to a polynucleotide which aids, for example, in expression and / or secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.

[0160] In embodiments, the polynucleotides can comprise the coding sequence for the protein fused in the same reading frame to a marker sequence that allows, for example, for purification of the encoded polypeptide. For example, the marker sequence can be a hexahistidine tag supplied by a vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA)tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. Additional tags include, but are not limited to, Calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag, Spy Tag, Biotin Carboxyl Carrier Protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green fluorescent protein tags), maltose binding protein tags, Nus tags, Strep-tag, thioredoxin tag, TC tag, Ty tag, and the like.

[0161] The isolated fusion proteins described herein can be produced in vitro by any suitable method known in the art. Such methods range from direct protein synthetic methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by sitespecific mutagenesis to provide functional analogs thereof. See, e.g. Zoeller et al, Proc. Nat'l. Acad. Sci. USA 81 .5662-5066 (1984) and U.S. Pat. No. 4,588,585.

[0162] In example embodiments, oligonucleotides encoding a fusion protein can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest is produced. In example embodiments, oligonucleotides encoding a fusion protein is inserted into an expression vector and optionally operatively linked to an expression control sequence appropriate for expression of the protein in a desired host.

[0163] Recombinant expression vectors may be used to amplify and express DNA encoding the fusion protein. Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a fusion protein operatively linked to suitable transcriptional or translational regulator elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail herein. Such regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader)is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. Generally, operatively linked means contiguous, and in the case of secretory leaders, means contiguous and in reading frame. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product. Useful expression vectors for eukaryotic hosts, especially mammals or humans include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Usefill expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli.

[0164] Suitable host cells for expression of a fusion protein include prokaryotes, yeast, insect or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram- negative or gram-positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems could also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known in the art (see Pouwels et al, Cloning Vectors: A Laboratory Manual, Elsevier, 1985).

[0165] Various mammalian or insect cell culture systems are also advantageously employed to express recombinant protein. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified and completely functional. Examples of suitable mammalian host cell lines include the COS -7 lines of monkey kidney cells, described by Gluzman (Cell 23: 175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa, HEK293, and BHK cell lines. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5 ' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0166] The proteins produced by a transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography, and the like), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence, glutathione-S-transferase, and the like can be attached to the fusion protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.In Vivo Peptide / Polypeptide Synthesis

[0167] The present invention also contemplates the use of nucleic acid molecules as vehicles for delivering immunogens to a subject, in vivo, in the form of, e.g., DNA / RNA vaccines (see, e.g., WO2012 / 159643, and WO2012 / 159754, hereby incorporated by reference in their entirety).

[0168] In one embodiment fusion proteins may be administered to a patient in need thereof by use of an mRNA vaccine (see, e.g., Sahin, U, Kariko, K and Tureci, O (2014). mRNA-based therapeutics - developing a new class of drugs. Nat Rev Drug Discov 13: 759-780; Weissman D, Kariko K. mRNA: Fulfilling the Promise of Gene Therapy. Mol Ther. 2015;23(9): 1416- 1417. doi:10.1038 / mt.2015.138; Kowalski PS, Rudra A, Miao L, Anderson DG. Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery. Mol Ther. 2019;27(4):710-728. doi:10.1016 / j.ymthe.2019.02.012; Magadum A, Kaur K, Zangi L. mRNA-Based Protein Replacement Therapy for the Heart. Mol Ther. 2019;27(4):785-793. doi:10.1016 / j.ymthe.2018.11.018; Reichmuth AM, Oberli MA, Jaklenec A, Langer R, Blankschtein D. mRNA vaccine delivery using lipid nanoparticles Ther Deliv. 2016;7(5):319- 334. doi:10.4155 / tde-2016-0006; and Khalil AS, Yu X, Umhoefer JM, et al. Single-dose mRNA therapy via biomaterial-mediated sequestration of overexpressed proteins. Sci Adv. 2020;6(27):eaba2422). In an exemplary embodiment, mRNA encoding for a fusion protein is delivered using lipid nanoparticles (see, e.g., Reichmuth, et al., 2016). In an exemplary embodiment, mRNA encoding for a fusion protein is delivered using biomaterial-mediated sequestration (see, e.g., Khalil, et al., 2020).

[0169] In one embodiment, a fusion protein may be administered to a patient by use of a plasmid. These are plasmids which usually consist of a strong viral promoter to drive the invivo transcription and translation of the gene (or complementary DNA) of interest (Mor, et al., (1995). The Journal of Immunology 155 (4): 2039-2046). Intron A may sometimes be included to improve mRNA stability and hence increase protein expression (Leitner et ai, (1997). The Journal of Immunology 159 (12): 6112-61 19). Plasmids also include a strong polyadenylation / transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410; Robinson et al ., (2000). Adv. Virus Res. Advances in Virus Research 55: 1-74; Bohmet al, (1996). Journal of Immunological Methods 193 (i): 29-40.). Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).

[0170] Because the plasmid is the “vehicle” from which the immunogen is expressed, optimizing vector design for maximal protein expression is essential (Lewis et al., (1999), Advances in Vims Research (Academic Press) 54: 129-88). One way of enhancing protein expression is by optimizing the codon usage of mRNAs for eukaryotic cells. Another consideration is the choice of promoter. Such promoters may be the SV40 promoter or Rous Sarcoma Vims (RSV).

[0171] Plasmids may be introduced into animal tissues by a number of different methods. The two most popular approaches are injection of DNA in saline, using a standard hypodermic needle, and gene gun delivery. A schematic outline of the construction of a DNA vaccine plasmid and its subsequent delivery by these two methods into a host is illustrated at Scientific American (Weiner et al., (1999) Scientific American 281 (1 ): 34-41). Injection in saline is normally conducted intramuscularly (IM) in skeletal muscle, or intradermally (ID), with DNA being delivered to the extracellular spaces. This can be assisted by electroporation by temporarily damaging muscle fibers with myotoxins such as bupivacaine; or by using hypertonic solutions of saline or sucrose (Alarcon JB, Waine GW, McManus DP. DNA vaccines: technology and application as anti-parasite and anti-microbial agents. Adv Parasitol. 1999;42:343-410). Gene gun delivery, the other commonly used method of delivery, ballistically accelerates plasmid DNA (pDNA) that has been adsorbed onto gold or tungsten microparticles into the target cells, using compressed helium as an accelerant (Alarcon et al., (1999), Adv Parasitol. 1999;42:343-410; Lewis et al., (1999), Advances in Virus Research (Academic Press) 54: 12.9-88). Alternative delivery methods may include aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa, (Lewis et al., (1999).Advances in Virus Research (Academic Press) 54: 129-88) and topical administration of pDNA to the eye and vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54: 129-88). Mucosal surface delivery has also been achieved using cationic liposome- DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors.

[0172] In example embodiments, the fusion protein of the invention may be encoded and expressed in vivo using a viral based system (e.g., an adenovirus system, an adeno associated virus (AAV) vector, a poxvirus, or a lentivirus). In one embodiment, the immunogenic composition may include a viral based vector for use in a human patient in need thereof, such as, for example, an adenovirus (see, e.g., Baden et al. First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (1PCAVD 001). J Infect Dis. 2013 Jan 15;207(2):240-7). Plasmids that can be used for adeno associated virus, adenovirus, and lentivirus delivery have been described previously (see e.g., U.S. Patent Nos. 6,955,808 and 6,943,019, a d U.S. Patent application No. 20080254008).

[0173] In another embodiment a Poxvirus is used in the immunogenic composition. These include orthopoxvirus, avipox, vaccinia, MVA, NYVAC, canarypox, AL VAC, fowlpox, TROVAC, etc. (see e.g., Verardiet al., Hum Vaccin Immunother. 2012 Jul;8(7):961-70; and Moss, Vaccine. 2013: 31(39): 4220-4222). Poxvirus expression vectors were described in 1982 and quickly became widely used for vaccine development as well as research in numerous fields. Advantages of the vectors include simple construction, ability to accommodate large amounts of foreign DNA and high expression levels.Vaccine or Immunogenic Composition Administration

[0174] Effective vaccine or immunogenic compositions advantageously include a strong adjuvant to initiate an immune response. An effective immune response advantageously includes a strong adjuvant to activate the immune system (Speiser and Romero, Molecularly defined vaccines for cancer immunotherapy, and protective T cell immunity Seminars in Immunol 22: 144 (2010); and Zhao, T., Cai, Y., Jiang, Y. et al. Vaccine adjuvants: mechanisms and platforms. Sig Transduct Target Ther 8, 283 (2023)).

[0175] Prime / boost regimens refer to the successive administrations of a vaccine or immunogenic or immunological compositions. In certain embodiments, administration of the immunogenic composition is in a prime / boost dosing regimen, for example administration of the immunogenic composition at weeks 1, 2, 3 or 4 as a prime and administration of the immunogenic composition is at months 2, 3 or 4 as a boost. In another embodiment DNAencoding the fusion protein is used to prime followed by a protein boost. In another embodiment, protein is used to prime followed by boosting with a virus encoding the fusion protein (see, e.g., Hutchings CL, Birkett AJ, Moore AC, Hill AV. Combination of protein and viral vaccines induces potent cellular and humoral immune responses and enhanced protection from murine malaria challenge. Infect Immun. 2007;75(12):5819-5826). In another embodiment a virus encoding the fusion protein is used to prime and another virus is used to boost. In another embodiment protein is used to prime and DNA is used to boost.

[0176] The pharmaceutical compositions can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients in need thereof, including humans and other mammals. For example, the immunogenic composition can include a pharmaceutically acceptable carrier and / or excipient. In example embodiments, the pharmaceutically acceptable carrier comprises water. In example embodiments, the pharmaceutically acceptable carrier further comprises dextrose. In example embodiments, the pharmaceutically acceptable carrier further comprises dimethylsulfoxide. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., ocular, oral, topical or parenteral, including gels, creams ointments, lotions and time released implantable preparations, among numerous others. Solutions or suspensions used for ocular, parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Liposomal suspensions may also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. In example embodiments, the vaccine or immunogenic composition is administered intravenously or subcutaneously.

[0177] The invention provides for pharmaceutical compositions containing a fusion protein as described herein. In embodiments, the pharmaceutical compositions contain a pharmaceutically acceptable carrier, excipient, or diluent, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to a subject receiving the composition, and which may be administered without undue toxicity. As used herein, the term “pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, EuropeanPharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. These compositions can be useful for treating and / or preventing viral infection and / or autoimmune disease.

[0178] A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (17th ed., Mack Publishing Company) and Remington: The Science and Practice of Pharmacy (21st ed., Lippincott Williams & Wilkins), which are hereby incorporated by reference. The formulation of the pharmaceutical composition should suit the mode of administration. In embodiments, the pharmaceutical composition is suitable for administration to humans, and can be sterile, nonparticulate and / or non-pyrogenic. Pharmaceutically acceptable carriers, excipients, or diluents include, but are not limited, to saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

[0179] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLESExample 1 - Human chimeric mAb 77.1Human chimeric mAb 77.1 potently inhibits MeV fusion and replication.

[0180] There are currently no approved antiviral treatments for measles virus infection, although the provision of such treatments would provide important medical defense to those who cannot be vaccinated or in whom vaccines didn’t take. Antibody is a crucial life-saving protective intervention for several other viruses69-80. Applicants previously isolated and sequenced the F-targeted, neutralizing mouse antibody mAb 77.446. To facilitate future use as a human therapeutic, Applicants grafted the 77.4 variable regions into a human IgGl framework to obtain a chimeric antibody termed mAb 77.1 As mAb 77.1 is thus far the only neutralizing antibody described against MeV F, Applicants wished to assess its in vivo efficacy against MeV exposure.

[0181] Applicants first evaluated the antiviral activity of mAb 77.1 using an in vitro viral entry assay in which cells, Vero-SLAM were treated with serial dilution of mAb 77.1 or previously described inhibitors, 3G and HRC4 and infected with MeV WT B3-GFP virus. Applicants found that mAb 77.1 inhibited fusion with a potency of 0.034 nM (5.1 ng / mL) (Fig. 1A). Notably, the activity of mAb 77.1 was substantially greater than that of two other known inhibitors evaluated side-by-side with the antibody. A fusion-inhibiting lipopeptide termedHRC4, derived from the C-terminal heptad repeat of F, blocked fusion with a potency of 1.17 nM while the small molecule 3G, which locks F in the pre-fiision conformation, had a potency of only 150.12 nM.

[0182] Applicants next assessed mAb 77.1 for in vivo efficacy against MeV exposure using the standard cotton rat model81,82. Cotton rats were treated with 0.1 mg / kg (n=5) or 1 mg / kg (n=10) of mAb 77.1 intraperitoneally 12 hours before challenge with 105TCID50(i.e., 50% tissue culture infectious) dose of MeV wt B3-GFP. Antibody pre-treatment resulted in a significant reduction in viral replication as compared to mock-treated animals (Fig. IB). The average titer in mock-treated animals was 3.6 log 10 TCID50 / g lung tissue, whereas three animals pretreated with 0.1 mg / kg body weight of monoclonal antibody were virus negative and two had reduced virus titers in lung tissue (2.4 and 2.6 loglO TCID50 / g). Eight animals treated with 1 mg / kg mAb were virus negative and two had reduced virus titers (2.9 and 3.6 loglO TCID50 / g). The activity of mAb 77.1 at even a low 0.1 mg / kg dose was striking. Therefore, Applicants next wished to determine its mechanism of action.77.1 prevents conformational changes required for fusion.

[0183] The F protein is primed to undergo substantial conformational change and refolding to drive the fusion of the viral and host membranes. This refolding is thought to be initiated by a signal from the stalk of the H protein transmitted after it engages the receptor83. As a first step in F conformational rearrangement, the N-terminal heptad repeat (contained in Domain III of the F1 head region) refolds from three short helices and beta-harpin motifs into an approximately 100 Å-long helix. This assembled and now extended helix projects the fusion peptide, previously concealed within Domain III, toward and into the host membrane. This conformation of F is often referred to as the 'pre-harpin intermediate’. The sequence of events leading to complete refolding remains elusive but involves substantial rearrangement and repositioning of the stalk region containing C-terminal Heptad Repeat (HRC). This movement enables HRC to collapse onto HRN into a 6-helix bundle that draws the viral and host membranes into proximity for fusion.

[0184] Previous studies demonstrated that mAb 77.1 inhibits fusion of viral and host cell membranes46, but the step at which it functions remains unclear. The possibilities were that it could (I) stabilize the F prefusion conformation to prevent F from triggering, (II) stabilize the intermediate state and interrupt the refolding sequence to prevent viral and host membranes from joining, or (III) functionally impair F refolding by inducing protomer separation or misfolding.

[0185] To elucidate the stage of the fusion process at which mAb 77.1 works, Applicants used an assay that measures fusion of red blood cells (RBC) with HEK293T cells bearing versions of H and F (Fig. 2A and B)68. In this assay, Applicants needed a version of H that would release its hold on receptor to discern anchoring by F from anchoring by H. Further, RBCs do not express the MeV receptors SLAM / CD 150 or Nectin-4 and are instead coated with the sialic acid receptor naturally targeted by the hemagglutinin-neuraminidase surface protein (HN) of the related human paramyxovirus parainfluenza virus 3 (HPIV3). Moreover, the HN- sialic acid interaction can be interrupted by addition of the small molecule drug Zanamivir84,85. Applicants thus employed a previously described chimeric receptor-binding protein which contains the MeV H stalk (the portion of H that interacts with MeV F) combined with the receptor-binding head domain of HPIV3 HN68. The resulting chimeric protein H-HN enables both releasable attachment via sialic acid receptors and MeV H-driven triggering of F. Applicants also employed a point mutation T461I in F, which destabilizes the protein and allows easy activation for fusion85. HEK293Ts co-transfected with FT461Iand the H-HN chimera were incubated with RBCs for 30 mins at 4°C to allow for receptor engagement but not fusion. The cells were washed to remove unbound RBCs and subsequently incubated with mAb 77.1 or diluent media. Two distinct MeV F viral entry inhibitors were included as additional controls. One inhibitor, N-(3-cyanophenyl)-2-phenylacetamide (compound 3G in86) binds to prefiision F and blocks H-mediated fusion activation87. The other inhibitor is an HRC- derived peptide termed HRC4, which binds to HRN in the pre-hairpin intermediate and blocks the conformational transition to the 6-helix bundle68.

[0186] After treatment with mAb, control inhibitor, or diluent media, the cells were transferred to 37°C for 60 min to permit F activation (and fusion of the RBCs with the HEK293T in the mock control). RBCs were then stratified into three distinct groups: (1) those that were reversibly bound to the HEK293T cells; (2) those that remained anchored to HEK293Ts but had not fused; and (3) those that had successfully fused with the HEK293Ts. The first group was quantified by the amount of RBCs that were released from the HEK293Ts (i.e., reversibly bound) via zanamivir; release by zanamivir indicates that the RBCs were anchored to the HEK293T target cells through H-HN interactions only and that no F-mediated fusion had commenced. The second group was quantified through osmotic lysis of RBCs; these were anchored to the HEK293Ts through F (irreversibly bound) but had not yet fused. The third group of RBCs - those which had fused with the HEK293T cells - were quantified by lysis of the remaining cells using a detergent. In each case, the released hemoglobin in thelysate was measured via absorbance at 410nm, with the amount of reversibly bound + irreversibly bound + fused RBCs set to 100%.

