EP4633675A1

EP4633675A1 - Modified measles viruses for treating coronavirus infections

Info

Publication number: EP4633675A1
Application number: EP23904651.9A
Authority: EP
Inventors: Miguel A. MUNOZ ALIA; Stephen J. Russell
Original assignee: Mayo Foundation for Medical Education and Research; Mayo Clinic in Florida
Current assignee: Mayo Foundation for Medical Education and Research; Mayo Clinic in Florida
Priority date: 2022-12-15
Filing date: 2023-12-15
Publication date: 2025-10-22
Also published as: WO2024130083A1

Abstract

This document relates to recombinant measles virus (MeV) vectors and methods and materials related to using recombinant MeV vectors. For example, recombinant MeV vectors that can be used to deliver one or more immunogens associated with SARS-CoV-2 to cells within a mammal such that the mammal produces an immune response against SARS-CoV-2 are provided.

Description

MODIFIED MEASLES VIRUSES FOR TREATING CORONAVIRUS INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application Serial No. 63/432,809, filed on December 15, 2022. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2162W01_SL.xml.” The XML file, created on December 12, 2023, is 57000 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This document relates to multimeric immunogens and methods and materials related to using multimeric immunogens. For example, multimeric immunogens provided herein can be administered to a mammal (e.g., a human) such that the mammal produces an immune response against the immunogen.

BACKGROUND INFORMATION

SARS-COV-2 is an enveloped, non-segmented positive-stranded RNA virus belonging to the family Coronaviridae, genus Betacoronavirus, (Coronaviridae Study Group of the International Committee on Taxonomy of V. Nat. Microbiol., 5(4): 536-44 (2020)) that was identified on January 7, 2020, as the cause of a cluster of pneumonia cases related to a seafood market in Wuhan city, Hubei province of China (Zhou et al., Nature, 579(7798):270- 3 (2020)). Rapid dissemination of the virus resulted in the declaration of pandemic by the World Health Organization on March 11, 2020. Vaccination is still the leading public health prevention strategy to forestall the Coronavirus Disease 2019 (COVID-19) pandemic posed by SARS-CoV-2. Even though highly immunogenic and efficacious CO VID-19 vaccines have been deployed, the continued emergence of immune evasive variants of SARS-CoV-2 combined with the waning efficacy of SARS-CoV-2 vaccines represent a major global health challenge (Israel et al., medRxiv, 2021.08.03.21261496 (2021); Puranik et al., medRxiv, 2021.08.06.21261707 (2021); Beams et al., Viruses, 13(5): 854 (2021); and Chemaitelly et al, medRxiv, 2021.08.25.21262584 (2021)).

SUMMARY

This document provides multimeric immunogens and methods and materials related to using multimeric immunogens. For example, a multimeric immunogen can include two or more (e.g., two, three, four, or more) polypeptides that each include an immunogen and a multimerization domain (e.g., an immunogen fused to a multimerization domain), such that the two or more polypeptides can form a multimeric immunogen in vivo. In some cases, a multimeric immunogen can be presented on a self-assembling nanoparticle. For example, each polypeptide within a multimeric immunogen also can include a scaffold polypeptide, such that the multimeric immunogens can assemble (e.g., self-assemble) into a nanoparticle in vivo. In some cases, compositions (e.g., vaccine compositions) that contain one or more multimeric immunogens provided herein can have the ability to increase immune responses against the immunogen within a mammal (e.g., a human). This document also provides methods and materials for using multimeric immunogens provided herein to induce immune responses within a mammal (e.g., a human). In some cases, multimeric immunogens provided herein can be administered to a mammal (e.g., a human) such that the mammal produces an immune response against the immunogen(s). For example, compositions (e.g., vaccine compositions) that contain one or more multimeric immunogens provided herein (or nucleic acid encoding a polypeptide including an immunogen and a multimerization domain such that two or more polypeptides can form a multimeric immunogen provided herein) can be administered to a mammal (e.g., human) to increase an immune response against the immunogen within the mammal. For example, compositions (e.g., vaccine compositions) that contain one or more multimeric SARS-CoV-2 immunogens provided herein (or nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen and a multimerization domain such that two or more polypeptides can form a multimeric SARS-CoV-2 immunogen provided herein) can be administered to a mammal (e.g., human) having or at risk of developing a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) to treat the mammal.

As demonstrated herein, multimeric SARS-CoV-2 immunogens have the ability to increase an immune response (e.g., a neutralizing immune response) against multiple members of the coronavirus family within a mammal (e.g., a human). For example, administration of recombinant measles virus (MeV) vectors designed to express a SARS- CoV-2 immunogen fused to a multimerization domain, such that a trimeric SARS-CoV-2 immunogen is formed in vivo, protected mice from lethal toxin challenge after passive immunization. In some cases, such multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens) can be presented on a nanoparticle (e.g., self-assembling nanoparticle). For example, administration of MeV vectors designed to express a SARS-CoV-2 immunogen fused to a multimerization domain and a scaffold polypeptide, such that a trimeric SARS- CoV-2 immunogen is formed in vivo and such that trimeric SARS-CoV-2 immunogens assemble (e.g., self-assemble) in vivo into a nanoparticle presenting the trimeric SARS-CoV- 2 immunogens can improve the immunogenicity of the SARS-CoV-2 immunogen. Also as described herein, recombinant MeV vectors including (1) nucleic acid encoding a SARS- CoV-2 immunogen and (2) nucleic acid encoding a multimerization domain can be administered to a mammal (e.g., a human) such that cells infected by the recombinant MeV vectors express a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain, and such that two or more (e.g., two, three, four, or more) of the polypeptides including a SARS-CoV-2 immunogen fused to a multimerization domain can form a multimeric SARS-CoV-2 immunogen in vivo to increase an immune response (e.g., a neutralizing immune response) against SARS-CoV-2.