[0187] Applicants found that treatment with diluent media resulted in the fusion of approximately 50% of total RBCs with the HEK293T cells (Fig. 2A and B, Fig. 7). The remaining RBCs had either begun, but not completed fusion (irreversibly bound through F),or had not yet initiated fusion (reversibly bound through HN only). Treatment with the 3G inhibitor prevented nearly all F-mediated anchoring, indicating, as expected, that this inhibitor prevents F activation. Meanwhile, treatment with the HRC4 peptide inhibitor resulted in most RBCs being irreversibly bound, indicating F was activated, but could not complete fusion. Treatment with mAb 77.1 had the same effect as HRC4, where the majority of RBCs were irreversibly bound. This suggests mAb 77.1 does not prevent the activation of F nor initiation of fusion and formation of the pre-hairpin intermediate, but instead prevents the collapse of this intermediate into the 6-helix bundle required for complete fusion.Stabilization of MeV Fin the pre-fusion state

[0188] Expression of the ectodomain of wild-type F (FECTO, comprising amino acids 1- 495) results in production of the post-fusion conformation only. Previous structural characterization of MeV F was achieved by engineering four cysteines in the stalk region which results in disulfide linkages between the three protomers87. However, Applicants found that protein produced from this construct aggregated, even at lower concentrations. As such, Applicants took a different approach and instead generated a MeV F ectodomain that incorporates two naturally occurring point mutations, E170G88and E455G89(Fig. 3A). These mutations were identified in independent central-nervous system isolates of MeV and stabilize F in the pre-fusion state88,89.When paired, stabilization was further enhanced (data not shown). This protein, termed F2MECTO, was -80% cleaved by furin and approximately 85% of the resulting particles were in the pre-fusion conformation (Fig. 8 A, B).

[0189] Although F2MECTOcan be purified in the pre-fusion state, the protein spontaneously springs to the post-fusion state over time. To enhance the stability of the pre-fusion conformation following purification, Applicants used differential scanning fluorimetry (DSF) to systematically explore the impact of pH and salt concentration on the apparent melting temperature (Tmapp) of F2MECTo(Fig. 8 C, D). Applicants found that while pH had little influence on the Tmappof F2MECTO, higher salt concentration increased the Tmapp, from 44.6°C at 60 mM NaCl to 54.8°C at 1460 mM NaCl (Fig. 8 D). Hence, buffering conditions of 50 mM Hepes pH 8.0 and 800 mM NaCl were used in subsequent purification of F2MECTO. Notably,Applicants do not observe any F2MECTOin the post-fusion conformation even after more than 2 weeks of storage in this buffer (Fig. 8 B).

[0190] Applicants also measured the binding affinity of the 77.1 Fab fragment to purified F2MECTOusing Bio-Layer Interferometry. In agreement with the potent activity of mAb 77.1 in the cell based assay, Fab 77.1 bound to F with a KDof 360pM (Fig. 8 E).Mab 77.1 binds to prefusion MeV F

[0191] The cell-based assays indicate that mAb 77.1 locks an intermediate state of F refolding after the fusion peptide has been released but before the collapse of the trimer into the 6-helix bundle required for fusion to complete. To elucidate the binding epitope of mAb 77.1 and further discern the mechanism of action, Applicants determined the structure of F2MECTOin complex with Fab 77.1. At the same time, Applicants solved the structure of F2MECTOin complex [FIP-HRC]2-PEG11(a tandem peptide conjugate of two copies each of FIP and HRC, linked by a PEG11moiety68. These structures were solved in order to 1) confirm that F2MECTOadopts the expected prefusion structure, and 2) identify any antibody-dependent conformational changes in F2MECTO. Note that this inhibitor contains both the tri-peptide FIP, which stabilizes prefusion F, and an HRC mimic, which binds to the pre-hairpin intermediate. Hence, this inhibitor could, in theory, be used to capture either state of F2MECTO, provided the correct state is available for binding.

[0192] F2MECTOwas incubated with either [FIP-HRC]2-PEG11or Fab 77.1 and purified via size exclusion chromatography (SEC) (Fig. 8 F). The resulting complexes were analyzed by single-particle cryogenic electron microscopy (cryo-EM). Applicants obtained a high- resolution structure of the F2MECTO-[FIP-HRC]2-PEG11complex at 2.3 Å (Fig. 3B). All F2MECTOparticles adopt a prefusion state with clear density for FIP. However, Applicants did not see density for the HRC-derived mimic, likely due to the high flexibility of the peptide when tethered to FIP (Fig. 12 A, B). Moreover, Applicants did not observe alternative conformations of F in 2D class averages suggesting that a fusion-intermediate state was not available for HRC to stably bind.

[0193] The overall structure of F2MECTOclosely resembles previous cysteine-stabilized prefusion F configurations, where the head region is a composite structure made up of segments from both F1 and F2, which are linked by disulfide bonds. The F2 subunit twists around the trimer of hetero dimers and intercalates between the N-terminal heptad repeat (HRN), DI, and Din of the F1 subunit. Meanwhile, the fusion peptide, located at the extreme N-terminus of F1, reaches out from one monomer to lay on top of the immunoglobulin-like domain II of aneighboring monomer. Together, these domains form a pyramidal shape. Finally, the C- terminal region of F1 (C-terminal heptad repeat, HRC) coils around domain II and extends downward to form the stalk of the trimer (Fig. 3 C). Applicants note, however, two differences in previous F structures as compared to those presented here. First, comparison of the previously published X-ray structures (Cys-stabilized, APO: 5YXW, FlP-bound: 5YZD and AS-48-bound: 5YZC) to these cryo-EM structures (no Cys stabilization, but two naturally occurring point mutations that confer stability, and bound to either Fab or inhibitoiy peptide), shows loop 248-265 in different conformations, with an RMSD of ~4 Å. This conformational variance is likely attributable to crystal contacts between loop 248-265 and F2 helix 75-86 from a symmetry mate (Fig. 12C, D, E). Second, Applicants observe differences in the conformation of the stalk regions between the structures. In the cryo-EM-based model, the alpha-helices of the HRC in the stalk region begin to super-coil around each other, starting at residue 473. Conversely, in the Cys-stabilized construct, the stalk region remains linear and does not exhibit supercoiling (Fig. 12C, D, E). Overall, the differences are minor and might be related to the construct stabilization (stalk conformation) or technique used for structure determination (loop 248-265) but the general protein structure remains the same.

[0194] Similar to the previous structure of MeV F in complex with a related peptide inhibitor here Applicants find that FIP stabilizes F by occupying the hydrophobic pocket created by F1 residues from all 3 protomers including residues FIA 311W, 370L, 451P, 4521, 453S, 459V and 466A, FIB 463L, 464G, 4671, 471E and F1c 352S and 353P. Together, these interactions lock MeV F in the prefusion state and prevent the refolding of heptad repeats68,87,90.

[0195] The final map of the F2MECTO-Fab 77.1 complex was resolved to 2.6 Å by masking the flexible constant regions of the Fab and conducting a final local reconstruction in C1 from the C3 symmetry-expanded particle set (Fig. 11 A). Three copies of Fab 77.1 are bound to the prefusion F2MECTOtrimer. The epitope is located at the base of the trimer and encompasses regions in F1 and F2 from one protomer and F1 from a neighboring protomer (Fig. 3 D, E, G). The heavy chain of Fab 77.1 contributes the bulk of the interaction, burying 889 Å2of surface area on FECTO, as compared to the 283 Å2buried by the light chain (Table 3). The predominant interactions between the antibody and F2MECTOoccur via the heavy chain complementaritydetermining region (CDR)-3, with additional contributions from CDR-H1, CDR-H2, and CDR-L2 (Fig 3B, Table 2).

[0196] CDR-H3 occupies a shallow cleft between domains I and II of FIB, forming six hydrogen bonds and one salt bridge. In particular, FIB residue R436 forms hydrogen bonds tothe backbone oxygens of CDR-H3 residues Y105 and Y107 and a salt bridge with CDR-H3 D109 (Fig. 3 G). CDR-L2 residues Y49 and E55 also bind to this region, making a hydrogen bond with the backbone nitrogen of R435 and the hydroxyl group of S434 (Table 4). Meanwhile, CDR-H2 interacts with F2 through contacts with F2 residues W27, G37, and S40. Notably, CDR-H1 residues Y27 and S31 form hydrogen bonds with the backbone of residues L123 and V125, located in the fusion peptide of the neighboring protomer, F1c.

[0197] Together, this extensive network of 16 hydrogen bonds and one salt-bridge anchor domains I and II of F1 in the prefusion state.77.1 binding interface is well preserved in MeV variants.

[0198] Although MeV has been generally considered a serologically monotypic virus, genetic characterization of wild-type viruses has identified eight clades (A-H), which have been further divided into 22 genotypes plus one additional proposed genotype91-94. To evaluate whether natural genetic variation in MeV might impact mAb 77.1 binding, Applicants examined sequence conservation across the available MeV F protein sequences in the NCBI database. Of the 550 residues in MeV F, 319 are fully conserved, while 231 (42%) have single or multiple amino acid substitutions (Fig. 5A). The signal peptide and cytoplasmic tail have the greatest sequence variability, but substitutions are distributed throughout the tertiary structures of pre- and post-fusion MeV F (Fig. 5A, B). Within the mAb 77.1 epitope, however, only two residues, G37 and R436, vary across the range of emerged MeV sequences (Fig. 5C). One single F sequence (0.12%) had a G37R substitution which could sterically hinder the F- 77.1 interface. The second mutation, however, R436K, occurs in more than ¼ of F sequences. Mutation of R436 to a lysine could impact association of mAb 77.1 with F, particularly the hydrogen bond network and salt bridge formed between R346 and mAb residue D109. To assess the impact of each of these substitutions, Applicants evaluated the ability of mAb 77.1 to block fusion of F variants bearing either the naturally occurring G37R or R436K or an artificially created charge reversal mutant of R436D or charge loss of R436G.

[0199] Applicants first evaluated the expression of each F mutant, as well as recognition by mAb 77.1. Both expression and mAb 77.1 binding to FR43GKwere comparable to that of FWT(Fig. 14A, B, C). In contrast, while FG37Rmaintained wild-type expression levels, recognition by mAb 77.1 was substantially reduced (Fig. 14 A, B, C). Lastly, the compromised expression of FR436Gand FR436Dat 37°C was only partially recovered at 32°C. Recognition of either mutant by mAb 77.1 was also reduced (R436G) or abrogated (R436D), likely due to the charge loss or reversal, respectively, at this site (Fig. 14A, B, C, F).

[0200] Notably, Applicants found that both fusion capacity and m Ab-mediated fusion inhibition of FR436Kparalleled that of FWT (Fig. 5D, Fig. 14D, E). FG37R, on the other hand, had ~8-fold less fusion as compared to FWT and escaped mAb-mediated inhibition (Fig. 5D, Fig. 14D, E). The lower fusogenicity of the FG37Rvariant likely explains its very low incidence in naturally 31 occurring MeV strains. As expected, FR436Gand FR436Dalso escaped mAb- mediated fusion inhibition (Fig. 14 D, E).

[0201] This data suggests that mAb 77.1 is likely to maintain efficacy towards currently circulating measles virus strains. However, it could be susceptible to infrequent mutations, such as G37R, or other emergent mutations not yet described, particularly if used as a therapeutic for active MeV infection.

[0202] To understand the potential impact of mAb 77.1 on viral evolution, Applicants attempted to select escape mutations. MeV was incubated with increasing concentrations of mAb 77.1 for five viral replication cycles. Viral proliferation ceased after cycle five likely due to the accumulation of deleterious mutations. Nevertheless, the viral population from cycles four and five was harvested and deep-sequenced to identify potential genetic changes. Intriguingly, despite the extended exposure of the virus to mAb 77.1, the analysis failed to uncover any significant point mutations (allele frequency ≥ 5%, sequencing depth ≥ 20) (Fig. 5E, Fig ISA, B). Together, the analysis of naturally occurring mutations and in vitro viral evolution suggests a high barrier to viral escape from mAb 77.1.F post-fusion structure

[0203] The structure of FECTOin complex with Fab 77.1 unequivocally demonstrates this mAb recognizes the pre-fusion conformation of F and anchors the fusion peptide through interactions with CDR-H1. However, the RBC fusion assay (Fig 2) suggests that mAb 77.1 prevents viral infection by arresting the fusion process after the fusion peptide has been released from the trimer and inserted into the host cell membrane.

[0204] To reconcile the divergence between the Fab 77.1 epitope and the cell biology results, Applicants determined the cryo-EM structure of F2MEcroin its post-fusion state, which was not yet available. In particular, direct structural comparison of the pre- and post-fusion states from the same protein sequence would provide a more comprehensive understanding of the conformational dynamics of F2MEcioand clarify how mAb 77.1 might selectively interact with the pre-fusion form or reveal conservation of the epitope in the post-fusion form.

[0205] As previously described, while storage of F2MECTOin a high-salt buffer results in conformational stability, storage in a low-salt buffer results in spontaneous refolding to thepost-fusion state. Hence, by incubation of the protein in low-salt buffer (i.e., 150 mM NaCI), Applicants were able to capture the 2.7 Å resolution cryo-EM structure of F2MECTOin its postfusion conformation using the same construct as represented in the pre-fusion F2MECTOstructures (Fig. 4 A).

[0206] Like post-fusion structures of the F proteins of other paramyxoviruses, the MeV FECTOpost-fusion structure adopts a distinct elongated shape with a refolded head domain and an extended 6-helix bundle composed of HRN and HRC (PDB: 8DMJ (unpublished),56,95-97). This conformational shift leads to an approximate 20% reduction in head radius (from ~110 Å to ~93 Å) and an extension of the protein's long dimension by 60 Å (Fig. 16 A, B). Despite the fusion peptide (FP) (residues 115-140) being part of the FECTOsequence, the absence of an observable density for the fusion peptide (FP) could be due to several factors: the FP's inherent structural disorder or its ability to adopt multiple conformations.

[0207] Alignment of the post-fusion structure of F2MECTOwith its pre-fusion counterpart revealed that like other paramyxoviruses, domain I (residues 290-373) and N-terminal part of F2 undergo the least rearrangement during fusion and provide a structural hinge about which the movement and rotation of other domains occur (60,98, Fig. 4C). In contrast, there is a substantial spatial and structural rearrangement of domains II (F1 residues 374-426) and I (F1 residues 115-287 and F2 residues 50-104) (Fig. 4D, E, Fig. 15 B). Although the majority of domain II retains the immunoglobulin-like fold present in the pre-fusion conformation, residues 434-446 of this domain as well as the linker connecting domain II to HRC (residues 447-456) elongate as HRC transitions from the “down” state to the “up” state as fusion proceeds. As a result, the entirety of domain II shifts 4.3 Å toward domain I.

[0208] The most profound conformational change from pre- to post-fusion occurs in the FP and HRN. Structural rearrangement within these regions includes the refolding of HRN from several disjointed helices and ^-strands to one elongated helix, exposure of the buried fusion peptide, and a 52° rotation around residue T287 (Fig. 4D). Overall, the conformational transition from pre- to post-fusion involves dramatic refolding of the FP and HRN, retention of the globular domain structure, but repositioning of the domains relative to their pre-fusion positions.

[0209] The significant conformational shifts and repositioning of the three components of the tripartite mAb 77.1 epitope clarify why 77.1 is unable to recognize F in its post-fusion state (Fig. 4F). Specifically, the spatial rearrangement between domain II and the C-terminal heptadrepeat (HRC), coupled with the movement of domain II relative to domain I, leads to the dislocation of the mAb 77.1 epitope and disrupts recognition of F. mAb 77.1 interrupts the fusion cascade, leading to trimer disintegration.

[0210] The structure of mAb 77.1 in complex with F, coupled with the post-fusion F structure, clearly demonstrated the prefiision conformational specificity of the epitope. However, the cell biology mechanistic assay suggested that mAb 77.1 acts on F at an intermediate state - after fusion has been activated but before forming the six-helix bundle.

[0211] To gain a clearer understanding of the mechanism by which mAb 77.1 inhibits viral entry, Applicants sought to investigate its impact on FECTOstructure and stability. Specifically, Applicants considered two main hypotheses: 1) mAb 77.1 destabilizes the prefusion state of FECTO, thereby facilitating its transition to the post-triggered state, or 2) binding of Fab 77.1 induces conformational changes in FECTO, influencing the refolding cascade. To test these hypotheses, Applicants investigated the structural effects of Fab 77.1 binding on FECTO, as well as assessed the influence of Fab 77.1 on thermal stability via DSF.

[0212] Alignment of the structures of FECTOin complex with either [FIP-HRC]2-PEG11or Fab 77.1 demonstrated high structural conservation, with an RMSD of 0.79 Å over all Cα or 1.23 Å over all atoms. Residues involved in the mAb 77.1 epitope showed only minor differences between structures, with fusion peptide residues L123 and V125 having an RMSD of ~2.2 Å over all atoms (Fig. 16 A, B). Hence, Fab binding only marginally alters the conformation of FECTO. In line with this comparison, Applicants found that Fab 77.1 increases the thermostability of F from a Tmappof 47.6 (± 0.1) °C to 51.6 °C (Fig. 16 C). While this stabilization was not as significant as that of [FIP-HRC]2-PEG11(56.2°C), it nevertheless suggests that mAb 77.1 does not destabilize F.

[0213] To further elucidate the mechanism of action mAb 77.1, Applicants turned to negative stain (NS) EM to directly visualize the molecular events following F activation in the presence of Fab 77.1. FECTOwas complexed with excess Fab 77.1 for 4 hours at room temperature. Subsequently, the mixture was incubated at 50 °C (above the Tmappof F but below that of the F-Fab 77.1 complex) for 1, 5, 7.5, or 10 minutes. Samples were then rapidly cooled on ice for 5 minutes and applied to negative stain grids.

[0214] NS EM imaging of the 1, 7.5, and 10-min time points showed a mixture of states including FECTO-Fab 77.1 complex, FECTO in post-fusion conformation and new, previously unobserved conformations and those were the most apparent in the 5-min time point (Fig. 6A, Fig. 17). Specifically, Applicants observed a subset of complexes where three Fab moleculesremained bound to FECTO, albeit with significant changes in the conformation of FECTOin which the head appears to adopt a relaxed, open state, leading to the separation of the protomers and potentially refolded HRN and FP domains. Additionally, another state was observed where one of the protomers had dissociated from the trimer with the remaining two protomers, each flanked by a Fab, forming a “V” -shaped structure. Lastly, Applicants also identified 2D classes where only a small rigid fragment of FECTOremained bound to Fab 77.1 (Fig. 6A).