Having the ability to display an immunogen (e.g., a SARS-CoV-2 immunogen) in a multivalent format as described herein (e.g., as a trimeric immunogen that can optionally be presented on a self-assembling nanoparticle) can markedly improve the immunogenicity of the immunogen. For example, multimeric SARS-CoV-2 immunogens provided herein can produce immune responses against multiple lineages, clades, and strains of SARS-CoV-2 in mammals (e.g., humans), and can improve survival and minimize the impact of the infection. For example, MeV vectors including (1) nucleic acid encoding a SARS-CoV-2 immunogen and (2) nucleic acid encoding a multimerization domain can be used as a robust vaccine in the COVID- 19 pandemic to generate humoral immunity against both primary SARS-CoV-2 infections and recurrences after only a single immunization (e.g., a single nasal immunization).

In general, one aspect of this document features polypeptides including (1) an immunogen, (2) a multimerization domain, and (3) a scaffold polypeptide, where two or more of the polypeptides multimerize in vivo within a mammal administered the two or more of the polypeptides to form a multimeric immunogen, where two or more of the multimeric immunogens assemble in vivo to form a nanoparticle, and where the mammal produces an immune response against the immunogen. The mammal can be a human. The immunogen can include a coronavirus amino acid sequence. The coronavirus amino acid sequence can include at least a portion of a coronavirus spike (S) polypeptide. The portion of the coronavirus S polypeptide can be at least 232 amino acids in length. The coronavirus amino acid sequence can be a full-length coronavirus S polypeptide. The coronavirus amino acid sequence can be a coronavirus S polypeptide having a modified furin cleavage site, where the modified furin cleavage site is resistant to proteolytic cleavage. The modified coronavirus S polypeptide fragment can include two or more proline substitutions selection from the group consisting ofF817P, A892P, A899P, K986P, V987P, and A942P. The modified coronavirus S polypeptide fragment can include proline substitutions at K986P and V987P. The modified coronavirus S polypeptide fragment can include proline substitutions at F817P, A892P, A899P, and A942P The modified coronavirus S polypeptide fragment can include proline substitutions at F817P, A892P, A899P, K986P, V987P, and A942P. The coronavirus amino acid sequence can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 14. The coronavirus can be a betacoronavirus. The betacoronavirus can be SARS-CoV-2. The multimerization domain can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:21. The immunogen also can include an N-terminal leader sequence. The leader sequence can be an IgG K leader sequence. Three of the polypeptides can form a trimeric immunogen within the mammal. The scaffold polypeptide can be a C- terminal scaffold polypeptide. The scaffold polypeptide can be a neutrophil-activating protein (NAP) polypeptide. The NAP polypeptide can be a Helycobacter pylori NAP polypeptide. A polypeptide linker can be located between the multimerization domain and the scaffold polypeptide. The polypeptide linker can be a GlySer linker. The nanoparticle can include 12 of the multimeric immunogens.

In another aspect, this document features multimeric immunogens having two or more polypeptides, each polypeptide comprising (1) an immunogen, (2) a multimerization domain, and (3) a scaffold polypeptide, where two or more of the multimeric immunogens assemble />/ vivo within a mammal administered the two or more of the multimeric immunogens to form a nanoparticle, and where the mammal produces an immune response against the immunogen. The mammal can be a human. The immunogen can include a coronavirus amino acid sequence. The coronavirus amino acid sequence can include at least a portion of a coronavirus S polypeptide. The portion of the coronavirus S polypeptide can be at least 232 amino acids in length. The coronavirus amino acid sequence can be a full-length coronavirus S polypeptide. The coronavirus amino acid sequence can be a coronavirus S polypeptide having a modified furin cleavage site, where the modified furin cleavage site is resistant to proteolytic cleavage. The modified coronavirus S polypeptide fragment can include two or more proline substitutions selection from the group consisting of F817P, A892P, A899P, K986P, V987P, and A942P The modified coronavirus S polypeptide fragment can include proline substitutions at K986P and V987P. The modified coronavirus S polypeptide fragment can include proline substitutions at F817P, A892P, A899P, and A942P. The modified coronavirus S polypeptide fragment can include proline substitutions at F817P, A892P, A899P, K986P, V987P, and A942P The coronavirus amino acid sequence can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 14. The coronavirus can be a betacoronavirus. The betacoronavirus can be SARS-CoV-2. The multimerization domain can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:21. The immunogen further can include an N-terminal leader sequence. The leader sequence can be an IgG K leader sequence. Three of the immunogens can form a trimeric complex within the mammal. The scaffold polypeptide can be a C-terminal scaffold polypeptide. The scaffold polypeptide can be a NAP polypeptide. The NAP polypeptide can be a H. pylori NAP polypeptide. A polypeptide linker can be located between the multimerization domain and the scaffold polypeptide. The polypeptide linker can be a GlySer linker. The nanoparticle can include 12 of the multimeric immunogens. In another aspect, this document features nucleic acid encoding a polypeptide including (1) an immunogen, (2) a multimerization domain, and (3) a scaffold polypeptide, where cells within a mammal administered the nucleic acid express the polypeptide, where two or more of the polypeptide multimerize in vivo to form a multimeric immunogen, where two or more of the multimeric immunogen assemble in vivo to form a nanoparticle, and where the mammal produces an immune response against a coronavirus. The mammal can be a human. The immunogen can include a coronavirus amino acid sequence. The coronavirus amino acid sequence can include at least a portion of a coronavirus S polypeptide. The portion of the coronavirus S polypeptide can be at least 232 amino acids in length. The coronavirus amino acid sequence can be a full-length coronavirus S polypeptide. The coronavirus amino acid sequence can be a coronavirus S polypeptide having a modified furin cleavage site, where the modified furin cleavage site is resistant to proteolytic cleavage. The modified coronavirus S polypeptide fragment can include two or more proline substitutions selection from the group consisting of F817P, A892P, A899P, K986P, V987P, and A942P. The modified coronavirus S polypeptide fragment can include proline substitutions at K986P and V987P. The modified coronavirus S polypeptide fragment can include proline substitutions at F817P, A892P, A899P, and A942P. The modified coronavirus S polypeptide fragment can include proline substitutions at F817P, A892P, A899P, K986P, V987P, and A942P. The coronavirus amino acid sequence can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 14. The coronavirus can be a betacoronavirus. The betacoronavirus can be SARS-CoV-2. The multimerization domain can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:21. The immunogen also can include an N-terminal leader sequence. The leader sequence can be an IgG K leader sequence. The scaffold polypeptide can be a C-terminal scaffold polypeptide. The scaffold polypeptide can be a NAP polypeptide. The NAP polypeptide can be a H. pylori NAP polypeptide. A polypeptide linker is located between the multimerization domain and the scaffold polypeptide. The polypeptide linker can be a GlySer linker. The nanoparticle can include 12 of the multimeric immunogens. The nucleic acid can be in form of a viral vector. The viral vector can be a recombinant MeV vector. The recombinant MeV can include a modified H polypeptide. The modified H polypeptide can comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO:4. The recombinant MeV can include a modified F polypeptide. The modified F polypeptide can comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO:6.