[0215] To further validate these findings, Applicants re-examined the cryo-EM dataset of the FECTO-Fab 77.1 complex, where similar classes were noted, particularly those of Fab bound to a small fragment of F (Fig. 6B, C Fig. 11 A). A map of this class was successfully resolved to 7 Å resolution. Fab 77.1 was first docked into the apparent Fab density. Then, the Fab portion of the FECTO-Fab trimer was aligned to this docked model. The density not attributable to Fab aligned well with the mAb 77.1 epitope on FECTO(i.e., domain II, part of domain I, and the N- terminal part of F2) (Fig. 6C).

[0216] To further dissect how mAb 77.1 modulates F conformational rearrangements, Applicants assessed the binding of a post-trigger-specific antibody (mAb 19GD6, anti-Trig) to cell-surface expressed F, in the presence and absence of mAb 77.1". Although the specific epitope of this mAb has yet to be elucidated, previous studies have shown it binds after triggering has occurred. F-transfected cells were incubated with mAb 77.1 or diluent media alone, heated to 55 °C for 30 or 60 mins, and stained with mAb 19GD6. The presence of mAb 77.1 led to increased detection of post-trigger F epitopes, corroborating the earlier findings from negative-stain electron microscopy (Fig. 6D). These collective results imply that the antibody's mechanism of action involves disrupting the F trimer upon activation, thereby revealing post-trigger epitopes possibly through trimer fragmentation.

[0217] Compiling these results with previously obtained data, Applicants propose a model detailing how mAb 77.1 effectively neutralizes MeV. In the canonical fusion cascade, the H-F complex engages either CD 150 or Nectin-4 receptors, triggering structural changes in the H protein that lead to F protein activation (Fig. 6E). Subsequent refolding steps lead to a postfusion conformation, driving membrane fusion. The data indicate that mAb 77.1, while binding to the pre-fusion state of F, does not prevent F activation. Structural changes in domain III and HRN and imposed conformational constraints of domain I and domain II by mAb 77.1 result in protomers separation and trimer relaxation which ultimately disrupt the FECTOtrimeric structure, except for the fragment stabilized by mAb 77.1 (Fig. 6F).

[0218] The model provides a mechanistic basis for the Fab 77.1 -bound pre-fusion state observed in cryo-EM data and explains the outcome of the cell biology mechanistic assay. Moreover, Applicants postulate that the post-fusion conformation observed in this study, a critical step in the canonical fusion pathway, is not part of the mAb 77.1 -modulated refolding cascade. This hypothesis is supported by the absence of any post-fusion states in the cryo-EM dataset for the FECTO-Fab 77.1 complex and the presence of Fab-stabilized FECTOfragments. Conclusion

[0219] MeV remains a global concern and source of considerable morbidity and mortality despite the availability of an effective vaccine. Notably, all MeV vaccines in use involve live- attenuated virus, which poses risks to those with weakened immune systems, such as cancer patients, those undergoing immunosuppressive therapy, pregnant women, and infants below 12-15 months. Furthermore, approximately 5% of those that can safely receive the live- attenuated vaccine do not develop protective anti-MeV responses. Alternatives to vaccination, such as antibody-based therapeutics, would provide an orthogonal line of defense that could be used in a broader population. However, such molecules are not yet available.

[0220] Nearly all anti-MeV monoclonal antibodies thus far described are directed against the receptor-binding protein H. The few anti-MeV F mAbs that have been isolated belong to one of two main antigenic groups, termed I and II100. Epitope I mAbs are potently neutralizing but susceptible to escape by naturally occurring MeV variants. Among epitope II mAbs, 77.4 was the most potent. Here, Applicants show the human-chimeric mAb 77.1 retains this potent inhibition of F-mediated fusion in vitro, and demonstrate that the mAb limits viral replication in vivo, even at a very low dose of O.lmg / kg (Fig. 1).

[0221] Several antibodies that bind the pre-fusion state of other paramyxovirus fusion proteins have been described (101-105PDB ID: 7UOP, 7UPA, 7UPB, 7UPK), and for the respiratory syncytial virus (RSV), one such antibody is in clinical use69,106,107(Fig 18). Comparison of mAb 77.1 and the 12 Fab-bound F structures currently deposited in the PBD reveals there are three, rather than two, major sites that anti-F antibodies target: the apex of the head domain, the face of the trimer where two protomers meet, and the base of the head domain (Fig 18 A-D). Like the two unpublished Nipah F-targeted mAbs, 2B12 and 1H1, mAb 77.1 binds the base of the head domain. However, its quaternary epitope spans across domains and protomers, rather than encompassing only domain n, as do 2B12 and 1H1. Regardless of the epitope, the suggested, and in some cases known, mechanism of action for each of thesepublished antibodies is to block fusion activation by locking F in the prefusion state. As such, Applicants hypothesized that mAb 77.1 inhibits viral entry similarly: by binding to pre-fusion F before it has been activated by H-receptor engagement on the target cell. This mechanism is in contrast to HRC peptide mimics, which interact with the F protein only after activation of fusion where the virus has attached to the cell membrane, but before fusion has been completed65,108. Unexpectedly, however, the cell biology mechanistic assay demonstrated that mAb 77.1 does not block the activation of fusion and the launch of conformational change, but instead, like HRC mimics, arrests the protein at an intermediate step along the fusion pathway (Fig. 2).

[0222] Paradoxically, the high-resolution cryo-EM structure of F2MECTObound to Fab 77.1 shows F is in the prefusion conformation instead of adopting a conformation more consistent with a fusion intermediate. Comparison of Fab 77.1-bound F2MECTOto other FECTOstructures, including the [FIP-HRC4]2-PEG11-F2MECTOcomplex detailed here, demonstrates little structural difference. The only exception is the fusion peptide, which is held in a slightly different position by the Fab (Fig. 16 B). Furthermore, the post-fusion structure of F2MECTO, the first available for MeV, shows that the tripartite mAb 77.1 epitope is fractured upon the collapse of HRC into the 6-helix bundle with HRN. Hence, mAb 77.1 cannot bind to postfusion F.

[0223] Curiously, the quaternary epitope of mAb 77.1 includes the fusion peptide of F, which extends from a neighboring protomer (e.g., FB) and onto the top of domain II of FA. However, the mechanistic assay indicates that the fusion peptide is released and inserted into the host membrane. Applicants note that the fusion peptide makes a relatively small contribution to the overall epitope. Only two of the sixteen hydrogen bonds are formed between the mAb and the fusion peptide (CDR Hl Y27 and S31 with L123 and V125, respectively). Based on PISA predictions, these interactions contribute just -1.5 kcal / mol of free energy upon complex formation as compared to the -8.6 kcal / mol released over the entirety of the epitope. Hence, the fusion peptide, which is located on the edge of the epitope, could potentially be extracted, leaving the antibody anchored to the N-terminal portion of F2 and the loop between domain II and HRC. The stronger remaining interactions would likely still preclude repositioning of HRC, which requires release of the domain II-HRC loop. The structures also suggest that the fusion peptide may not be the first region to refold upon fusion triggering. Instead, refolding of HRN from several disjointed sub-sections into an elongated helix couldbe the driving force for the refolding that, once initiated, pulls the FP from its location atop the neighboring domain n, like a ribbon from a box.

[0224] Indeed, using NS EM, Applicants are able to capture several distinct conformations of F-Fab complexes, including a relaxed and open trimer, still bound by three Fabs, disassembled trimer with only two promoters bound by Fab 77.1 and a single Fab bound to small, structured fragment. Upon further data mining of the cryo-EM data set used to solve the trimeric F-Fab structure, Applicants also identified a population of Fab bound to an ordered fragment of F, corresponding to the epitope, minus the fusion peptide.

[0225] Based on these biochemical and structural findings, Applicants propose a model outlining how mAb 77.1 provides protection against MeV. mAb 77.1 recognizes the F protein while it is in its pre-fusion state, implying that binding could occur while the virus circulates within the host, rather than during the virus-host engagement phase, unlike HRC derived peptides. Despite this, the F protein can still be activated by the H-receptor and initiate the canonical pathway for membrane fusion. However, the rigidity introduced by the binding of mAb 77.1 to the epitope, which includes domains I and n, as well as the linker region between domain II and HRC, prevents F from following its standard refolding route. Instead, the F trimer adjusts by separating its protomers to accommodate this rigidity. As the refolding progresses to undefined intermediate states, the F protomers continue to separate until only one remains, stabilized by mAb 77.1. This stabilized state may even induce in vivo specific neutralizing antibodies against the intermediate state, a hypothesis that Applicants can test in the future.

[0226] This newly understood mechanism of antibody-mediated defense may also apply to other antibodies specific for pre-fusion states. Previous studies focusing on such antibodies against Nipah, Hendra, or Parainfluenza viruses have primarily examined binding kinetics, neutralization efficiency, and structural aspects without delving into the mechanistic actions of these antibodies101-103,105. Therefore, it is plausible that some of these previously studied antibodies may function similarly to mAb 77.1 (Fig. 18 D), although the exact mechanism of action has not been explored. In the case of the HPIV3 neutralizing antibody PIA174 Applicants have shown that it blocks fusion by stabilizing the pre-fusion state of the protein and escape variants are easily obtained104. Additionally, the only other antibodies shown to recognize a viral glycoprotein refolding intermediate state are the HIV-1 gp41 -specific monoclonal antibodies, 2F5 and 4E10. These rare antibodies were known to neutralize by targeting a transient conformation during the gp41 conformational changes involved inmembrane fusion. However, the precise epitopes and structures contributing to their specific binding to the intermediate state had been unresolved109.

[0227] In conclusion, the research thoroughly characterizes a novel, highly efficacious prophylactic antibody against the MeV that uses a unique neutralization mechanism. This study is the first to provide a comprehensive analysis of the mechanism for antibody-mediated neutralization. The findings could have broader implications for understanding other antibodies that target the pre-fusion state of proteins. Furthermore, leveraging high-resolution cryo-EM reconstructions, Applicants have elucidated the two states of the MeV F protein, thereby paving the way for the next generation of MeV therapies that can be precisely tailored to accommodate the dynamic nature of F.Material and Methods

[0228] Plasmids. The genes of MeV IC323 or B3 hemagglutinin, fusion protein, nectin 4, and CD 150 (or SLAM) proteins were codon optimized, synthesized, and sub-cloned into the mammalian expression vector pCAGGS. A codon-optimized fusion protein sequence derived from the Measles virus strain Ichinose-B95a (Taxonomy ID: 645098) was synthesized by GeneScript and subcloned into the pcDNA3.1 vector. Stabilizing point mutations (E170G and E455G) previously characterized in SSPE and MIBE Measles strains were sequentially introduced using the Q5 Site-Directed Mutagenesis kit. The ectodomain construct was generated by removing the transmembrane region and cytoplasmic tail residues (496-550) using the Q5 Site-Directed Mutagenesis kit. To generate Drosophila S2 stable cell lines, fusion protein ectodomain construct E170G / E455G was subcloned from the pcDNA vector to the pMT-Puro vector, including a C-terminal enterokinase cleavage site-twin-strep-tag via NEB HiFi DNA assembly.

[0229] Cells. HEK293T (human kidney epithelial), Vero, and Vero-SLAM / CD150 (African green monkey kidney) cells were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Life Technologies; Thermo Fisher Scientific) and antibiotics at 37°C in 5% CO2. The Vero-SLAM / CD150 culture media were supplemented with Img / ml Geneticin (Thermo Fisher Scientific). Drosophila S2 cells were cultured in complete Schneider's Drosophila Medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U / mL penicillin and 100 jig / mL streptomycin) at 25°C without CO2or Lonza Insect Xpress Medium. ExpiCHO cells were maintained in ExpiCHO ExpressionMedium (Gibco) supplemented with 8 mM L-glutamine in a humidified incubator at 37°C with 8% CO2.

[0230] In vitro viral entry: Vero-SLAM cells were plated in 96-well plates (1 x 104cells / well). The following day, cells were treated with serial dilutions of antibody 77.1 (mAb 77.1); N-(3-cyanophenyl)-2-phenylacetamide, referred to as 3G, and MeV F-derived HRC4 dimeric fusion inhibitory lipopeptide (i.e., [HRC-PEG4]2-chol). Simultaneously, cells were infected with MeV WT B3-EGFP for 3 h at 37°C (150-200 pfu / well). After 3h, the medium was replaced with medium containing 2% of methylcellulose Avicel / 2x complete medium (1:1). After 24 h, GFP-infected cells were counted using a Cytation 5 (BioTek) and analyzed with Gen5 3.11 software (min object size 25 μm, max object size 135 μm, threshold 3,000). The samples were tested in three separate experiments performed with technical duplicates in five-fold dilutions in 96 well plates. The measurements from each plate were normalized using 100 nM of mAb 77.1, 25 μM of 3G compound, and 500 nM concentration of HRC4 as 100% inhibition. Data was analyzed in OriginPro 2023, where all biological and technical replicates were fitted with logistic regression function with shared IC50 and Hill slope values, and Al and A0 (highest and lowest signals) were used to normalize the data. Based on the normalized data, the percent of inhibition was calculated using the following equation: [1 - X]*100, where X is the fraction of the normalized signal.

[0231] Beta-Galactosidase (Beta-Gal) complementation-based fusion assay. The Beta- Gal complementation-based fusion assay was performed as previously described68. Briefly, HEK293T cells transiently transfected with nectin 4 or CD150 receptor-bearing cells expressing the omega subunit of β-Gal are mixed with cells co-expressing glycoproteins F and H and the alpha subunit of β-Ga. Cell fusion leads to alpha-omega complementation and is measured in the presence or absence of fusion inhibitory compounds. Fusion is stopped by lysing the cells and, after addition of the substrate (®The Tropix Galacto-Star™ chemiluminescent reporter assay system, Applied Biosystems), fusion is quantified on a Tecan Ml 000 microplate reader. Three biological replicates were recorded, and globally fitted with a logistic function in OriginPro 2023. Obtained parameters were used to normalize the data per sample and plotted with standard error calculated from all data points.

[0232] Cell surface staining with conformation-specific anti-F mAbs. HEK293T cells transiently transfected with viral glycoprotein constructs. The transfected cells were incubated at the temperatures and times indicated in the figures. Specific MeV F conformations were detected with mouse monoclonal antibodies (mAbs) followed by Alexa-488 anti-mousesecondary antibody (Life Technologies) and DAPI (4',6-diamidino-2-phenylindole; Thermo Fisher) staining. Plates were imaged with a Cytation5, and the relative fluorescence of antibody-bound cells was determined using BioTek Gen5.

[0233] Viral evolution assay: Vero-SLAM cells were infected with 100 PFU / well of MeV B3-EGFP in 96-well plates then incubated for 2 h at 37 °C and further treated with several concentrations of inhibitors to promote the emergence of escape variants. Viruses were collected after five days and passaged similarly several times as indicated.

[0234] In vivo experiments: Cotton rats. Inbred cotton rats (Sigmodon hispidus) were purchased from Envigo, Inc., Indianapolis. Both male and female cotton rats aged 5 to 7 weeks were used. For i.n. infection, 105TCID50of MeV (strain B3 wt EGFP) was inoculated intranasally to isoflurane-anesthetized cotton rats in a volume of 100 pl. Animals received either the indicated amount of mAb 77.1 or vehicle 12 hours prior to infection. Four days after infection, the animals were euthanized by CO2inhalation, and their lungs were collected and weighed. Lung tissue was homogenized with a glass dounce homogenizer. Serial 10-fold dilutions of supernatant fluids were assessed on Vero SLAM cells for the presence of infectious virus in 48-well plates using cytopathic effect (CPE) as the endpoint. Plates were scored for CPE microscopically after 7 days and the TCID50was calculated. The results are presented as TCID50 / g of lung tissue.

[0235] RBC fusion assay. RBC fusion assays were performed on HEK 293T cells transiently expressing MeV H Y17H HPIV3 T193A chimera and MeV F (T461I). Cell monolayers were washed three times with serum-free medium, placed at 4°C with 1% RBC in CO2-independent media for 30 min, then treated with different concentrations of inhibitors and placed at 37°C for 60 minutes. Zanamivir was added at a final concentration of 10 mM, and the cells were incubated at 4°C for 30 min, rocked, and the liquid phase was collected in V- bottomed 96-well plates to measure reversibly bound RBCs. The cells were then incubated at 4°C for 30 min with ACK-Lysing buffer, and the liquid phase was collected in V-bottomed 96- well plates to measure irreversibly bound RBCs. The cells were then lysed in lysis buffer and transferred to flat-bottom 96 well plates to quantify fused RBCs. The amount of RBCs in each of the three compartments (reversibly bound, irreversibly bound, and fused) was determined by absorption measurement at 410 nm.

[0236] FECTOpurification: Drosophila S2 cells were transfected with a plasmid encoding the ectodomain construct (pMT-puro-Fecto2M-EK-twin-Strep-TagII) following the manufacturer's protocol. On day 1, S2 cells were seeded in a 6-well plate at a density of 1.0 x105- 1.5 x 105cells / mL in 3 mL of complete Schneider's medium. On day 2, the cells were transfected with 2 μg of the plasmid using the Effectene (QIAGEN) transfection protocol. Selection with puromycin (6 μg / mL) started on day 5, at which point cells were transferred to a T25 flask. The cells were then gradually expanded to a T75 flask, with the medium replaced by Lonza Insect Xpress medium containing 6 μg / mL puromycin. Subsequently, cells were expanded to 2 x 1 L in 2 L shaking flasks, induced with 500 μM CuSO4 at a 1 x 107cells / mL density, and harvested four days post-induction. Cells were removed from the culture medium by centrifugation at 5,000 x g for 20 minutes. The medium was adjusted to pH 8.0 with 1 M HEPES, and Biolock (IB A) (2.4 mL / L of medium) and NaCl (to a final concentration of 800 mM) were added. The medium was loaded onto a StrepTrap HP 5 mL column (Cytiva), and the column was washed with high-salt HBS buffer (50 mM HEPES pH 8.0, 800 mM NaCl) until a stable UV absorbance at 280 nm was achieved. The protein was then eluted with a high- salt HBS buffer containing 5 mM d-desthiobiotin. Next, the eluted protein fractions were pooled, and the buffer was exchanged to high-salt HB S using a HiPrep 26 / 10 Desalting column. Finally, the purified protein was flash-frozen in liquid nitrogen. Protein concentration was estimated using the molar extinction coefficient at 280 nm (48,820 M-1cm-1).

[0237] Antibody expression: Monoclonal antibody mAb 77.1.1 was produced in the ExpiCHO cell line by following the manufacturer's high-titer transfection protocol using the pTRIOZ-mAb 77.1 plasmid, which encodes the heavy and light chains of the antibody. After seven days of expression, cells were removed from the culture medium by centrifugation at 4,000 x g. The medium was then adjusted to pH 8.0 with 1 M HEPES (pH 8.0) buffer and incubated overnight at 4°C with protein A agarose beads to capture the mAb 77.1. On a subsequent day, the beads were washed with 20 ml of HBS buffer, and the bound antibody was eluted using 15 ml 0.2 M glycine (pH 2.0). The eluate was immediately neutralized by adding 10% (v / v) 1 M HEPES buffer (pH 8.0).

[0238] To generate Fab fragments, mAb 77.1.1 was digested at Img / ml with 1% (w / v) cysteine-activated papain for 4 hours at 37°C in a water bath. The enzymatic reaction was halted by adding 0.5 M iodoacetamide to a final concentration of 50 mM.

[0239] Following the digestion, the reaction mixture was diluted 10-fold with 10 mM HEPES buffer (pH 8.0) to decrease salt concentration and subsequently loaded onto a 1 mL Mono S column pre-equilibrated with 20 mM HEPES buffer (pH 8.0). The Fab fragments' flowthrough was collected, while the Fc fragments were eluted using a single step with 20 mM HEPES buffer (pH 8.0) containing 2 M NaCl. The isolated Fab fragments were concentrated,and their buffer was exchanged for BBS buffer. The Fab fragments were stored at 4°C for further analysis and experimentation. Protein concentration was estimated using the molar extinction coefficient at 280 nm (mAb 77.1: 240360 M-1cm -,1Fab 77: 84,270 M-1cm-)1.

[0240] Formation of Fecto2M-Fab 77.1 Complex: The Fecto2M and Fab 77.1 complex was prepared by incubating approximately 5 μM of Fecto2M with a three-fold molar excess of Fab 77.1 at 4°C overnight. Following the incubation, the mixture was subjected to sizeexclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (Cytiva) in HBS buffer (50 mM HEPES, pH 8.0, and 150 mM NaCl). The fractions containing the FECTO-Fab 77.1 complex were collected, concentrated, and used on the same day.

[0241] Differential Scanning Fluorimetry (DSF) Assay: Differential Scanning Fluorimetry (DSF) was used to determine protein stability and evaluate variations in thermal denaturation profiles under distinct buffer conditions110. Each analysis was conducted using a 25 μL volume containing 2.5 μg of protein and 2.5 μL of a 1% SYPRO Orange solution (Thermo Scientific), with the remaining volume composed of the buffer being tested. Stability measurements for FECTO, FECTO-Fab 77.1, FECTO-[FIP-HRC]2-PEG11were performed in the HBS buffer. For pH screening, buffers of Sodium Acetate (pH 5.0), Bis-Tris (pH 6.0), MOPS (pH 7.0), HEPES (pH 8.0), and CAPSO (pH 9.0) were used, all at 50 mM. The salt screening was performed using 50 mM HEPES buffer (pH 8.0) with NaCl concentrations ranging from 0 to 2 M.

[0242] The samples were dispensed into a 96-well PCR plate (BIO-RAD Multiplat 96- Well PCR Plates, low profile, unskilled), and the plate was then sealed with a heat-sealing film (Bio-RAD Microseal 'B' PCR Plate Sealing Film) to prevent evaporation during the heating process. A thermal cycling procedure was initiated on a Bio-Rad CFX 96 thermocycler, with the temperature incrementally elevated from 25°C to 95°C at a rate of 1°C per minute.

[0243] Throughout the thermal cycling, SYPRO Orange fluorescence was monitored continuously with the wavelengths for excitation and emission set at 520 nm and 540 nm, respectively. Data analysis was automatically carried out in Bio-RAD CFX Manager software, and the first derivative of the melting curve was graphed against temperature. Each experimental condition was performed in quadruplicate, and the average derivative signal was calculated. The minimum value in the averaged derivative signal represented the apparent melting temperature (Tmapp) of the protein.

[0244] Bio-layer interferometry: BLI-based kinetic analysis was conducted using the OctetRed384 system. Anti-human Fab (FAB2G, Sartorius) capture sensors were loaded withFab at 15 μg / mL in 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, supplemented with 0.5 mg / ml BSA (1xHBSTE / BSA). Binding of F2MECTOto Fab-loaded sensors was performed with two-fold serial dilutions of F. Baseline and dissociation steps were carried out in IxKinetics buffer. Regeneration was carried out using 10 mM Glycine, pH 2.5. Reference sensors (those not loaded with Fab but dipped into F-containing wells) were subtracted from each dataset to account for non-specific binding of F. Fab-only sensors were used as sample references. Kinetic parameters (Konand Koff) and affinities (KD) were calculated from a nonlinear fit based on the 1:1 binding kinetic model of the data using the Octet Data Analysis HT software version 11 (Fortebio). Three replicates for each binding curve were performed.

[0245] Structural mapping of protein variants: The sequences for Measles morbillivirus Fusion (MeV F) proteins were acquired using a custom Python script using the BioPython library111,112. This script was used to query the Entrez protein database for “Measles morbillivirus Fusion.” All results obtained from this process were consolidated into a single fasta file.

[0246] The sequences were then filtered based on size, retaining only those longer than 400 and shorter than 600 amino acids. Subsequently, the Needleman-Wunsch algorithm inside BioPython was employed for pairwise alignment with the F WT sequence (Uniprot: Q786F3) to further curate the sequences. Any sequences with low alignment scores (lower than 0) were removed from consideration.

[0247] After this curative process, the filtered fasta file was globally aligned using the Clustal Omega software113. This global alignment was stored in a clustal format file, which was then used to calculate the sequence conservation for each position using the AL2CO program114. This primary sequence conservation was visualized and graphed using Matplotlib and Seaborn Python packages115,116.

[0248] Furthermore, structural sequence conservation was mapped onto the pre- and postfusion structures of FECTOusing ChimeraX 1.6, which also uses AL2CO117. Statistics regarding the point mutations at the 77.1 epitope were computed in Python based on the Clustal alignment.

[0249] Cell Surface Biotinylation: HEK293T cells were transfected with Measles B3 F variants (wildtype-wt, 436K, 436G, 436D, 37R) or Measles H and by incubating 2 μg / well of cDNA in OPTIMEM with 4 μg / well of Lipofectamine 2000 (Invitrogen 11668019) in OPTIMEM (Gibco 31985088) for 20 minutes at room temperature and then adding to a wellof a 6 well plate for 3 hours at 37° C. After transfection, cells were incubated with DMEM (Gibco 10569044) with 10% FBS (Gibco 16000044) and 1% Penicillin-Streptomycin (Gibco 15140122) at 37°C or 32°C for 24 or 48 hours. Cells were then incubated at 4°C with 3.3 mM NHS-S-S-dPEG4-biotin (Quanta Biodesign 10194) in DPBS (Gibco 14287080) for 1 hour before lysing with DH Buffer [50 mM HEPES (Gibco 15630080), 100 mM NaCl, 5 mg / mL dodecyl maltoside (Thermo Scientific 89903) in Milli-Q Water] supplemented with complete Protease Inhibitor Cocktail (1 tablet / 50 mL; Roche 11836145001). Biotinylated proteins were pulled down with Streptavidin Sepharose (Invitrogen 434341) for 16 hours at 4° C. Protein was eluted from the beads in reducing Laemmli SDS Sample Buffer (Boston BioProducts BP- 110R), boiled for 10 minutes, and run on a 4-20% Novex Tris-Glycine Protein Gel (Invitrogen WXP42026BOX). The gel was transferred to nitrocellulose using iBlot quick transfer method (Invitrogen IB23001). The blot was blocked (Invitrogen WB7050), treated with Anti-Measles F HRC (GenScript; Rabbit) and alkaline phosphatase-conjugated anti-rabbit secondary antibody (Invitrogen WB7105). The blot was then developed using NBT / BCIP Substrate (Invitrogen WP20001). Analysis of the blot was performed in GelAnalyzer 23.1 and the integrated intensity of the bands was normalized to the expression level of F wt at 37°C.

[0250] Post-triggered antibody recognition with and without 77.1: HEK293T cells were transfected using pCAGGS MeV B3 wt F. Specifically, 0.1 μg / well of plasmid was mixed with 0.3 μg / well of Lipofectamine 2000 (Invitrogen, Cat# 11668019) in OPTI-MEM (Gibco, Cat# 31985088). The mixture was incubated for 20 minutes at room temperature before being added to a 96-well plate and incubated for 3 hours at 37°C. Post-transfection, the cells were cultured in DMEM (Gibco, Cat# 10569044) supplemented with 10% FBS (Gibco, Cat# 16000044) and 1% Penicillin-Streptomycin (Gibco, Cat# 15140122) at 37°C for 24 hours. Cells were then exposed to either 250 nM of 77.1 antibody (Creative Biolabs, Cat# Custom-211420; human origin) or left untreated, both at 4°C. After a 30-minute incubation, the media was replaced. Subsequently, one plate was maintained at 4°C, another was incubated at 55°C for 30 minutes, and the last one was incubated at 55°C for 60 minutes. Afterward, the cells were cooled to 4°C and treated with a previously characterized mouse monoclonal antibody specific to the posttriggered state of measles F". Following a 60-minute incubation, the cells were stained with goat anti-mouse conjugated with Alexa Fluor 488 (1 μg / mL; Invitrogen, Cat# A32723) and goat anti-human conjugated with Dylight 594 (1 μg / mL; Abeam, Cat# ab97005). Cells were then counterstained with DAPI (1 μg / mL; Thermo Scientific, Cat# 62248) and imaged using an Agilent Cytation 5 system. Total fluorescence per well was quantified using BioTek Gen5Software. Background fluorescence was calculated from untreated wells and subtracted from the measurements. Data was plotted against the time of incubation at 55°C with standard error and fitted with logistic regression function.Grid preparation

[0251] Negative stain: Copper grids (400 mesh) coated with carbon film (CF400-Cu, Electron Microscopy Sciences) were used for negative staining. The grids were first subjected to glow discharge treatment using the easyGlow Glow Discharge Cleaning System (Pelco) for 15 seconds at 15 mA to enhance their hydrophilicity. Next, the protein sample concentration was adjusted to 0.01 absorbance units at 280 nm, and 3 μL of the sample was applied to the grid for a 1 -minute incubation. Subsequently, the grid was blotted to remove an excess sample and washed three times with 3 μL deionized water, blotting between each washing step. The grid was incubated with 3 μL of 1% uranyl acetate solution for negative staining. After two blotting steps, a final drop of the staining solution was added, followed by a 1 -minute incubation. The grid was then blotted once more to remove excess stain and allowed to air-dry.

[0252] Graphene oxide grids: In this study, Applicants used custom-made graphene oxide (GO) grids. The GO grids were prepared by diluting a commercial GO solution (2 mg / mL, Sigma-Aldrich) in deionized water to a final concentration of 0.2 mg / mL. This diluted solution was sonicated for 5 minutes in a water bath to ensure proper dispersion of GO layers. Next, large aggregates were removed via centrifugation at 500 x g for 1 minute. Finally, the supernatant containing the dispersed GO was collected by transferring the upper 50% solution to a fresh 1.5 mL microcentrifuge tube.

[0253] Quantifoil R2 / 2 grids were washed with ethyl acetate overnight and glow- discharged under the same conditions as the negative stain grids. Following this, 3 μL of the GO solution was applied to the grids and incubated for 4 minutes. The grids were subsequently blotted, rinsed with 20 μL of deionized water, blotted once more, and air-dried for at least 10 minutes.

[0254] On the same day of grid preparation, the dried GO grids were used for the vitrification of the protein samples. The Vitrobot Mark IV plunge-freezing system was operated at 4°C and a relative humidity of 100%. A 3 μL aliquot of the protein sample (0.2 - 0.5 mg / ml) was deposited onto the GO grid, followed by a 12-second incubation period. After the incubation, the excess sample was blotted away (blot force 0) for 3 seconds. The grid was immediately plunge-frozen in liquid ethane cooled by liquid nitrogen.

[0255] C-flat grids: C-flat 1.2 / 1.3400 mesh grids were glow discharged like GO grids and immediately used for sample freezing. A 3 μL aliquot of 0.5 mg / ml protein sample was deposited onto the grid, immediately blotted for 6s, and plunge-frozen in liquid ethane cooled by liquid nitrogen.Cryo-EM data processing

[0256] FECTO2M-Fab 77: Movies were collected on Titan Krios operated at 300 KV using a K3 Direct electron detector (Gatan) in counting mode (0.66 Å pixel-1) at a nominal magnification of ×130,000 using a defocus range of -0.5 μm to -2.0 μm with Bioquantum energy filter slit set to 20 eV. Movies (LZW compressed TIFF files) were collected in EPU (Thermo) over a 3 s exposure with an exposure rate of ~15 e- pixel- 1 s-1, resulting in a total exposure of ~50 e- Å-2(1 e- Å-2frame-1). Downstream processing was done using Relion, homemade Relion scripts, and cryoSPARC118-121. Frame alignment was done in Relion 4 using MotionCor2 version 1.4.0 with 6x5 patches and a B factor of 150122. CTF determination was performed using CTFFIND4 version 4.1.14 using dose-weighted micrographs123.

[0257] Particles were auto-picked in Relion from randomly selected micrographs using the Laplacian-of-Gaussian algorithm, extracted at 512 px box size and Fourier-cropped to 64 px (5.28 A / px), and used for the 2D classification step. Next, particles resembling glycoprotein were selected and subjected to further rounds of 2D classification. The best classes showing different glycoprotein orientations were selected and used for the Topaz model training over 10 epochs124. Finally, the optimized model was used to pick particles in the whole dataset. ~1.1 M particles were extracted in Relion, imported in cryoSPARC, and subjected to 2 rounds of 2D classification. Classes resembling glycoprotein with bound fab were selected (148k particles), and particles corresponding to Fab and Fab2 were discarded. Selected particles from cryoSPARC were transferred back to Relion using a homemade script and re-extracted at 512 px with Fourier-cropping to 160 px (2.112 A px-1). Particles were again imported to cryoSPARC, where the initial model was generated. The obtained model was used for the 3D classification in Relion with 3 Classes.

[0258] During the first 25 iterations, global angular searches were performed using 15- degree sampling intervals. Once the classification reached equilibrium (followed by monitoring class distribution using Follow Relion Gracefully125), the 3D classification job was resumed with local angular searches of 7.5-degree sampling intervals and a 30-degree local angular search range. This procedure was repeated with a decreasing angular sampling interval and search range (kept double the angular sampling) until there was no improvement inestimated angle accuracy. Two classes showed high-resolution features and were selected for the 3D Refine protocol (142k particles). Next, refined particles were re-extracted at 512 px with Fourier-cropping to 320 px (1.056 A px-1) with re-centering coordinates. Finally, particles were refined, post-processed, and CTF and aberration refinement were performed. This procedure was repeated until there was no improvement in the reported post-processing resolution. Then, the Polishing step was applied, first by training on 10000 particles with a Fraction of Fourier pixels for the testing set to 0.5. The obtained trained parameters were used for particle polishing, and particles were extracted at 320 px (1.056 A px-1). Particles were again 3D refined, and CTF and aberration refinement were performed until there was no further improvement in the reported post-processing resolution. Finally, polished particles were imported to cryoSPARC, and a non-uniform refinement protocol was used to generate the final cryo-EM map in C3 symmetry. Then, particles were C3 symmetry expanded and refined using the Local Refine protocol in cryoSPARC to obtain the final map.

[0259] FECTO[FIP-HRC4]2-PEG11: Similarly, as for the FECT-OFab 77 structure, FECTO [FIP-HRC4]2-PEG11structure was recorded and processed following the same workflow. Number of particles and workflow overview is presented in Fig. 11C.

[0260] FECTOpost-fusion: FECTOwas incubated in lower salt buffer (50 mM HEPES pH 8.0, 150 mM NaCl) for 1 week in 4°C. In contrast to the other two datasets, Quantifoil R1.2 / 1.3 grids were used to freeze FECTO in post-fusion confirmation. 3 μL of 0.5 mg / ml FECTO was mixed with 1μL of 0.36 mM LMNG, blotted for 6s, and frozen in liquid ethane. The data processing was similar to the other two datasets, and detailed processing is shown in Fig 11 B. Model building

[0261] For the FEcro-Fab 77.1 structure, the obtained cryo-EM map was used to dock the previously published F X-ray structure (PDB ID: 5XYW87) using ChimeraX. Next, the antibody model was generated with an in-house implementation of ColabFold based on AlphaFold2126. Once the models were positioned, manual model building was conducted in Coot 0.9.8.7, sourced from the CCP4 package127,128. An automatically sharpened and density- modified map from PHENIX ResolveEM (using only half-maps) was the basis for model building129. A real-space refinement protocol followed manual model building in the PHENIX package, consisting of 10 macrocycles without any enforced restraints, and increased the number of Max iterations to 800130. Finally, water placement was carried out manually on a density-modified map. Water molecules were positioned where spherical density was visible at map RMSD > 5.0, no more than 2.7 Å from protein atoms. Interfaces were analysed usingChimeraX and PISA server131. Electrostatic maps, as well as the generation of F variants, were calculated in ChimeraX. Local map scales for FECTOwere calculated in OccuPy132.

[0262] A refined model of FECTO from FECTO-Fab 77.1 was used to dock in the FECTO[FIP-HRC4]2-PEG11map. FECTO in post-fusion conformation was first generated using homology modeling using SWISS-MODEL software (133-137) and manually rebuilt in Coot. Refinement and evaluation of the model was done as described above.