In another aspect, this document features compositions including polypeptides provided herein, multimeric immunogens provided herein, and/or nucleic acid provided herein. The composition can include an aluminum hydroxide adjuvant.

In another aspect, this document features methods for inducing an immune response against a coronavirus in a mammal. The methods can include, or consist essentially of, administering polypeptides provided herein, multimeric immunogens provided herein, and/or nucleic acid provided herein to a mammal (e.g., a mammal having or suspected of having a coronavirus infection) under conditions where the nanoparticle in the mammal leads to induction of the immune response. The mammal can be a human. The coronavirus can be a betacoronavirus. The betacoronavirus can be SARS-CoV-2. The SARS-CoV-2 can be a B.1.17 (alpha), a Bl.351 (beta), a Pl (gamma), a B.1.617.2 (delta), a B.1.1.529 (omicron), a B.1.526 (iota), a B.1.617.1 (kappa), a C.37 (lamda), a B.1.621 (mu), a B.1.427/B.1.429 (epsilon), P2 (zeta), or any combination thereof. The administering can include a single administration. The administering can be a nasal administration. The immune response can be an IgG antibody response. The immune response can be an IgA antibody response. The immune response can be a Th 1 cell-mediated response. The immune response can be a Th2 cell-mediated response.

In another aspect, this document features uses of a composition comprising polypeptides provided herein, multimeric immunogens provided herein, and/or nucleic acid provided herein to induce an immune response against a coronavirus in a mammal.

In another aspect, this document features the polypeptides provided herein, multimeric immunogens provided herein, and/or nucleic acid provided herein for use in the preparation of a medicament for inducing an immune response against a coronavirus in a mammal.

In another aspect, this document features polypeptides provided herein, multimeric immunogens provided herein, and/or nucleic acid provided herein for use in inducing an immune response against a coronavirus in a mammal. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show that a full-length SARS-CoV-2 spike ectodomain protein elicits poor pseudovirus-neutralizing antibodies and non-existent T-cell responses. FIG. 1A) SARS- CoV-2-spike binding responses. Type-I interferon, human CD46 transgenic mice IFNAR - CD46Ge were vaccinated intraperitoneally at day 0 and 21 with 5 pg of purified SARS-CoV- 2 proteins adjuvanted with aluminum hydroxide gel (Alum-adjuvanted): full-length spike ectodomain (S1+S2), spike receptor binding domain (Sl-RBD), spike SI domain (SI), spike S2 domain (S2), and nucleocapsid (N). Serum samples were collected on days 21 (before second vaccination) and 49 to be assessed for spike ectodomain IgG binding antibodies by enzyme-linked immunosorbent assay (ELISA). FIG. IB) The IgG binding of serum from mice vaccinated twice with SARS-CoV-2 spike proteins and domains (SI, Sl-RBD, Sl+Sl, and S2) were also assessed by ELISA for binding to homologous or heterologous antigens. Serial five-fold dilutions were run and data was computed as area under the curve. FIG. 1C) Pseudovirus-neutralizing antibody responses. Neutralizing-antibody titers in mice vaccinated once (day 21) or twice (day 21 and day 49) with the indicated SARS-CoV-2 proteins were determined using pseudotyped viruses expressing the SARS-CoV-2 spike encoding the D614G amino acid change. Virus neutralization was plotted as percentage of relative virus infection over inverse of serum dilution. The inverse of serum dilution at with 50% inhibition of infection was achieved (EC50) was determined and plotted. Antibody titers below the lower limit of detection (LLoD) were replaced with 0.5x LLoD. FIG. ID) T-cell responses were elicited against SARS-CoV-2 spike. An enzyme-linked immunosorbent spot (ELISPOT) for IFN-y was performed on splenocytes isolated from mice vaccinated twice (on day 0 and day 21) and stimulated ex vivo with phorbol 12-myristate 13-acetate (PMA)Ziomycin or two separate pools of 15-mer, 11 -amino acid (aa) overlapping peptides comprising the SARS-CoV-2 spike (SI, aa 1-632; S2, aa 632-1273). The data are shown as IFN-y -secreting cells or spot-forming cells (SFC) per IxlO⁶ splenocytes. The values represent the geometric mean ± geometric standard deviation with each data point representing an individual mouse. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparison test (ns, not significant *, p<0.05; **, p<0.003, ***, p<0.0001).

FIGS. 2A-2C show that multimerization of SARS-CoV-2 spike enhances neutralizing antibody responses. FIG. 2A) A schematic diagram of the full-length SARS-CoV-2 spike and engineered full-length ectodomain spikes. Shown are some of the structural domains that include: the cleavable signal peptide (SP), N-terminal domain (NTD), receptor binding domain (RBD), S2 cleavage (685) fusion peptide (FP), heptad repeat 1 and 2 (HR1 and HR2), transmembrane domain (TM) and cytoplasmic tail (CT). The native furin cleavage site NSPRRARSVAS (SEQ ID NO:7) containing the amino acid sequence RRAR (SEQ ID NO:8) was altered to the sequence NSPGSASSVAS (SEQ ID NO:9) containing the amino acid sequence GSAS (SEQ ID NOTO) to resist proteolytic cleavage and six prolines were also introduced to increase stability. For example, a wild type (WT) sequence can include the amino acid sequence SRLDKVEAEV (SEQ ID NO: 11) and two prolines can be introduced to alter the amino acid sequence to SRLDPPEAEV (SEQ ID NO: 12) to increase stability. For example, amino acid substitutions F817P, A892P, A899P, and A942P can be introduced to increase stability. Further modifications include the C-terminal domain of the T4 fibritin (FoldON) placed at the C-terminus of the spike, and the neutrophil-activating protein (NAP) polypeptide of//, pylori preceded by a GlySer linker. FIG. 2B) SDS-PAGE and BN-Native gel analysis of the purified proteins. 1 pg of protein was separated by SDS-PAGE (4-12% Bis-Tris gel) or BN-Native (4-16% Bis-Tris) followed by Coomassie staining. FIG 2C) Representative electron micrographs of negatively stained purified proteins. Scale bars, 10 nm. FIG. 2D and FIG. 2E) Pseudovirus-neutralizing antibody responses. Mice were vaccinated once with either I pg or 5 pg of Alum-adjuvanted proteins, and neutralizing antibodies were determined using LV-SARS-CoV-2 (FIG. 2D) or VSV-SARS-CoV-2-S (FIG. 2E) pseudoviruses on serum samples collected 21 days post-vaccination. Antibody titers below the LLoD were replaced with 0.5x LLoD. Black dots represent individual mouse and bars and error bars depict geometric mean ± geometric standard deviation, respectively. Statistical analysis between groups was calculated with Two-way ANOVA with Bonferroni’s post-test (ns, p>0.05; ****, p<0.0001).

FIGS. 3A-3B show the generation of recombinant measles virus (rMeV)- Moraten resurfaced (MR) expressing SARS-CoV-2 spike antigens. FIG. 3A) Schematics ofthe MeV- MR vector with SARS-CoV-2 spike-based constructs inserted as an additional transcript unit (ATU). The top schematic shows the MeV genome consisting of the following genes from the Moraten vaccine strain: nucleoprotein, phosphoprotein, V and C accessory proteins, matrix, and large polymerase protein. The envelope glycoproteins were substituted for canine distemper virus fusion protein and a wild-type hemagglutinin (H) protein with deletion of 8 antigenic sites. The bottom schematics show modifications to the SARS-CoV-2 spike protein, including deletions of the transmembrane and/or cytoplasmic tail region as well as the substitution of the SARS-CoV-2 spike signal peptide by the murine IgG kappa leader sequence, followed by an HA tag. Among other modifications, H. pylori NAP was genetically fused at the extreme C-terminus of the spike and either preceded or not by a stop termination codon. Alternatively, a foldON trimerization domain was inserted between the spike and NAP. The amino acid sequence GSAS (SEQ ID NO: 10) can resist proteolytic cleavage. FIG. 3B) Expression of SARS-CoV-2 spike-based constructs from the rMeV-MR vector. Vero cell lysates and supernatants were analyzed by western blot two days after infection with the various spike-based measles vector constructs at multiplicity of infection (MOI) of 0.03. The antibodies used for immunodetection as well as the molecular weight of a standard are indicated.

FIGS. 4A-4D show that trimerization and stabilization of SARS-CoV-2 spike constructs augment the humoral antibody response. FIGS. 4A-4B) IFNAR -CD46Gc mice were vaccinated intraperitoneally at day 0 and 21 with IxlO⁵ plaque-forming units (pfu) of either rMeV or Vesicular Stomatitis Virus (VSV) expressing various spike-based constructs. Serum samples were collected on day 21 (before second vaccination) and day 42, and were assessed by ELISA for IgG binding to MeV-bulk antigen (FIG. 4A) and spike ectodomain (FIG. 4B). FIG. 4C) Pseudovirus-neutralizing antibody responses. Neutralizing-antibody titers in mice vaccinated once (day 21) or twice (day 21 and day 42) with the indicated recombinant virus were determined using pseudotyped viruses expressing the SARS-CoV-2 spike D614G as shown in FIG. 1C. Virus neutralization was plotted as percentage of inhibition of virus infection relative to virus incubated with negative mouse serum over inverse of serum dilution. The inverse of serum dilution at with 50% inhibition of infection was achieved (EC50). FIG. 4D) Shows the ELISPOT for IFN-y on splenocytes isolated from mice vaccinated twice (day 0 and day 21) and stimulated ex vivo with PMA/iomycin or antigen-specific peptides. The number of SFC per IxlO⁶ splenocytes is plotted. Values represent the geometric mean ± geometric standard deviation with each data point representing an individual mouse.