[0263] Heat-Induced Refolding of FECTO-Fab 77.1: A 7.5 μM solution of FECTOin HBS containing 800 mM NaCl was combined with a 1.5-fold molar excess of Fab 77.1. This mixture was then diluted fourfold using 50 mM HEPES (pH 8.0), resulting in a final NaCl concentration of approximately 150 mM. The complex was allowed to form at room temperature over 4 hours. A 15 μL aliquot of the formed complex was transferred to a PCR tube and subjected to heat treatment in a PCR thermocycler. The incubation times tested were 1, 5, 7.5, or 10 minutes at 50°C and 1 or 5 minutes at 55°C. After incubation, the samples were rapidly cooled by placing the tube on ice for 5 minutes. Negative stain grids were then prepared using the described protocol.

[0264] The acquired data were processed using cryoSPARC 4.3. After motion correction and estimation of CTF values, particle picking was performed using the blob picker. Subsequent 2D classification was performed using the previously described deep 2D classification protocol to segregate the particles into distinct classes.

[0265] Sequence analysis: Approximately 1400 MeV genomic sequences were obtained from the nucleotide NCBI database. CDS regions were manually annotated and curated to include only those between 400 and 600 amino acid residues in length with pairwise alignment scores greater than 0 as compared to the F WT sequence. The resulting ~820 sequences were aligned using Clustal Omega, and the conservation score was computed using AL2CO114. Conservation scores were placed on the 3D models of FECTOPREand FECTOPOSTin ChimeraX using AL2CO. Sequence variants were determined using a homemade Python script based on the multiple sequence alignment file.

[0266] mNGS and variant calling; mNGS was performed as previously described 138(PMID: 29970463). Briefly, RNA was extracted from 50 μL of viral culture using the Quick- RNA Viral Kit (Zymo) and treated with TURBO DNase I (ThermoFisher). cDNA was generated from the DNase-treated RNA using Superscript IV Reverse Transcriptase (Thermo Fisher) and random hexamers (IDT), followed by second-strand synthesis via Sequenase Version 2.0 DNA Polymerase. The resulting double-stranded cDNA was then purified with theDNA Clean & Concentrator Kit (Zymo). Libraries were constructed from 2 μL of cDNA using the Nextera XT kit (Illumina) and sequenced on 1x192 bp Illumina MiSeq runs. Sequencing reads were adapter and quality trimmed using Trimmomatic v0.38 (PMID: 24695404). Variants present at a frequency greater than 10% and coverage greater than 10x were identified with LAVA (github.com / greninger-lab / lava) using the MeV reference genome. All variants were manually confirmed by mapping sequencing reads to the same MeV reference strain in Geneious vl 1.1.4139. Those variants present in intergenic region between the matrix and fusion proteins as well as in homopolymeric tracts were excluded from the analysis. Sequencing reads are available under NCBI BioProject number PRJNA594952.

[0267] Statistical analysis: All data analysis and visualizations were conducted using a Python notebook integrating SciPy, Matplotlib, Pandas, Seaborn, and Statannotations packages 140-142 statistical significance was determined using the Mann-Whitney test. For p-value annotations, the following symbols were used: 'ns' indicates p <= 1,* represents 0.01 < p <= 0.05,** signifies 0.001 < p <= 0.01, and *** stands for 0.0001 < p <= 0.001.

[0268] Chemicals and peptides: N-(3-cyanophenyl)-2-phenylacetamide (also known as 3G) was commercially acquired from ZereneX Molecular Limited (UK). The purity of 3G was tested by high-pressure liquid chromatography (HPLC) and shown to be >95% pure.

[0269] Peptides were purchased from CPC, Ltd. Bromoacetyl cholesterol and bismaleimide cholesterol derivatives were custom made by Chamwood Molecular Ltd. Dimethylsulfoxide (DMSO), tetrahydrofuran (THF), N,N-diisopropylethylamine (DIPEA) were purchased from Sigma Aldrich, bismaleimide-PEG11from Quanta BioDesign. HPLC purification was performed on a 1100 Series Agilent HPLC system equipped with a UV diode array detector and a fraction collector using a reverse phase (RP.) Phenomenex Jupiter C4 LC column 300Å (150 x 21.2 mm, particle size 5 μm). MALDI-TOF analysis was performed on Bruker UltrafleXtreme MALDI-TOF-TOF instrument.The Antibody’s Complementarity Determining Regions (CDRs)

[0270] In the antibody against measles virus F protein, Applicants identified the CDRs in the heavy chain variable region and the light chain variable region, as follows (with the CDRs identified by underlining):Heavy chain:Light chain:DVOITQSPSYLAASPGETITINCRASKSISKYLAWYOEKPGKTNELLIYSGSTLOSGIPSRFRGSGSGTDFTLTISSLEPEDFAMYYCOOHNEYTLTFGGGTKLELKR

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[0272] Previously, Applicants assessed the neutralization properties of mouse monoclonal antibodies that specifically target the MeV F protein in its pre-fusion state. From the most potent antibody Applicants derived a human monoclonal antibody, referred to as 77.1 mAb, and Applicants show that it has viral neutralizing activity in vitro and in vivo. The 77.1 mAb, with variable domains (VL and VH) derived from 77.4 (mouse) was cloned in pTRIOZ-hlgGl vector (FIG. 19).77.4 VL Sequence77.4 VH Sequence77.1 mAb inhibits spread of MeV wild type (wt) and central nervous system (CNS)- adapted MeV variant

[0273] The antiviral activity of 77.1 mAb was assessed in MeV B3-eGFP-F wt and F L454W (CNS-adapted variant) live virus infection (Fig. 20). Since the virus bearing L454W F spreads in the absence of a known MeV entry receptor, Applicants compared the inhibitory efficacies in Vero cells expressing the known receptor SLAM / CD150 (Vero-SLAM) (Fig.l Panel A) and Vero cells (Fig. 20 Panel B) that express no known MeV receptor. After a 120- min infection with either MeV F_wt or F L454W viruses, cells were treated with the antibody and visualized at several time points (24, 48, and 72 h). Without treatment, the two viruses spread and formed large syncytia after 72 h in Vero-SLAM cells. In Vero cells (without receptor), MeV F_wt did not spread, while MeV F L454W formed syncytia that were smaller than those formed in the presence of CD150 / SLAM. The 77.1 mAb treatment blocked both viruses in the presence or absence of receptor.In vivo prophylaxis and post-exposure efficacy of 77.1 mAb

[0274] Antiviral efficacy for MeV in vivo was assessed in cotton rats (CR) as an infection model. The 77.1 mAb was administered subcutaneously 12 hours before infection (0.1 mg / kg or 1 mg / kg) or 24 hours post infection (1 mg / kg). Four days after infection, the animals wereeuthanized and the viral titers in the animals’ lungs were quantified. Treatment with 77.1 mAb reduced the viral titer in the lungs compared with the untreated control (FIG. 21).Materials & Methods

[0275] Recombinant 77.1 mAb Expression and Purification (Creative Biolabs, CBL).The gene for the 77.1 (human monoclonal antibody) mAb derives from 77.4 mAb (mouse monoclonal antibody) and it was synthesized using PCR-based methods and cloned into the pTRIOZ-hlgGl vector. The expression vectors of the antibody were transiently transfected and expressed in mammalian cells with chemically defined culture media. The mAb was purified by affinity chromatography, then ultrafiltration, and then subjected to 0.2 micron sterile filtration.

[0276] Viral spread inhibition in vitro. Vero or Vero-SLAM cells were plated in 12-well plates (2 x 105cells / well). The following day, cells were infected either with MeV B3-eGFP- F wild type and MeV B3-eGFP-F L454W (200 PFU / well for Vero-SLAM and 500 PFU / well for Vero cells) for 2 h at 37°C. The medium was replaced with complete medium and humanized monoclonal antibody referred to as 77.1 mAb (100 ug / ml for Vero-SLAM and 10 ug for VERO cells). After 72 h pictures were obtained using a Cytation 5 Cell Imaging MultiMode Reader (BioTek). Areas of infection were measured using BioTek Gen5 software on images randomly acquired from one experiment in duplicate. (Unpaired t test with Welch’s correction).

[0277] In vivo experiments in cotton rats. Inbred cotton rats (Sigmodon hispidus) were purchased from Envigo, Inc., Indianapolis. Both male and female cotton rats aged 5 to 7 weeks were used. For i.n. infection, 105TCID50of MeV B3-eGFP-F wild type in PBS was inoculated intranasally to isoflurane- anesthetized cotton rats in a volume of 100 μl. Four days after infection, the animals were euthanized by CO2inhalation, and their lungs were collected and weighed. Lung tissue was minced with scissors and homogenized with a glass dounce homogenizer. Serial 10-fold dilutions of supernatant fluids were assessed for the presence of infectious virus in 48-well plates using cytopathic effect (CPE) in Vero SLAM cells as the endpoint. Plates were scored for CPE microscopically after 7 days. The TCID50was calculated as described previously.Example 3 - Thermostable measles fusion glycoprotein as a new vaccine strategy

[0278] This grant can assess the efficacy of thermostable forms of the measles fusion protein to elicit neutralizing antibodies and induce a protective immune response in vivo. The proposed work can address two Specific Aims:

[0279] 1. Engineering thermostable measles F protein immunogen. The preliminary results illustrate that while the two initial stabilizing mutations yield prefusion F, additional mutations further anchor F and offer a remarkable level of thermostability. Hypotheses: Applicants hypothesize that these stabilized, pre-fiision measles fusion proteins can elicit strong neutralizing antibodies. Second, Applicants hypothesize that immunization with full- length, stabilized measles F protein can result in superior protection over immunization with ectodomain alone [e.g., by the inclusion of membrane-proximal external region (MPER) and absence of an immunogenic, but non-neutralizing “stump / base” at the bottom]. In this aim, Applicants can produce and characterize stabilized (full-length and ectodomain) measles fusion proteins with the improved thermostability substitutions (at CUIMC / LJI), and measure neutralizing antibodies against full-length and ectodomain forms of these measles fusion protein, including antiviral activity, structures and functions (at CUIMC / LJI).

[0280] 2. Evaluate the protection afforded by anti-F protein immune responses.Hypothesis: Applicants propose that stabilized measles fusion can induce protective immunity comparable to the current live vaccine (without risk of vaccine-enhanced disease). Proof of concept is first demonstrated by passive administration of anti-F neut. Ab.

[0281] 1. Passive immunization with neutralizing antibodies (at CUIMC / OSU).

[0282] 2. Assessment of in vivo immunization with stabilized measles fusion proteins(at CUIMC / OSU / LJI).

[0283] Research Strategy

[0284] A. SIGNIFICANCE

[0285] Al. Re-emergence of measles virus (MeV) in high-income countries. Infection by MeV remains a major cause of morbidity and mortality among young children worldwide, despite the availability of safe and effective MeV vaccines14. MeV was presumed to be eliminated in the U.S. in 2000 (elimination means ≥12 months without transmission)15. However, reintroduction of the virus has resulted in a resurgence of cases in recent years, causing repeated outbreaks16-20. Outbreaks in the U.S. and other countries with high vaccination coverage are often attributed to unvaccinated individuals. For example, the influence of antivaccination proponents in (social) media lead to vaccine hesitancy among parents, resulting in pockets of unvaccinated children28. However, a significant number of cases also occur in previously vaccinated people20-22, since up to 5% of vaccine recipients do not develop adequate protective antibodies, even after the recommended two-dose vaccination regimen, and also because vaccine-elicited immunity likely wanes over time23-27.

[0286] There is no effective antiviral regimen currently available for MeV, and insufficient coverage (defined as ≤95% vaccination rate by WHO) can lead to a rise in incidence in vulnerable populations. Indeed, MeV has already undergone a global resurgence: estimated global MeV deaths rose from 89,780 in 2016 to 207,500 in 2019. The situation may worsen since the SARS-CoV-2 pandemic reduced routine childhood vaccination coverage and may expose more vulnerable individuals to infection29. Without global eradication, a significant increase in number of MeV cases can be unavoidable30-33.

[0287] Of particular concern during this resurgence in MeV cases are the rising number of individuals with compromised immune systems, such as cancer patients, transplant recipients, persons with autoimmune disorders on immunosuppressive therapies, and human immunodeficiency virus (HIV)-infected individuals, in addition to pregnant women. In such immunocompromised people, MeV vaccination is often risky and inefficient9, 34-38; antibody responses are impaired, and even pre-existing immunity wanes39-46. Measles resurgence in other parts of the world, combined with increased vaccine hesitancy in the U.S., increases the risk of future measles outbreaks in the U.S. thus posing a risk to vulnerable patient populations (see CDC reports11, 45, 47-53)

[0288] A2. MeV pathogenesis. MeV is the most infectious virus known to humans. transmits via the air, and causes systemic infection. The wild-type (wt) virus can use two cellular receptors: CD 150 expressed by subsets of immune cells54and nectin-4 expressed by epithelial cells55, 56. MeV infection of a susceptible host starts in the respiratory tract. Alveolar macrophages and dendritic cells expressing the cellular receptor CD 150 are the initial targets57,58. The first MeV-infected cells then transmit the virus to bronchus-associated lymphoid tissues and / or draining lymph nodes, where the virus replicates and is disseminated to CD 150-positive B and T lymphocytes, with subsequent viremia59, 60. This stage of the disease leads to widespread infection of immune cells in primary, secondary, and tertiary lymphoid tissues. Infection of epithelial cells, which happens at the late stage of the viral life cycle in the host, is associated with aerosol and droplet transmission61, 62.

[0289] In healthy individuals, MeV infection paradoxically elicits both a strong MeV- specific immune response and a transient immune suppression. Studies in non-human primates have implicated that MeV preferential infection of memory lymphocytes is a direct cause of T- and B-lymphocyte depletion, causing the ‘immune amnesia’ in which pre-existing immune memory is partially erased and replaced by MeV-specific and bystander lymphocytes63, 64These observations were recently corroborated in an observational MeV cohort study inunvaccinated children13, 65, 66. Most virus-infected cells are cleared by cellular immunity within two to three weeks after infection. However, viral genomes can persist for several months after infection, even in the face of protective immunity67'69.

[0290] As a result of viral persistence, central nervous system (CNS) complications may occur soon after acute MeV infection in acute encephalomyelitis (AME), or years after infection in subacute sclerosing panencephalitis (SSPE)67, 70. Despite presumably functional cell-mediated immunity and high antiviral antibody titers, immune control of CNS infection is not achieved in patients suffering from SSPE71, 72. The third form of MeV-induced CNS disease - progressive infectious encephalitis or measles inclusion body encephalitis (MIBE) - occurs in immunosuppressed patients several months following MeV infection2, 3, 73, 74. MeV- associated CNS complications are highly lethal. Due to the perceived elimination of MeV from populations in high-income countries over the last few decades, there has been little incentive to develop novel intervention strategies. Further, there are currently no licensed antivirals for acute or persistent MeV infections.

[0291] A3. Scientific premise: Mechanism of MeV virus membrane fusion and its inhibition: the “fusion complex” in the CNS. This application builds on the recently published data and on several new findings from Applicant’s laboratories. The MeV hemagglutinin (H, the receptor binding protein) and fusion (F) envelope glycoproteins work together to mediate virus attachment and entry into target cells. Applicants refer to the HZF pairs of MeV as the viral “fusion complex” since these proteins, in the wild-type virus, act in concert. F is synthesized as a precursor (Fo) that is cleaved within the cell to yield the prefusion F complex comprising three C-terminal Fi subunits associated covalently with three N- terminal F2 subunits. This trimeric F structure is kinetically trapped in a metastable conformation, primed for fusion activation upon engagement of the H glycoprotein by the cellsurface receptor (discussed in A.2)54-56, 75'77. After receptor engagement by H, the pre-fusion F undergoes a structural transition, extending and inserting its hydrophobic “fusion peptide” into the target cell. During entry, F refolds into a “trimer of hairpins” post-fusion structure that brings together the N-terminal heptad repeat (HRN) and the C-terminal heptad repeat (HRC), triggering subsequent fusion of viral and cell membranes78'85. Before refolding to the postfusion state, the F protein undergoes a series of intermediate states. One of these intermediates is the putative target of antiviral HRC-derived peptides. Applicants have shown that in MeV field isolates from the CNS, the fusion complex of MeV can mediate viral spread in the CNS in the absence of canonical receptors2, 3, 74, 86, and that this specific CNS adaptation is due tomutations in the MeV F protein. The combination of acquired mutations in the F protein derived from the CNS includes a balance between those mutations that lead to decreased thermostability and others that partially “compensate” for the decreased stability. Applicants previously reported that the introduction of these compensatory mutations alone into the wildtype fusion protein increases the fusion protein stability2, 3, 74, 86.

[0292] Based on these results, Applicants produced MeV F proteins with an abolished cleavage site containing the first two identified stabilizing mutations (E170G3and E455G2), in full-length and ectodomain versions. Applicants describe the full-length protein in Fig.22 and the ectodomain version in Fig. 23.

[0293] The full-length MeV fusion protein bearing the mutations E170G and E455G maintains the pre-fusion state both in nanodisc and amphipol (Fig.22A). In the stabilized full- length structure (Fig.22B), the trans-membrane and intracytoplasmic domains are not yet ordered, buttheir presence is confirmed. Immunization with this full-length version resulted in protective immunity in vivo: Animals received 5 μg of the stabilized full-length MeV F twice. Immunized animals were protected against the subsequent viral challenge (Fig. 22C) and developed neutralizing antibodies (not shown). No vaccine-enhanced disease after the subsequent challenge with the virus was observed (not shown).