FIGS. 5A-5C show that MR-CoV-S6p312 elicits a Thl-oriented humoral immune response that is sensitive to amino acid substitutions present in SARS-CoV-2 variants. FIG. 5 A) Isotype analysis of anti-SARS-CoV-2 spike antibodies. Serum samples from IFNAR - CD46Ge vaccinated once (day 21) or twice (day 21 and day 42) with MR-Co V-S6p312 were analyzed by ELISA for IgG and IgG2a binding antibodies to SARS-CoV-2. Serum from mice vaccinated twice with purified SARS-CoV-2 Spike adjuvanted with alum was used as a control for Th2 bias humor response. FIG. 5B) Cytokine production from splenocytes of vaccinated mice. Splenocytes isolated from mice vaccinated were stimulated as indicated in FIG. ID, and cytokine secretion in the supernatant was analyzed by multiplex cytokine analysis. Dots represent individual animals, horizonal bars and error bar are mean ± SD. IL- 1P LLoD: 1.45 pg/mL; IL-12 LLoD: 1.68 pg/mL; TNF-oc LLoD 3.48 pg/mL; IFN-y LLoD 2.19 pg/mL; GM-CSF LLoD: 3.20 pg/mL;IL-6 LLoD: 5.52 pg/mL; IL-5 LLoD: 2.19 pg/mL; IL-2 LLoD 1.88 pg/mL;IL-4 LLoD: 1.37 pg/mL; IL-13 LLoD: 2.86 pg/mL. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparison test (ns, not significant *, p<0.05; **, p<0.003, ***, p<0.0003; ****, p<0.0001). FIG. 5C) Neutralizing activity response against SARS-CoV-2 variants. Serum samples from animals vaccinated once with MR-CoV-S6p312 were assessed for neutralizing antibody responses against pseudoviruses-bearing the SARS-CoV-2 spike from different variants. Black dots represent individual mouse serum and bars and error bars depict geometric mean ± geometric standard deviation, respectively. Statistical analysis between groups was calculated by One-way ANOVA with Dunn’s post-test (ns, p>0.05; *, p<0.05; ****, p<0.0001).

FIGS. 6A-6D show that a booster dose of an omicron BA.1-matched MeV-C0VID19 vaccine candidate enhances neutralizing activity and confers protection in K18-hACE2 mice. FIGS. 6A-6B) IFNAR -CD46Ge mice were vaccinated on week 0 with D614G-based MeV/SARS-CoV-2S6p312 and boosted on week 10 with either the same D614G-based MeV/SARS-CoV-2S6p312 or with Omicron BA.1 MeV/SARS-CoV-2S6p312 vaccine. Serum samples were collected at weeks 3, 10, and 13, and were analyzed for pseudovirus neutralizing antibodies using Wuhan-based VSV/SARS-CoV-2 pseudovirus based on Wuhan spike (FIG. 6A) or Omicron-BA-1 (FIG. 6B). FIG. 6C) Protection from body weight lost in mice challenged with SARS-CoV-2 (WA1/2020). K18-ACE2 mice were passively immunized intraperitoneally with serum samples from the previous IFNAR -CD46Ge vaccinated animals after homologous (Wuhan) or heterologous (Omicron) boost. Serum samples from animals vaccinated twice with an empty MeV-MR vector were used as shamvaccination (empty). Two-hours later, K18-ACE2 mice were challenged intranasally with WA1/2020 and monitored for body weigh lost. FIG. 6D) Virus burden 6-days post-challenge with WA1/2020 or BA.l virus as assessed by plaque assay on homogenates from lung and nasal turbinates.

FIGS. 7A-7D shows that pre-existent MeV antibodies do not blunt the anti-SARS- CoV-2 spike immune response elicited upon vaccination with MR-CoV-6p312. FIG. 7A) A schematic of the experimental design. IFNAR -CD46Ge mice were passively immunized on days 0 and 21 with 400 milli-international units (mIU) of anti-measles neutralizing antibodies before each vaccination dose of IxlO⁵ pfu of MR-CoV-6p312. MeV Moraten was used as a control for vaccination. Serum samples were collected three weeks after the second vaccination dose and tested for MeV neutralizing antibodies (FIG. 7B) and SARS-CoV-2 spike pseudovirus-neutralizing antibodies (FIG. 7C). FIG. 7D) ELISPOT assays were also run on splenocytes collected three weeks after the second vaccination. Dots represent individual mouse and bars and error bars depicts geometric mean ± geometric standard deviation. Statistical analysis between groups was calculated by unpaired Two-tail T-test (ns, p>0.05; *, p<0.05). FIGS. 8A-8D show the impact of age and virus dose on C0VID19 vaccination efficacy. IFNAR -CD46Ge mice were vaccinated twice at a three-week interval with 2x10⁶ pfu ofMR-CoV-S2p312. Serum samples were collected three weeks after each vaccination dose to assess for IgG binding antibody responses to MeV bulk antigens (FIG. 8A) and SARS-CoV-2 spike (FIG. 8B). FIG. 8C) Neutralizing antibody responses against SARS- CoV-2 were also determined using pseudotyped viruses expressing spike D614G spike. FIG. 8D) Splenocytes were harvested three weeks after the second vaccination dose and subjected to ELISPOT analysis with SARS-CoV-2 spike, MeV-nucleocapsid peptides, or control medium. Each dot represents a single mouse. Bars and error bars depict geometric mean ± geometric standard deviation. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparison test (ns, not significant, p<0.05).

FIGS. 9A-9E show the effect of dose of MR-SACT on the humoral response to SARS-CoV-2 spike. FIG. 9 A) Multi-step growth kinetics of rMeVs on Vero cells infected at MOI of 0.03. FIG. 9B) Time-course western blot analysis of rMeV-infected Vero cells. FIGS. 9C-9D) Serum samples from mice vaccinated twice (day 0 and day 21) with rMeV expressing SARS-CoV-S2ACT or firefly luciferase (Flue) were assessed by ELISA for MeV- IgG binding antibodies (FIG. 9C) and SARS-CoV-2 spike IgG binding antibodies (FIG. 9D). Data are plotted as absorbance over inverse of serum. FIG. 9E) Neutralizing antibody responses against SARS-CoV-2 were also determined using pseudotyped viruses expressing spike D614G spike.

FIGS. 10A-10C show that polypeptides containing a scaffold polypeptide can form nanoparticles. FIG. 10A shows a schematic diagram of the full-length measles virus H glycoprotein and engineered constructs. For the soluble MeV-H (aa 179-617), an IgG k murine signal sequence was added preceded by a HA tag. For the MeV-H-NAP, a H. pylori NAP polypeptide was genetically fused at the C-terminus of the glycoprotein. The apparent molecular weight for the constructs is indicated. FIG. 10B shows a structural model of the full-length MeV H polypeptide. The different domains are indicated on the right, with the numbers referring to the amino acid position. FIG. 10C shows a structural model of the MeV H polypeptide globular head displayed on H. pylori NAP. Each nanoparticle can display 12 copies of the MeV H polypeptide. FIGS. 11A-1 ID shows that different MeV-H polypeptides were expressed from recombinant MeVs and that MeV-H polypeptides were recognized by MeV-H-specific mAbs. FIG. 11A shows the analysis of MeV-H and MeV N protein expression in cell lysates (MeV-N) and supernatants (MeV-H, anti-HA) by Western blot. Vero cells in 6-well plates were infected with each recombinant virus at MOI of 0.1. At 22 hours or 44 hours, cell supernatants were collected, and the remaining cells were lysed with lysis buffer before they were used for immunoblotting analysis. Blots were probed using rat mAb reactive against the HA peptide (used as a proxy of the engineered MeV-H, upper blot) or rabbit polyclonal against MeV-N (lower blot). Migration and size of molecular mass markers are indicated FIG. 11B shows the recognition of soluble MeV-H by monoclonal antibodies. 100 μL of cell supernatant collected 44 hours post infection was subjected to immunoprecipitation with 1 pg of the indicated mAbs against MeV-H. Immunoprecipitated MeV-H was revealed with anti-HA mAbs. Anti-HA mAb was alternatively used as positive control for immunoprecipitation, and supernatant from mock infected cells served as negative control. FIGSs 11C and 1 ID show electron micrographs of negatively stained soluble MeV-H (FIG. 11 C) and MeV-H-NAP (FIG. 1 ID). MeV-H supernatants from infected cells were affinity purified with HA tagged protein purification kit (MBL 3320) and stained with 1% phosphotungstic acid, pH 7.2. Micrographs were taken on a FEI Tecnai 12 operating at 80KV

FIG. 12 shows that a soluble MeV-H polypeptide elicits only binding antibodies whereas a MeV-H-NAP elicits both binding and neutralizing antibodies. IFNAR -CD46Ge mice were immunized once with MeV-MR expressing firefly luciferase (negative control), soluble MeV-H (sMeV-H) or (MeV-H-NAP). As positive control, animals were immunized with a Moraten vaccine strain expressing a full-length MeV-H as an additional transcript unit (Julik and Reyes-del Valle, J. Virol., 90(11): 5270-5279 (2016)). At week three, serum samples were collected and analyzed for neutralizing antibodies (left axis) and binding antibodies (right axis)). Neutralizing antibodies were determined by a focus reduction neutralization test using GFP-expressing measles virus Moraten vaccine strain. Binding antibodies were determined using cells stably expressing vaccine-derived MeV-H.

FIGS. 13A-13E shows that a rMeV-MR expressing MeV-H-NAP elicited humoral and cellular immune responses in the presence of pre-existent MeV antibodies. FIG. 13A shows the experimental design. IFNAR -CD46Ge were passively immunized on days 0 and 21 with MeV-IgG before each vaccination with a dose of le5 pfu of the MeV Moraten vaccine strain or the MeV-MR expressing MeV-H-NAP. Mouse MeV-IgG was produced by immunizing a group of five HuCD46Ge-IFNarKO mice with MeV Moraten. Serum samples and splenocytes were collected on day 42 (three weeks post-boost). FIG. 13B shows the MeV-H-specific binding antibodies elicited after vaccination with each virus in the presence of anti-MeV antibodies. Medium-inoculated mice served as mock vaccination controls. Binding antibodies were determined by ELISA via the specific optical density (OD) 450 nm value. Depicted are the means and respective standard deviation of the mean of each group (N=5), with the dilution of serum samples in log 10. Horizontal bars are mean per group. FIG. 13C shows neutralizing antibodies as determined by a focus reduction neutralization test using a luciferase-reported microneutralization assay based on the Moraten vaccine. Each dot represents an individual animal. FIGS. 13D-E show the ELISpot quantification of IFN-v producing T cells. Spot forming cells were quantified after the cells were stimulated with pools of 15-mer peptides derived from the MeV-H (FIG.13D) or MeV-N proteins (FIG.13E)