[0294] In addition to the full-length MeV F, Applicants have also produced an ectodomain form of the stabilized MeV F protein (Fig. 23). In Fig. 23A, Applicants report the structural information of the extra cytoplasmic domain (ectodomain, aa 1 to 495) of the MeV F in complex with the variable region (Fab) of a monoclonal antibody (mAb77.1, the humanized version of a mouse mAb 77.4 Applicants previously described1) which is protective in vivo (Fig. 23B). The ectodomain is thermostable (Fig. 23C), and its thermostability is further improved by a previously described fusion inhibitor8. Note that Fab 77 (77.4 is the mouse mAb and 77.1 is the humanized version) recognizes the prefusion state, and arrests fusion at the pre- hairpin intermediate state.

[0295] Based on the success of these two initial mutations (E170G and E455G), Applicants explored other stabilizing substitutions as well. Applicants report here (Table 6) that when these mutations are combined in the wild-type F background, their effect is additive to the point that the most stable F protein can be exposed at 60° C for a prolonged time and still maintain the pre-fusion state (assessed by recognition of pre-fusion antibody1, 8). For the experiment in Table 6, cells were transfected with F containing the indicated substitutions and 24 hours post-transfection, cells were exposed to either 55°C or 60°C over time. The presence of the prefusion epitope assessed (as in2, 3, 5) and the time in minutes at which 50% of the pre-fusion epitope was still observed (TSso) are reported.Table 6. Thermal stability of the MeV F mutant proteins detected by the binding of a mouse monoclonal antibody (mAb 77.41) specific for the pre-fusion conformation of F. Data are reported as an average of three independent experiments and are expressed as the time (minutes) at 55°C or 60°C that decrease the fraction of pre-fusion epitope to 50% (TSso) compared to time 0 (100%, after overnight at 37°C).

[0296] Based on the data from Table 6 and more recent findings5, Applicants can introduce additional mutations with the aim of obtaining highly stable and highly immunogenic MeV fusion protein. Note that the wild-type F are in post-fusion state (data not shown).

[0297] In summary, Applicants have obtained structural data for both full-length and ectodomain forms of stabilized MeV fusion F, both in complex with and without a neutralizing antibody, and demonstrated the preliminary efficacy of the stabilized fusion proteins as a safe, recombinant subunit vaccine strategy.

[0298] A4. Current gaps and. medical need: passive immunotherapy and vaccination modalities. Applicants propose developing both an active immunization strategy (Fig. 22C) and passive immunization strategy (Fig. 23B), based on educating the immune system to target the pre-fusion state of measles F.

[0299] The availability of the live attenuated vaccine means that most of the U.S. population has not been exposed to wild-type MeV. In the absence of exposure, the level of neutralizing sera in the overall population (herd) is decreasing over time25, 87leaving highly immunized countries actually susceptible to larger outbreaks45, 47-50, 53, 88, 89. This increasing susceptibility poses a particular threat to those who are too young or too vulnerable immunologically90to be protected by the live vaccine. “America’s success in eliminating measles produced a paradoxical increase in the vulnerability ” (quote from50).

[0300] The existence of an efficacious, live-attenuated MeV vaccine has almost led to the successful eradication of MeV. However, several political and socioeconomic factors have prevented complete eradication. The growing number of immuno-compromised people, who are most at risk of MeV infection, cannot be vaccinated themselves and are at risk during MeV outbreaks. A safer form of the vaccine, from stabilized recombinant subunit, is needed for these individuals.

[0301] Successful proof-of-concept for the stabilized, recombinant F approach can be found in the related respiratory syncytial virus (RSV). Several stabilized forms of fusion protein are in the clinical pipeline for evaluation of efficacy and safety, and one was recently approved for clinical use (see FDA news release May 03, 2023). In this application, Applicants assess a stabilized MeV F proteinaceous vaccine and test whether the unique stabilization strategy has the additional advantage of thermostability: maintaining the right structure and shape, even at high temperatures. This thermostability would also be a boon to MeV global eradication where poor or inconsistent cold chain compromises vaccine access and delivery, an issue that has not been solved by the dry formulation of the current vaccine91. A cold chain is a temperature-controlled supply chain that includes all vaccine-related equipment and procedures. The cold chain begins with the cold storage unit at the manufacturing plant, extends to the transport and delivery of the vaccine and correct storage at the provider facility, and ends with administration of the vaccine to the patient. With this project, Applicants expect to fill specific unmet needs for people at risk for MeV infection, both at home and abroad, who cannot be vaccinated or for whom a thermostable vaccine would improve access.AS. Scientific Rigor and Consideration of Relevant Biological Variables.

[0302] Scientific rigor: All experiments can be performed with robust sample numbers and repeated multiple times on different days. Appropriate statistical tests can be applied to evaluate efficacy, and all virological readouts. Orthogonal assays can be used to confirm the results. All reagents can be validated as outlined in “Authentication.” Detailed experimental protocols and primary data can be maintained and available to interested parties. Positive and negative controls (e.g., infected vs. non-infected, immunized vs. naive) are included in all experiments, as evidenced in the prior body of research and preliminary data.

[0303] Relevant biological variables: Some proposed experiments can be performed in cell culture systems, where the gender for established cell lines is not a variable. Where primary cells are used as test systems, the gender of origin can be noted and tracked as an experimentalvariable (the same applies for human induced pluripotent stem cells - hiPSC), and in vivo experiments can include both males and females.

[0304] B. INNOVATION

[0305] This proposal embodies three levels of innovation:1) Mechanistic: The in vitro mechanistic experiments revealed that several mutations, which arose to counterbalance F-destabilizing mutations in CNS-adapted fusion complexes, effectively stabilize the prefusion MeV virus F. By combining the stabilizing mutations in the protein, Applicants successfully obtained a novel, thermostable, prefusion MeV F, which can be made in full-length and ectodomain forms and have both been confirmed by cryo-EM.2) Conceptual: A previous attempt to stabilize the MeV F in its pre-fusion state involved disulfide stapling of MeV F ectodomain92. However, this artificial disulfide F protein aggregates even at 4°C (data not shown). Here, instead of artificially introduced mutations, Applicants used a combination of natural mutations isolated in human lethal cases of MeV encephalitis from viruses adapted to the human central nervous system2, 3, 74, 86. Among the natural mutations are some that decrease and others that increase thermal stability213, 74, 86. By combining only the naturally evolved stability-increasing mutations, Applicants created a remarkably thermostable form of MeV F (see Table 6; stable at 60°C). Additionally, Applicants can test the hypothesis that inclusion of the membrane-proximal external region (MPER), which is present only in the full-length protein, can lead to more potent neutralization activity than immunization with ectodomain alone, as has been shown for RSV93, HIV94-99, and Influenza100-102.3) Translational: The stabilized full-length MeV F elicits a protective immune response in a validated animal model for vaccine evaluation (cotton rat). Applicants propose that the new forms of MeV F incorporating the additional stabilizing mutations can retain immunogenicity (see Tables 6 and 7) over time and temperature to obtain a shelf and temperature-stable vaccine. Additionally, Applicants have developed a fusion inhibitor that can further stabilize the pre-fusion F protein (8and Fig. 23C), which could be used as an additional pre-fusion state stabilizer. Finally, Applicants propose that Applicants can obtain several human mAbs, for post-exposure prophylaxis as an alternative to the currently use of IgG preparation purified from hyperimmune sera. In contrast, monoclonal antibodies preparations consist of IgG only, have defined targets and optimized neutralizing activity per amount protein.

[0306] C. APPROACH

[0307] CC11..00 Engineering thermostable MeV F protein immunogen. Hypothesis:Applicants hypothesized that pre-fusion MeV fusion protein would elicit strong neutralizing antibodies. Applicants propose that immunization with full-length MeV stabilized F protein can result in superior protection vs. immunization using ectodomain forms of MeV, by (i) the inclusion of membrane-proximal external region (MPER) antibody epitopes, (ii) superior quaternary structure, and (iii) lack of potentially distracting “stump” at the very base of the ectodomain.

[0308] Rationale: The current MeV vaccine is based on live-attenuated MeV that has been proven safe and efficacious to prevent MeV infection and disease in the immune-competent host. Using this vaccination strategy, MeV infections could be controlled in the early 2000s. However, the resurgence of MeV infection in advanced countries, the persistently incomplete vaccine coverage in developing countries, and the growing number of vulnerable immunocompromised individuals without vaccination who can no longer rely on herd immunity for protection, in aggregate underscore the need for an additional vaccine that complements and solves the shortcomings of the current vaccine. The two major drawbacks of the current live-attenuated vaccine strategy are that (1) severely immunocompromised people cannot be vaccinated with the live vaccine and (2) that distribution of the vaccine requires a cold-chain and careful handling during reconstitution91. Additionally, the current vaccine elicits mostly antibodies against hemagglutinin (H): 80% of the human sera antibodies are against H, and only 20% against F, and those anti-F antibodies that are elicited by the current vaccine are poorly neutralizing103-105. Applicants believe that this demonstrates a gap in vaccine coverage. Applicant’s preliminary data show that antibodies against pre-fusion F are neutralizing and offer strong in vivo protection (Figs. 22 and 23). During CNS infections (which are most often associated with lethal outcomes) antibodies blocking F protein may be most effective by preventing viral spread early on1, 2, 74. Applicants hypothesize that a neutralizing anti-F response can be beneficial. Here, Applicants can test the hypothesis that antibodies elicited by immunization with F are protective. Applicants propose to develop a strategy based on the use of a stable pre-fusion F protein that could be stored and transported at room temperature and which could be safely used in the immune-compromised population. This strategy could boost existing immunity and develop de novo protection in those who have not been able to receive any current vaccine. F could be combined with H in an eventualvaccine; here, Applicants begin by testing the hypotheses that a stabilized F can lead to protective immunity, as has been observed for RSV.

[0309] Based on the published2, 3, 5, 74, 86and unpublished data (see Table 6) Applicants have identified several mutations that stabilize the pre-fusion state of MeV F and render the recombinant subunit thermostable. In aim Cl, Applicants can prepare stabilized F proteins, determine the efficacy of these highly stabilized F as immunogens, and compare full-length to ectodomain versions to evaluate the role of the membrane-proximal, transmembrane, and intracytoplasmic domains as neutralizing epitopes. For the related respiratory syncytial virus (RSV), a stabilized pre-fusion F ectodomain, lacking the membrane-proximal, transmembrane and intracytoplasmic domains, and stabilized using introduced disulfide bonds, has been recently approved for clinical use (see FDA site106, 107), providing a strong rationale for the strategy here. For RSV, only the pre-fusion state of the F protein induced neutralizing antibodies, whilst the post-fusion state does not93, 108-113. This finding is in stark contrast to the related human metapneumovirus (HMPV), for which neutralizing antibodies in vivo are directed against both the pre- and post-fusion state of the HMPV F114-118. Here, Applicants can also determine whether MeV neutralization requires antibodies only against the pre-fusion state of F (as in the RSV case) or if both pre- and post- fusion states contain valid neutralizing epitopes (as in the HMPV case).

[0310] Strategy and Plan: In the published2, 3, 74, 86and unpublished data (see, Figs. 22, 23 and Table 6), Applicants have shown that several naturally occurring amino-acid mutations in the MeV F protein stabilize the pre-fusion state of MeV F. These naturally occurring mutations are superior in their stabilizing properties compared to the previous artificial disulfide bonds introduced into the MeV F ectodomain92. In the preliminary data presented here, Applicants show that the combination of two previously published natural mutations (E170G3and E455G2) supports production of pre-fusion MeV F (Figs. 22 and 23), and that this stabilized prefusion F can be efficiently used to induce protective immunity in vivo (Fig. 22C). Newer data (Table 6) show that two additional mutations further stabilize the pre-fusion F. In this aim, Applicants can produce full-length and ectodomain versions of MeV F with these new mutations and assess their stability over time (up to 12 months) and temperature (up to 60°C).

[0311] Applicants have also shown that a humanized anti-pre-fusion F antibody is efficacious in vivo (see Fig. 23B). Using the current and newly generated MeV F protein (full- length and ectodomain versions), Applicants can elicit fully human monoclonal antibodies andassess the antiviral activity in ex vivo models that represent the main targets of MeV infection and complication (immune system, lung epithelia, and central nervous tissue) using both wt and central nervous system (CNS)-derived recombinant viruses1-3, 5, 8, 74, 86, 119-124to ensure that the antibodies Applicants are eliciting are neutralizing in the appropriate target tissue and could block CNS-adapted viruses. Additional viral evolution in the three ex vivo models described above can test the barrier to resistance to the neutralizing antibodies. Applicants hypothesize that the full-length version of the stabilized MeV F can induce stronger immunity (in terms of neutralizing activity and with a higher barrier to resistance) compared to the ectodomain version. In C1.1 and C1.2a, cryo-EM, and biochemical characterization can determine the precise structure of the stabilized F and determine the specific epitopes recognized by the neutralizing antibodies. In C2, the efficacy of the neutralizing antibodies and the vaccine strategy can be assessed in vivo in a relevant animal model for MeV infection.

[0312] Cl.l Production and characterization (biochemical and structural) of stabilized (full-length and ectodomain) MeV fusion proteins (at CUIMC / LJI). Hypothesis:Highly stabilizing mutations in the MeV F can allow for a long shelflife even in the absence of cold-chain. Applicants can utilize a total of six different MeV F: Wild-type F in ectodomain and full-length (post-fusion); E170GZE455G in ectodomain and full-length (pre-fusion); and two additional combinations of stabilizing mutations to be selected here, also in ectodomain and full-length (pre-fusion). The first four (blue highlight in Table 7) have been described and characterized above and are ready to be used (see Figs. 22 and 23). The fifth and sixth prefusion F proteins can be selected based on the results of this subaim. Applicants can begin with the four available MeV F proteins (pre- vs. post-fusion; ectodomain vs. full-length) to determine whether only pre-fusion F elicits / selects neutralizing antibodies, or if the post-fusion state can also induce neutralizing immunity. In Table 6 Applicants show a series of mutations that, when combined, further stabilized the MeV F. Applicants can produce F proteins from this set of mutations as follows: E170G / Y442H / E455G ectodomain and full-length; and E170G / Y442HZE455G / G506E full-length (yellow highlight in Table 7). The ectodomain ends at aa 495; therefore, the G506E stabilizing mutations can be used only for the full length. For the next versions of F, Applicants can test whether a V459I5that Applicants have also recently identified to stabilize F offers improved stability when combined with the other mutations. Applicants can produce the F proteins that incorporate stabilizing mutations as described in Table 7. Applicants can then assess the stability of the different F proteins and based on stability data (tested as in Fig. 23C; requiring small-scale preparation), Applicants can produce6 new F proteins (three full-length and three ectodomains) plus the two original ones (blue highlight in Table 7) for a total of 8 different proteins. The wt F proteins adopt the post-fusion conformation and can be tested only in immunization studies. Applicants can prepare multiple aliquots of the F proteins. Aliquots can be either lyophilized or left in solution. Both groups can be kept at different temperatures (-80, -20, 4, 30, 40, 50, 60°C) and for several times (1, 3, 6, 9, 12 months). Applicants expect -80°C to be the control for maintaining stability, and thus, Applicants can have several aliquots at -80°C to use for each time point and temperature. At the indicated time points and temperatures, Applicants can assess the amount of pre-fusion vs. post-fusion both structurally (using single particle negative stain and / or cryo-EM as in Figs. 22 and 23) and using conformation-specific monoclonal antibodies1-3, 74, 86. Preference can be given to the two MeV F proteins that show the highest stability of pre-fusion epitope after incubation at high temperature and for the longest time (see metrics in milestone).Table 7. F proteins to be produced and tested. F proteins Applicants have already in hand, tested in CRYO-EM and ready to use are wt and E170G / E455G. The E170G / E455Gfull-length and ectodomain are described in Figs. 22 and 23. E170G / Y4442H / E455G and E170G / Y442H / E455G / G506E have been assessed for stability (see Table 6).

[0313] Immunogenicity in mice: The most stable F proteins (one full-length and one ectodomain version), together with the wt F in its post-fusion state, as well the E170G / E455G pre-fusion full-length and ectodomain F that Applicants have already produced, can be used here and in C2.2 to assess their potential to elicit protective immunity in vivo. A total of 6 F proteins can be used for immunization: [3 full length and 3 ectodomain to include: one each post-fusion (wt), one each of the original 2 pre-fusion (E170G / E455G), plus 2 selected from the stability studies]. Mice can be immunized twice intramuscularly (i.m.) with 1 and 5 μg of protein. In Fig. 22 Applicants used 5μg of F protein given twice 21 days apart (day 0 + 21) togenerate a strong immune response. Adjuvant (for both mice and cotton rats): In the current experiments, Applicants used alum as an adjuvant. AS03 from GSK would be ideal125, 126. Applicants can attempt to obtain AS03 from GSK, or alternatively, Applicants can commercially acquire a similar formulation (i.e, AddaS03™, InvivoGen). Two weeks after last immunization (day 35) the neutralizing activity of the sera can be tested using live virus (both wt and CNS-adapted variants,2, 3, 74, 86. ). Sera can also be analyzed by ELISA for binding reactivity to the antigens used for immunization. Number of animals required: 7 proteins (6 F proteins + mock control) x 10 (5 each sex) x 2 doses (1 and 5μg) x 3 treatments (kept at -80°, kept at 4° for 1 month in solution, and incubated at the highest temperature and longest time and still be at 90% pre-fusion) = 420 mice. Note that in the case of the wt F, proteins can be in post-fusion state all the time and serve as control. If the sera are not neutralizing, the antigen and the immunized mice cannot be progressed further.

[0314] C1.2. Neutralizing antibodies against MeV F: antiviral activity, structural and biological characterization (at CUIMCZLJD. In this sub aim, Applicants can obtain monoclonal antibodies (mAb) by either mouse immunization (from Cl.l) and subsequent antibody humanization (as done above for 77.1), or by screening a de-identified human healthy donor library of hybridoma (which is currently frozen at CUIMC, from PBMC of an individual who was previously infected with MeV wt virus and now has a high neutralization titer). The overall objective is to obtain fully human monoclonal antibodies.

[0315] C1.2a mAbs against MeV F. Hypotheses: (1) Full-length MeV F can elicit more potent antibodies than the truncated ectodomain. (2) Post-fusion epitopes are not neutralizing. These hypotheses can be tested directly via mouse immunization and indirectly by assessing neutralizing activity of human hybridoma derived from PBMC.(i) Mouse: After assessing the neutralizing activity of the sera, a total of 12 mice from C1.1, two mice per antigen (one male and one female) with the most neutralizing sera against live virus can be used for hybridoma production.(ii) Human: Applicants have prepared a library of human hybridomas from a deidentified human donor with high neutralizing titer for MeV. The antibodies against F can be analyzed in this aim in order to address the specific hypotheses about protection from anti-F and anti-pre-fusion vs. anti-post fusion F.

[0316] The supernatant fluids from these hybridomas (murine and human) can be assessed for inhibition of viral entry. The supernatant fluids from human hybridoma (which may containa majority of anti-H antibodies103) can be further screened using a specific fusion assay mediated by MeV F in the absence of MeV H3, 8, 86. This can allow detection of specific F mAb and exclude H specific antibodies. The binding reactivity of the supernatant antibodies to the pre-fusion, or post-fusion, full-length or ectodomain antigens used for immunization (see Cl.l) can be assessed by ELISA. Applicants can use these results to determine if neutralization activity correlates with the recognition of pre-fusion and / or post-fusion F in cell-based ELISA, as described in2, 3, 86. Briefly, cells expressing F (full-length wild-type, full-length E170G / E455G, and the most stable full-length F selected in C1.1 from Table 7) can be kept at 4° C (a condition which maintains the pre-fusion state) or incubated at 55° C for 60’ (a condition which drives wild-type F into the post-fusion state). The supernatant fluids from the hybridomas can be assessed for their ability to bind to the resulting F on the cell surface. The data from these screens can indicate which hybridoma offers the most potent neutralizing activity and the viral target to which it binds (e.g., F in pre-fusion, and F in post-fusion). The most potent mouse and human antibodies identified can be sequenced. Mouse mAb can be humanized as Applicants have done for 77.41to obtain the 77.1 human antibody (Fig. 23). Synergy-additivity-antagonism between mAbs can be assessed as in1. The most potent humanized mouse- and human antibodies can be scaled up and tested (1) for potency ex vivo (as Applicants have done before1, 2) and (2) imaged using single particle cryo-EM to map their epitopes (see Fig. 23 for proof-of-concept using 77.1). The mechanism of action can also be assessed using established assays2, 5, 81 74, 119, 127, 128. Based on the ex vivo potency and epitope recognition, Applicants can select 8-10 mAbs for viral evolution studies in C1.2b.

[0317] C1.2b Ex vivo viral evolution under selective pressure. Hypothesis: Assessing the determinants of viral resistance early on in the drug discovery process can identify the most promising candidates prior to in vivo testing.

[0318] Rationale: Applicants have previously described a murine mAb 186a that although potent, was specific only for a subset of MeV strains1. Applicants have also described a mAb, 77.4, with a broader spectrum of activity, including efficacy against CNS-adapted MeV variants1. Applicants have humanized the broadly active mAb 77.4 into a fully human version termed 77.1 (Fig. 23). Consistent with its broad activity, Applicants have been unable to generate resistance mutations to 77.1 in cell culture despite several attempts. Applicants want to determine the potential for the new mAbs to be identified here to elicit resistance. Applicants propose here ex vivo (using B-cells, lung organoids, and brain organoids2, 119, 128) virus evolution to search for resistance mutations. The use of the ex vivo models has two mainadvantages: (1) the potential for viral evolution in a more authentic tissue, and (2) the time course of viral evolution can be of several weeks (especially for the organoids) allowing more time for the virus to evolve.

[0319] Strategy and overall plan: Applicants can attempt to elicit MeV variants resistant to the mAb in B-cells128, lung organoids119, and brain organoids2. Applicants can use wt virus and a virus bearing the L454W F (as example of CNS-adapted virus) mutation which spread most effectively in brain organoids and in vivo2, 5, 73, 74, 86. This knowledge can be used to identify the mAb(s) least likely to elicit resistance (for C2.1), and those able to block CNS- adapted variants, a feature important to avoid mAbs that may induce CNS-adapted viruses. Applicants can correlate escape mutation sites, lack of escape mutation emergence, epitope, and the form of F best recognized by the mAb (pre / post-fusion; stabilized or not), together with the data from C2.2 in order to select F best suited to passive immunization. Cotton rats can be used to assess the in vivo pathogenesis of viral variants that arise under the selective pressure of mAb treatment (e.g., whether resistance to mAb leads to a more pathogenic virus or not). The 8 mAb that performed best in C1.2a (e.g., lowest IC50and IC90ex vivo, with the most diverse epitope recognition) can be tested here for efficacy.

[0320] Generation of MeV variants resistant to mAb: Applicants can attempt to elicit MeV viruses resistant to the inhibitory effect of mAbs using protocols routinely performed in the lab5, 127, 129, 130. In these experiments, Applicants can use the B3 strain, which is the currently circulating strain of wt virus, labelled with GFP. Briefly, several dilutions of MeV B3-eGFP wt and B3-eGFP L454W (both viruses are in hand) can be passaged ex vivo in the presence of several concentrations of mAb (ranging between 5x and 20x the IC50) for four days in the B- cell ex vivo model, or for several weeks in the lung organoid and brain organoid models. Note that mAbs can be added after the initial infection to allow the viral polymerase complex to replicate and produce phenotypic variants for selection. Resistant viruses would spread even in the presence of mAb; spread of virus can be monitored by fluorescence microscopy. Yields of virus can be determined in immunoassays and / or by RT-qPCR5, 124, 127As the virus spreads in the presence and absence of antibody, the concentration of the mAb can be gradually increased, to obtain a population of resistant viruses. This strategy of applying selective pressure for viral evolution is similar to the informative experiments performed in the lab for neuraminidaseresistant variants129, small molecule inhibitor-resistant variants5, and antibody resistant viruses127.

[0321] Analysis of resistant variants in vitro: Mutant, resistant viruses can be plaque purified (if possible - see potential pitfalls below), then analyzed for their phenotype (plaque formation, resistance to inhibition by other small molecules and / or fusion inhibitory peptides5,127). Applicants can determine whether the fitness of each variant is similar to that of the parent virus, or whether the variants require the presence of inhibitor for viability. Viral RNA can be extracted and sequenced. Applicants can analyze resistant virus mutants by high-throughput sequencing and obtain complete genomic sequences. Sequences of the plaque-purified viruses and viruses from ex vivo growth can be compared to population-derived sequences generated during the duration of the selection experiments. This way Applicants can avoid neglecting potentially important sub-populations that may co-exist during or after the selection process. The method enables specific selection of viral sequences from complex sample types, yielding sequencing depth and coverage through orders of magnitude increases in specific read numbers2, 5, 127

[0322] If F (and / or H, or other viral proteins e.g., the matrix protein) contain mutations, Applicants can introduce the mutated genes into the expression vectors and evaluate the glycoprotein functions in the functional assays (F stability, F-triggering, fusion assays86, 131-134). If multiple mutations are found, site-specific mutagenesis can be used to introduce the mutations into the F (or H) background, and singly mutated genes can be analyzed for their phenotypes using the same in vitro assays. Location and conservation of the mutations can tell the extent to which the resistance mechanism(s) for different mAb are similar. If the mutants derived from the different mAbs are markedly different, Applicants can analyze the contributions of the specific mutations (in either F or H, or other genes if mutated) to dissect each contribution. Applicants also have the reverse genetics system in hand in case Applicants want to introduce selected mutations into the live virus.

[0323] Analysis of resistant variants in vivo: If Applicants identify resistant variants that grow well in vitro and ex vivo, Applicants can assess their in vivo fitness. Resistant variants’ pathogenicity can be compared in vivo to the parent MeV virus. Applicants can use 6 adult cotton rats (CR) to assess the in vivo fitness of each resistant variant74. Applicants anticipate testing a maximum of 5 variants plus control for a total of 6 viruses, (6 animals per group, n=36 CR, 18 each sex).

[0324] Viral titer: Virus isolation can be done from lungs as described above and in8, 74,135. Virus titration can be performed by plaque assay124. Samples can also be sequenced to assess viral evolution in vivo74.

[0325] Anticipated results and potential pitfalls: Applicants already have four of the six MeV F proteins required for the work proposed here and in C2.2. In Cl.l, Applicants can express additional F proteins based on the mutations in Tables 6 and 7 and select the additional two for C2.2. If stabilization at high temperature for prolonged times (e.g., 90% pre-fusion stability) is not possible by simply using the mutations from Table 6, Applicants can also add a specific fusion inhibitor that can further stabilize the pre-fusion ectodomain of F protein (8and Fig. 23C). Applicants also have in hand one fully human antibody (mAb 77.1) for C1.2b and C2.1. Applicants anticipate generating additional mAbs, but propose to start C1.2b with mAb 77.1 while Applicants wait for the new mAbs identified in C1.2a. Applicants present ample evidence that single-particle cryo-EM work can proceed seamlessly and that specific epitope recognition can be identified (Fig. 22 and 23). Applicants suggest that Applicants can correlate epitope recognition to antiviral potency and test the working hypothesis that mAbs that recognize the membrane-proximal domain (present in the full-length, but not ectodomain F) can be potent and offer a higher barrier to resistance. Applicants have a set of functional assays that can identify the specific mechanism of action (e.g., F-stabilization or destabilization, or blockage of transient intermediates). Since the recombinant MeV viruses are in hand and ex vivo models are established, the efficacy study should proceed seamlessly. Doseresponse assays can determine the dose-response in cell lines and ex vivo of all the mAb to move forward into ex vivo efficacy studies. The potency of the mAbs and their likelihood of eliciting resistance can be revealed using relevant ex vivo models. Since Applicants have already used these models, Applicants anticipate minimal technical difficulties2, 119, 124, 128Applicants considered the possibility that the matrigel in the lung organoid model might prevent full diffusion of the antibody; in this case Applicants could make a single fragment antibody to facilitate diffusion1or use human airway epithelial cells as in the previous work119,136-138

[0326] Mutations arising under the selective pressure of mAbs ex vivo should be highly informative about the relationship between viral fitness in authentic host models and the role of F-mediated entry, i.e., which features of the MeV glycoproteins are conducive to fitness. Data on the molecular basis of resistance can derive from the analysis of mutations in plaque- purified resistant viruses as well as the population analyses of potential viral quasi-species generated during the selection process. Selection in organoids may differ from selection in B- cells, and the unbiased approach can capture this eventuality as well.

[0327] The sequence alterations that arise through H and F mutation can be linked to functional alterations, and this information used to understand resistance mechanisms. For human parainfluenza virus type 3, enhanced fusion kinetics led to partial resistance to peptide inhibitors in vitro139. However, Applicants showed that mutations that increase fusion kinetics have a concomitant negative impact on growth in natural host tissue (human airway) and such resistance mutations are likely to significantly reduce fitness in vivo140. Applicants propose that by assessing these mechanisms early on in antiviral development, Applicants can avoid advancing antiviral strategies that could lead to more pathogenic viruses. It is possible that no infectious particles can be recovered from the ex vivo models (since MeV tend to spread via cell-to-cell infection in this tissues) and in this case Applicants can simply use the sequence data and introduce selected mutations in the reverse genetics system to obtain recombinant viruses bearing the mutated viral genes. Determining the ease of generation of variants and the fitness of MeV-containing resistance mutations can permit Applicants to predict the likelihood of evolution of clinically relevant resistant MeV (in future work). Applicants also consider the possibility that no resistance can be elicited. This is unlikely, since in the presence of small molecules, resistant variants of MeV have previously arisen5, 141-145. However, lack of resistance would suggest that the fitness cost is too high to generate a viable variant resistant to that specific mAb, and this mAb would therefore be an ideal candidate to move forward. The information gained here can be used to identify mAbs with the lowest likelihood of eliciting resistance, and to infer the best antigen for active immunization.

[0328] Discussion: From aim Cl.l, Applicants can determine the stability and longevity of the stabilized MeV F proteins, as well as their potential to induce neutralizing antibodies. Applicants can determine whether neutralization activity is endowed only by the pre-fusion state of the F protein, or if the post-fusion state can also elicit neutralization activity (disproving the working hypothesis). In C2.2, the same question can be asked in the context of viral infection in vivo. The hypothesis that the membrane-proximal domain and / or other determinants in the full-length F may elicit an additional or superior neutralization activity can be tested here in terms of neutralizing antibodies and can be correlated with the in vivo protection data in C2.1 (as shown in the preliminary data above see Figs. 22 and 23). The stabilized F protein that elicits the most potent response and that is most stable can be identified. Applicants can also obtain a set of diverse mAbs directed against several epitopes of the F protein. By assessing mAbs in ex vivo tissue, Applicants can identify viral inhibitors that are active in authentic tissue, and Applicants can also identify ideal probes for use in future studiesof pathogenesis. In C1.2b, Applicants can attempt to elicit viral resistance to the neutralizing antibodies obtained in C1.2a, to select the most promising neutralizing antibodies to move to C2.1. Since Applicants propose that ideally the final use of the mAbs can be in a cocktail, synergy and non-overlapping epitope binding are ideal features. Applicants expect to move to C2.1 a total of 4 mAbs (77.1 plus additional 3). The data from Aim C1 can directly inform the work of aim C2.1, and therefore aims C1 and C2 are interconnected. However, the availability of mAb 77.1 can allow aim C2.1 to proceed even in the unlikely eventuality that no better mAbs can be identified in Cl. Aim C2.2 can start with the four available F proteins and await the results of Cl for the addition of two F proteins of potentially greater stability.

[0329] C2. Evaluate the protection afforded by anti-F protein immune responses.Hypothesis: Applicants propose that immunization with stabilized MeV F can induce protective immunity comparable to the current live vaccine (without risk of vaccine-enhanced disease).

[0330] Rationale: Applicants have shown that immunization with pre-fusion, full-length MeV F efficiently protects cotton rats from MeV infection (see Fig. 22) and that a specific mAb against the prefusion state of F is highly efficacious in pre-exposure prophylaxis (see Fig. 23). Importantly no hyperresponsiveness (enhanced disease) was observed in the preliminary immunization studies. Absence of enhancement is critical to avoid the safety issues caused by vaccination with formalin-inactivated MeV (Fig. 24). Here, in aim C2.1, Applicants can evaluate mAb against prefusion F in passive immunization, assessing whether specific mAbs against the prefusion state of F are superior to general polyclonal human hyperimmune serum, the current standard of treatment, in blocking MeV infection. Next, in aim C2.2, Applicants can evaluate activity against pre-fusion F in active immunization, comparing immunization with stabilized, pre-fusion F to the current live attenuated vaccine in both immune-competent and immune-suppressed animals. Of paramount importance is that the proposed strategy does not cause hyperresponsiveness (a.k.a. vaccine-enhanced) disease, a feature which can be carefully tested here.

[0331] Virus-induced lung damage or vaccine-enhanced disease. Immunization of children with formalin (FA)-inactivated MeV virus has led to more severe disease after infection with wildtype virus (so-called atypical or vaccine-enhanced disease146). Atypical measles was characterized histologically by severe pneumonitis. The analysis of lung damage after primary infection or vaccine-enhanced disease after challenge of immunized cotton rats can be performed stepwise. On day 4 after infection, the left lung lobe is secured for virustitration. The right lung lobe is inflated with formalin, fixed for 3 days, processed and embedded in paraffin. After processing, slides are stained with H&E. For vaccine-enhanced disease, lung pathology is scored on a scale from 0 (nothing) to 100 (severe) for alveolitis, peribronchitis, perivasculitis and interstitial infiltrates147Using this method, Applicants have obtained data for vaccine-enhanced disease after immunization with FA-inactivated MeV in cotton rats. In Applicant’s hands, immunization of cotton rats with FA-inactivated MeV leads to the development of T cells as well as neutralizing antibodies which decline towards the time of challenge (data not shown). After MeV challenge, viral titers in lung tissue were 100-fold reduced (2.6±0.31og10 in immunized versus 4.7±0.21og10 / g lung tissue in naive animals). However, histologically severe inflammation was observed on days 2 and 5 (Fig. 24). If in the experiments proposed here, histological damage is observed, Applicants can establish a more detailed cytokine profile similar to that already established for atypical disease after RSV vaccination in cotton rats148. Based on the RSV study, Applicants can measure IFN alpha, beta, and gamma; TNF alpha; TGF beta; and IL-6 by qPCR (LightCycler RNA amplification kit with SYBR green I (Roche) using plasmids containing cloned cotton rat cytokine genes as standards. Data can be analyzed using LightCycler software version 3. Quantification can be based upon fit point analysis with arithmetic baseline adjustment. Melting peak and melting curve analyses can be performed using the polynomial calculation method149, 150.

[0332] Justification of the proposed animal model: MeV naturally infects only humans and NHPs63, 151-153_ Although Applicants have previously used small animal models to test in vivo efficacy of fusion inhibitory peptides, including a suckling mouse model transgenic for human CD 150, none of these models recapitulates the complex pathogenesis of morbillivirus infections74, 123, 124In another approach, Applicants have used cotton rats (CR), susceptible to wild-type MeV infection8, 122, 123. While neither model is ideal for MeV pathogenesis, cotton rats are suitable for MeV vaccination studies (both active and passive) because the virus grows in the respiratory tract, and they develop antibody and T cell responses, allowing measurement of the reduction of virus growth and development of antibodies. Therefore, Applicants propose that this model can likely reflect the efficacy of the proposed strategies and their potential shortfalls (e.g., vaccine-enhanced disease, see Fig. 24).

[0333] C2.L Passive immunization with neutralizing antibodies (at CUIMC / OSUZLa Jolla). Applicants have shown that the first humanized monoclonal antibody is highly efficacious in preventing MeV infection in cotton rats (see Fig. 23B). Here Applicants can test additional mAbs and conditions to evaluate the efficacy of the strategy in pre-exposureprophylaxis (C2.1a), post-exposure treatment (C2.1b), and in severely immunocompromised hosts (C2.1c).