DETAILED DESCRIPTION

This document provides multimeric immunogens and methods and materials related to using multimeric immunogens. In some cases, a multimeric immunogen can include two or more (e.g., two, three, four, or more) polypeptides that each include an immunogen and a multimerization domain (e.g., an immunogen fused to a multimerization domain), such that the two or more polypeptides can form a multimeric immunogen having the ability to increase an immune response (e.g., a neutralizing immune response) against the immunogen within a mammal (e.g., a human). For example, a multimeric SARS-CoV-2 immunogen can include two or more (e.g., two, three, four, or more) polypeptides that each include a SARS- CoV-2 immunogen and a multimerization domain, such that the two or more polypeptides can form a multimeric SARS-CoV-2 immunogen having the ability to increase an immune response (e.g., a neutralizing immune response) against multiple members of the coronavirus family within a mammal (e.g., a human). In some cases, a multimeric immunogen can be presented on a self-assembling nanoparticle. For example, each polypeptide within a multimeric immunogen also can include a scaffold polypeptide, such that the multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens) can assemble (e.g., self-assemble) into a nanoparticle in vivo. In some cases, compositions (e.g., vaccine compositions) that contain one or more multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens) provided herein can have the ability to increase immune responses against the immunogen within a mammal (e.g., a human). This document also provides methods and materials for using multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens) provided herein to induce immune responses within a mammal (e.g., a human). In some cases, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens) provided herein can be administered to a mammal (e.g., a human) such that the mammal produces an immune response against the immunogen(s). For example, compositions (e.g., vaccine compositions) that contain one or more multimeric immunogens (e g., multimeric SARS-CoV-2 immunogens) provided herein (or nucleic acid encoding a polypeptide including an immunogen and a multimerization domain such that two or more polypeptides can form a multimeric immunogen provided herein) can be administered to a mammal (e.g., human) to increase an immune response against the immunogen within the mammal. For example, compositions (e.g., vaccine compositions) that contain one or more multimeric SARS-CoV-2 immunogens provided herein (or nucleic acid encoding a polypeptide including a SARS- CoV-2 immunogen and a multimerization domain such that two or more polypeptides can form a multimeric SARS-CoV-2 immunogen provided herein) can be administered to a mammal (e.g., human) having or at risk of developing a coronavirus infection (e.g., a SARS- CoV-2 infection such as COVID-19) to treat the mammal.

A polypeptide including an immunogen and a multimerization domain that can form a multimeric immunogen provided herein can include any appropriate immunogen. In some cases, an immunogen can be a soluble immunogen. In some cases, an immunogen can be a derived from a membrane glycoprotein of a virus. Examples of immunogens that can be included in a polypeptide including an immunogen and a multimerization domain that can form a multimeric immunogen provided herein include, without limitation, immunogens derived from SARS-CoV-2, immunogens derived from a respiratory syncytial virus (RSV; e.g., immunogens derived from a RSV F protein), immunogens derived from an influenza virus (e.g., immunogens derived from an influenza HA polypeptide), immunogens derived from an metapneumovirus (e.g., immunogens derived from a metapneumovirus (HMPV) such as a HMPV F protein), immunogens derived from an Ebola virus (e.g., immunogens derived from an Ebola GP protein), immunogens derived from an human immunodeficiency virus type I (HIV-1; e.g., immunogens derived from an HIV-1 gpl20 protein), and immunogens derived from a hepatitis C virus (HCV; e.g., immunogens derived from a HCV E2 protein).

In some cases, a polypeptide including an immunogen and a multimerization domain that can form a multimeric immunogen provided herein can include a SARS-CoV-2 immunogen. A SARS-CoV-2 immunogen can be derived from any appropriate SARS-CoV-2 polypeptide. In some cases, a SARS-CoV-2 immunogen can be derived from a structural SARS-CoV-2 polypeptide (e.g., a SARS-CoV-2 spike (S)-protein such as a receptor-binding domain (RBD) of a SARS-CoV-2 S-protein, a SARS-CoV-2 nucleocapsid (N)-protein, or a SARS-CoV-2 membrane (M)-protein).

When a SARS-CoV-2 immunogen (e.g., SARS-CoV-2 immunogen fused to a multimerization domain) is derived from a SARS-CoV-2 S-protein, the SARS-CoV-2 immunogen can be derived from any appropriate SARS-CoV-2 S-protein. Examples of SARS-CoV-2 S-proteins include, without limitation, those set forth in the National Center for Biotechnology Information (NCBI) databases at, for example, accession no. QUO955381.1, accession no. URN54379.1, accession no. QQX12069.1, and accession no. BCN86353.1. In some cases, a SARS-CoV-2 S-protein can have an amino acid sequence as set forth in SEQ ID NO:1 (see, e.g., Example 2).