[0334] Strategy and Plan: Testing in vivo antiviral efficacy of new human monoclonal antibodies directed toward MeV F protein can be performed using a tiered approach, compliant with 3R (Replacement, Reduction and Refinement) principles in animal experimentation, with tiers being separated by Go / No Go decisions. In C2.1.a, Applicants can perform a prophylaxis study. Antibodies that do not achieve adequate in vivo protection compared to the 77.1 (see Fig. 23) cannot be advanced to C2.1b. In C2.1b, Applicants can assess post-exposure antiviral efficacy by dosing antibody after exposure. Primary outcome parameters can be levels of viremia in the lungs. Antibodies that do not show efficacy in these experiments cannot be advanced to C2.1c. In C2.1c, Applicants can assess prophylaxis and therapeutic efficacy in immune compromised animals. Primary outcome parameters can be identical to those used in C2.1b. The work in aim 2 can start with the humanized anti-MeV F 77.1 (see Fig. 23 and1) and can proceed with additional 3 antibodies selected in C1.2.

[0335] C2.1a. Pre-exposure prophylactic efficacy. Groups of three (immunocompetent) cotton rats can be dosed with anti-MeV F antibody (using the humanized 77.1 mAb used in Fig. 23B as a positive control). Antibodies can be i.p. injected 1 day before inoculation with recombinant B3-eGFP wt virus. Four days after inoculation, the animal can be sacrificed, and viral titers can be assessed. The experiment can be performed twice to bring the number of animals per treatment group to six. The repeat experiment can not be performed if all treatments fail in the first experiment (i.e., the dose or time point does not prevent infection). Number of animals required: 4 antibodies (3 new mAbs plus mAb 77.1 as positive control) x 6 (3 each sex) x 2 doses (0.2 mg / kg body weight and 1.0 mg / kg body weight) = 48 CR.

[0336] C2.1b. Early post-exposure treatment Groups of three animals can be inoculated with recombinant B3-eGFP wt virus and treated with the most potent mAb (based on C2.1a, the one most effective at the lowest dose) and can be inoculated 1 or 2 days after infection. Sampling and follow-up can be identical to C2.1a, and the complete experiment can be performed twice to generate groups of six. Number of animals required: 4 Ab (2 most potent mAb from C2.1a+77.1 + immune serum) x 6 (3 for each sex) x 1 dose (highest efficacious dose from C2.1a) x 2 (time points) = 48 CR.

[0337] C2.1c. Prophylactic and therapeutic efficacy in immunocompromised animals.Cotton rat immune suppression: The protocol for immune suppression (I-S) in cotton rats is well established and has been extensively used for testing immunoprophylaxis for RSV154, 155.Briefly, CR can be treated with intraperitoneal (i.p.) injections of cyclophosphamide at a dose of 50 mg / kg three times weekly for 21 days prior to viral challenge, and I-S can continue for an additional 6 days (sufficient for the proposed experiments). To monitor the level of I-S during these experiments, CR can be bled for white blood cell counts and differential cell counts one day before viral challenge.

[0338] Prophylactic and therapeutic efficacy can be assessed by comparing viral titers four days post-infection (same as described in C2.1a) between mock- and antibody-treated animals. The experiment can be performed twice to bring the number of animals per group to six. Number of animals required: 4 samples (2 most potent mAbs from C2.1a + 77.1 + mock control) x 6 animals (3 for each sex) x 1 (route) x 2 (time points, before and after infection) = 48 CR.

[0339] Anticipated results and potential pitfalls: In this sub aim Applicants can define the prophylactic and therapeutic efficacy of passive immunotherapy in the cotton rat animal model (immune competent and immune suppressed) using antibody specific for MeV F. The primary outcome parameters are virological data obtained from the treated animals combined with the assessment of infection as in Fig. 23B. Applicants expect that prophylactic administration of the antibody can protect against infection (C2.1a) and can determine whether the new mAbs are superior to the 77.1 described in Fig. 23B and \ In C2.1b, Applicants can test whether passive immunoprophylaxis is also effective in a post-exposure regimen. In C2.1c, Applicants can test whether passive immunoprophylaxis is effective in immunosuppressed animals. Note that the main objectives of this sub-aim are to provide evidence that the strategy is effective in (i) prophylaxis, (ii) early post-exposure treatment and (iii) in immune- compromised hosts. The overarching goal of this sub-aim is to establish a passive treatment based on human mAbs to provide a well-defined composition without the inherent variability of depending on human hyperimmune serum obtained from healthy donors. A cocktail is likely an ideal eventual treatment. Here, Applicants can begin by testing them individually to assist in cocktail component selection. In future work Applicants propose to test such a cocktail of mAbs against MeV F that recognize different epitopes.

[0340] C2.2. Assessment of in vivo immunization with stabilized MeV F proteins (atCUIMC / OSU / LJI). Applicants have shown that the full-length pre-fusion MeV F protein in its pre-fusion state, given twice at 5μg, induces protective immunity in immune-competent cotton rats (see Fig. 22C). Here Applicants can test additional MeV F derived from aim C1.1.Applicants propose to compare a total of six MeV F proteins (four in pre-fusion and two in post-fusion, from aim C1.1, note that two pre-fusion (E170G / E455G, Figs. 22 and 23) and two post-fusion (wt) are already in hand and one full length in pre-fusion has shown to be protective, (see also Tables 6 and 7) given twice at two dosages (1 and 5 μg) in (C2.2a). The F that elicits the most potent neutralization without adverse effects can be tested in an additional range of dosages with the aim of decreasing to a single inoculation (C2.2b). In parallel, the F with the best protection can be assessed in severely immune compromised hosts (C2.2c). In addition to the virological data, sera from the cotton rats before and after immunization can be evaluated for neutralization activity and epitope recognition.

[0341] C2.2a. Pre- and post-fusion F protein immunization efficacy. Groups of three(immunocompetent) cotton rats can be dosed with MeV F in either the pre- or post-fusion state after i.p. administration, using the current live attenuated MeV vaccine as a positive control. The immunization can use two dosages (1 and 5 μg) and can be repeated twice (as Applicants have done for Fig. 22). Two weeks after the last immunization, animals can be infected and sacrificed four days post-infection. Viral titer can be assessed as in Figs. 22 and 23. Sampling of naive animals can be identical to that of the immunized animals, and infection can be detected based on virological data (including seroconversion). Sera of the animals can be collected before immunization, before infection, and after infection, and the neutralization titer can be measured. The experiment can be performed twice to bring the number of animals per treatment group to six. The repeat experiment cannot be performed if all treatments fail in the first experiment (i.e., the dose or time point does not prevent infection). Half of the lung can be used to assess viral titer, and the other half for the presence of hyper-responsiveness (Fig. 24). Number of animals required: 8 (6 F proteins + mock control + MeV antigen as in Fig. 22) x 6 (3 each sex) x 2 (doses: 1 and 5 μg) = 96 CR + 6 CR with live attenuated vaccine= 102 CR. Post-fusion F can be used here as well even in the eventuality that non-neutralizing sera is produced in mice in Cl.l. This is to avoid missing the possibility that post-fusion F vaccination may result in vivo protection despite not inducing neutralizing antibody156-161

[0342] C2.2b. Frequency of administration and stability testing. Groups of three animals can be inoculated with the two most efficacious F proteins (based on C2.2a, one full- length and one ectodomain, each the most effective at the lowest dose, highest neutralization titer, and without negative effect, based on the histology and cytokines and chemokineprofiles). Additionally, Applicants aim to assess whether a single dose would suffice to generate a response. Sampling and follow-up can be identical to C2.2a, and the complete experiment can be performed twice to generate three groups. Number of animals required: 5 (4 fusion proteins + mock control) x 6 (3 each sex) x 2 doses (5 μg, once or twice) x 3 (F preparations: kept at - 80, kept at room temperature, and incubated at the highest temperature and longest time based on C1.1) = 180 + 6 with live attenuated vaccine= 186 CR).

[0343] C2.2c. Efficacy in immunocompromised animals. Immunization efficacy can be assessed by comparing viral titer four days post-infection between mock- and immunized animals. The experiment can be performed twice to bring the number of animals per group to six. Number of animals required: 2 (most efficacious F protein from C2.2a and C2.2b+ mock control) x 6 (3 each sex) = 12 CR.

[0344] Note that the total number of animals listed is a maximal number (480). Applicants expect that certain immunization, dosage or frequency may fail at the first set and cannot be carried over to the next level of testing.

[0345] Anticipated results and potential pitfalls: In this sub aim, Applicants can define the efficacy of active immunization in the cotton rat animal model using MeV F in either the pre- or post- fusion state, in either full-length or ectodomain versions. The primary outcome parameters are virological data obtained from the immunized animals combined with the assessment of infection, neutralizing activity of the sera collected from the animals and lack of hyperresponsiveness (see, Fig. 24). Based on the earlier observation, there is no evidence of hyperimmune response, but it is possible that by increasing the dosage (to test activity of a single-shot immunization), this could be observed, and in this case, Applicants would return to the safer lower dosage. Applicants expect that administration of the pre-fusion state can be superior to the post-fusion state. Applicants expect some of the pre-fusion F described in Tables 6 and 7 to be superior to the current two-substitution F used in Figs. 22 and 23. These experiments can determine if immunization with recombinant F is as, more, or less protective than the current live attenuated vaccine. Many of the antibodies elicited against the live- attenuated vaccine are elicited against H, and antibodies against both H and F may be desirable in the final second-generation vaccine. This focused program analyzes F in order to begin with the fundamental questions of whether anti-F antibody is equally or more protective compared to the current vaccine. While a dry formulation of the current vaccine that does not require cold chain exists91the issue of the use of live attenuated vaccine persists for immune-compromisedpeople. In C2.2c, Applicants can test whether the strategy could be efficacious in immune- suppressed animals. It is particularly important to measure the response in the immune- suppressed animals because they would represent an indication of efficacy in the immune- compromised population. The main goal of this sub aim is to establish an active immunization strategy that could be given to both immune-compromised and -competent people and would not require cold-chain for distribution. How long the immunity derived from F immunization can last in comparison to the current vaccine can be addressed in future work, once Applicants determine the most effective subunit vaccination strategy in this application.

[0346] Aim 1 : Generate truncated and full-length pre-fusion F obtain structures to identify candidates that elicit more potent immune responses vs. first-generation stabilized proteins by a Y3Q2 Go / No-Go. Quantifiable metrics: Antigen stability / production quality: >10 mg / liter; stability at 4°C (>90% retention of activity at 30 days). If go, Applicants can test stability at 40°C (>45% retention of activity at 30 days). Stability can be assessed for lyophilized and solution forms of the antigen. Antigen epitope display: Retains all key complex, conformational epitopes (by ELISA or BLI) that are defined using neutralizing or protective mAbs. Immunogenicity characteristics: Elicits >1 / 10,000 titer of serum IgG binding to immunogen; >0.5% of cells produce IFNγ after peptide restimulation (T cell response); no reactogenicity in mice and cotton rat (weight loss of < 5% after immunization). If stabilized immunogens do not meet these metrics, Applicants can focus on...

Claims

CLAIMSWe claim:

1. A thermostable full-length paramyxovirus fusion protein (FFL), wherein the FFLcomprises at least one mutation that stabilizes the pre-fusion conformation, wherein the at least one mutation comprises at least one substitution mutation at a position corresponding to residue 170, residue 442, residue 455, residue 459, or residue 506 of measles virus (MeV) F protein.

2. The FFLof claim 1, wherein the FFLfurther comprises at least one mutation that removes or inactivates the furin cleavage site corresponding to the furin cleavage site of MeV F protein.

3. The FFLof claim 1 or 2, wherein the FFLcomprises the following mutations corresponding to MeV F protein:E455G; orE170G / E455G; orE170G / E455G / G506E; orE170G / E455G / V459I / G506E; orE170G / Y442H / E455G / G506E; orE170G / Y442H / E455G / V459I / G506E.

4. The FFLof any of claims 1-3, wherein the paramyxovirus is measles virus (MeV).

5. A paramyxovirus vaccine comprising the FFLof any of claims 1-4.

6. A method of inducing protective immunity and neutralizing antibodies, the method comprising administration of a therapeutically effective dose of the paramyxovirus vaccine of claim 5 to a subject in need thereof.

7. A paramyxovirus vaccine composition comprising paramyxovirus F protein or its ectodomain, wherein the paramyxovirus F protein or its ectodomain comprises at least one mutation that stabilizes the pre-fusion conformation, wherein the at least one mutation comprises at least one substitution mutation at a position corresponding to residue 170, residue 442, residue 455, residue 459, or residue 506 of measles virus (MeV) F protein.

8. The paramyxovirus vaccine composition of claim 7, wherein the at least one mutation corresponds to E170G, Y442H, E455G, V459I, or G506E in MeV F protein.

9. The paramyxovirus vaccine composition of claim 7 or 8, wherein the paramyxovirus F protein or its ectodomain comprises the following mutations corresponding to MeV F protein: E455G; or E170G / E455G; or E170G / E455G / G506E; or E170G / E455G / V459I / G506E; or E170G / Y442H / E455G / G506E; or E170G / Y442H / E455G / V459I / G506E.

10. The paramyxovirus vaccine composition of any of claims 7-9, wherein the paramyxovirus F protein or its ectodomain further comprises at least one mutation that removes or inactivates the furin cleavage site corresponding to the furin cleavage site of MeV F protein.

11. The paramyxovirus vaccine composition of any of claims 7-10, wherein the vaccine composition is a measles virus (MeV) vaccine composition.

12. A method of producing neutralizing antibodies against a paramyxovirus, the method comprising: administering to a subject the paramyxovirus vaccine composition of any one of claims 7-11; confirming the subject produces neutralizing antibodies from the paramyxovirus vaccine composition; isolating B cells from the subject; culturing the B cells to form hybridomas; and isolating the hybridoma that produces neutralizing antibodies against the paramyxovirus.

13. The method of claim 12, wherein the paramyxovirus is measles virus (MeV).

14. An antibody against measles virus F protein, comprising: one or more light chain variable region complementarity determining regions (CDRs) selected from the group consisting of: a first CDR comprising the sequence RASKSISKYLA, a second CDR comprising the sequence SGSTLQS, and a third CDR comprising the sequence CQQHNEYTLT; orone or more heavy chain variable region CDRs selected from the group consisting of: a first CDR comprising the sequence DY AWN, a second CDR comprising the sequence ISYTLTTGYNPSLKSRI, and a third CDR comprising the sequence RSGWLLPYWYFDV.

15. The antibody of claim 14, wherein the one or more light chain variable region CDRs are grafted onto a human antibody, and / or the one or more heavy chain variable region CDRs are grafted onto a human antibody.

16. The antibody of claim 14 or 15, wherein the light chain variable region comprises a first CDR comprising the sequence RASKSISKYLA, a second CDR comprising the sequence SGSTLQS, and a third CDR comprising the sequence CQQHNEYTLT, and / or the heavy chain variable region comprises a first CDR comprising the sequence DY AWN, a second CDR comprising the sequence ISYTLTTGYNPSLKSRI, and a third CDR comprising the sequence RSGWLLPYWYFDV.

17. The antibody of any one of claims 14-16, wherein the sequence of the light chain variable region is:DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNELLIYSGSTLQSGIPSRFRGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYTLTFGGGTKLELKR, and / or the sequence of the heavy chain variable region is:DVQLQESGPGLVKPSQSLSLTCTVSGYSITSDYAWNWIRQFPGNKLEWMGYISYTLT TGYNPSLKSRISITRDSSKNQFFLQLNSVTTEDTATYYCARSGWLLPYWYFDVWGAG TTVTVSS.

18. The antibody of any one of claims 14-17, wherein the antibody is encoded by GS66077- 1 pTRIOZ-hIgG1_HRV3C_77.1.

19. An antibody against measles virus F protein, comprising: one or more light chain variable region complementarity determining regions (CDRs) selected from the group consisting of: a first CDR comprising the sequence RATSSVVYMH,a second CDR comprising the sequence ATSNLAS, and a third CDR comprising the sequence QQWNSNPYT; or one or more heavy chain variable region CDRs selected from the group consisting of: a first CDR comprising the sequence NYWIE, a second CDR comprising the sequence EILPGNSYTNYNEKFRGKA, and a third CDR comprising the sequence PLSGNY.

20. The antibody of claim 19, wherein the one or more light chain variable region CDRs are grafted onto a human antibody, and / or the one or more heavy chain variable region CDRs are grafted onto a human antibody.

21. The antibody of claim 19 or 20, wherein the light chain variable region comprises a first CDR comprising the sequence RATSSVVYMH, a second CDR comprising the sequence ATSNLAS, and a third CDR comprising the sequence QQWNSNPYT, and / or the heavy chain variable region comprises a first CDR comprising the sequence NYWIE, a second CDR comprising the sequence EILPGNSYTNYNEKFRGKA, and a third CDR comprising the sequence PLSGNY.

22. The antibody of any one of claims 19-21, wherein the sequence of the light chain sequence is:the sequence of the heavy chain sequence is:

23. A humanized antibody comprising one or more CDRs from a single antibody selected from the group consisting of: C6, 9E1D3, H8, D9, and 10H2G3.

24. A method of neutralizing postfusion transition of measles virus (MeV) fusion protein in a subject, the method comprising administration of an effective amount of a first monoclonal antibody (mAb) selected from the group consisting of: G3, C6, F3, E11, 9E1D3, G4, H8, 10H2G3, D9, and Y10F, wherein the antibody is administered alone or in combination with mAb 77 to a subject in need thereof.

25. The method of claim 24, wherein the administration allows initial activation and refolding of the MeV F ectodomain and interrupts the refolding process of the F to prevent conformational changes required for membrane fusion and viral entry.

26. The method of claims 24 or 25, wherein the first mAb, alone or in combination with mAb 77, forms a complex with F.

27. A method of inhibiting measles virus spread through cells or treating measles virus infection in a subject, the method comprising administering to the cells or to the subject the humanized monoclonal antibody of any of claims 15-23.

28. The method of claim 27, wherein the humanized monoclonal antibody is administered onto a mucosal surface, such as by intranasal administration.

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