In some cases, a SARS-CoV-2 immunogen (e.g., SARS-CoV-2 immunogen fused to a multimerization domain) derived from a SARS-CoV-2 S-protein can be a fragment of a SARS-CoV-2 S-protein. When a SARS-CoV-2 immunogen is a fragment of a SARS-CoV-2 S-protein, the SARS-CoV-2 immunogen can be any appropriate SARS-CoV-2 S-protein fragment. In some cases, a SARS-CoV-2 S-protein fragment can be derived from the amino acid sequence set forth in SEQ ID NO: 1, provided that it maintains at least some immunogenicity). In some cases, a SARS-CoV-2 S-protein fragment can have an amino acid sequence set forth in SEQ ID NO:2 (see, e.g., Example 2)

A SARS-CoV-2 S-protein fragment can be any appropriate length (e.g., can include any number of amino acids). In some cases, a SARS-CoV-2 S-protein fragment can be from about 232 amino acids in length to about 1273 amino acids in length (e.g., from about 232 amino acids to about 1200 amino acids, from about 232 amino acids to about 1100 amino acids, from about 232 amino acids to about 1000 amino acids, from about 232 amino acids to about 900 amino acids, from about 232 amino acids to about 800 amino acids, from about 232 amino acids to about 700 amino acids, from about 232 amino acids to about 600 amino acids, from about 232 amino acids to about 500 amino acids, from about 232 amino acids to about 400 amino acids, from about 300 amino acids to about 1273 amino acids, from about 400 amino acids to about 1273 amino acids, from about 500 amino acids to about 1273 amino acids, from about 600 amino acids to about 1273 amino acids, from about 700 amino acids to about 1273 amino acids, from about 800 amino acids to about 1273 amino acids, from about 900 amino acids to about 1273 amino acids, from about 1000 amino acids to about 1273 amino acids, from about 1000 amino acids to about 1273 amino acids, from about 250 amino acids to about 1250 amino acids, from about 350 amino acids to about 1150 amino acids, from about 450 amino acids to about 1050 amino acids, from about 550 amino acids to about 950 amino acids, from about 650 amino acids to about 850 amino acids, from about 250 amino acids to about 450 amino acids, from about 350 amino acids to about 550 amino acids, from about 450 amino acids to about 650 amino acids, from about 550 amino acids to about 750 amino acids, from about 650 amino acids to about 850 amino acids, from about 750 amino acids to about 950 amino acids, from about 850 amino acids to about 1050 amino acids, from about 950 amino acids to about 1150 amino acids, or from about 1050 amino acids to about 1250 amino acids in length).

In some cases, a SARS-CoV-2 immunogen (e.g., SARS-CoV-2 immunogen fused to a multimerization domain) derived from a SARS-CoV-2 S-protein can have one or more (e.g., one, two, three, four, five, six, seven, eight, or more) modifications (e.g., relative to the SARS-CoV-2 S-protein from which its derived). For example, a SARS-CoV-2 immunogen can have one or more modifications relative to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2. A modification can be any type of modification including, without limitation, an insertion of one or more amino acids, a deletion of one or more amino acids, a substitution of one or more amino acids, and combinations thereof. In some cases, a modification can render a SARS-CoV-2 immunogen (e.g., SARS-CoV-2 immunogen fused to a multimerization domain) resistant to proteolytic cleavage. In some cases, a modification can increase stability of a SARS-CoV-2 immunogen (e.g., SARS-CoV-2 immunogen fused to a multimerization domain).

In some cases, a modification in a SARS-CoV-2 immunogen (e.g., SARS-CoV-2 immunogen fused to a multimerization domain) derived from a SARS-CoV-2 S-protein can be within a ftirin site within a SARS-CoV-2 S-protein. For example, the amino acid sequence RRAR (SEQ ID NO: 8) within a native furin cleavage site of a SARS-CoV-2 S-protein can be modified to replace that sequence with the amino acid sequence GSAS (SEQ ID NO: 10).

In some cases, a modification in a SARS-CoV-2 immunogen (e.g., SARS-CoV-2 immunogen fused to a multimerization domain) derived from a SARS-CoV-2 S-protein can be within a S2 domain within the SARS-CoV-2 S-protein. For example, a SARS-CoV-2 immunogen can include one or more proline substitutions at F817 (F817P), A892 (A892P), A899 (A899P), A942 (A942P), K986 (K986P), and V987 (V987P), as numbered in SEQ ID NO: 1. In some cases, a SARS-CoV-2 immunogen can include proline substitutions at K986 (K986P) and V987 (V987P), as numbered in SEQ ID NO: 1. In some cases, a SARS-CoV-2 immunogen can include proline substitutions at F817 (F817P), A892 (A892P), A899 (A899P), and A942 (A942P), as numbered in SEQ ID NO: 1. In some cases, a SARS-CoV-2 immunogen can include proline substitutions at F817 (F817P), A892 (A892P), A899 (A899P), A942 (A942P), K986 (K986P), and V987 (V987P), as numbered in SEQ ID NO: 1.

In some cases, a SARS-CoV-2 immunogen (e.g., SARS-CoV-2 immunogen fused to a multimerization domain) can comprise, consist essentially of, or consist of an amino acid sequence set forth in Table 1.

Atorney Docket No. 07039-2162WO1 / 2021-355

In some cases, a SARS-CoV-2 immunogen provided herein can include the amino acid sequence set forth in any one of SEQ ID NOs: 13-15 with zero, one, or two amino acid substitutions within the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 13-15), with zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 13-15), and/or with zero, one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier (e.g., any one of SEQ ID NOs: 13-15), provided that the SARS-CoV-2 immunogen retains at least some immunogenicity). Examples of such SARS- CoV-2 immunogens can be those set forth in Table 2.

A polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) and a multimerization domain that can form a multimeric immunogen provided herein can include any appropriate multimerization domain. A multimerization domain can be a synthetic polypeptide or can be obtained from a naturally-occurring polypeptide. In some cases, a multimerization domain can be obtained from a fibritin polypeptide (e.g., T4 bacteriophage fibritin polypeptide). In some cases, a multimerization domain can be obtained from a cholaramphenicol acetyl transferase polypeptide. In some cases, a multimerization domain can be obtained from a transcription factor GCN4 polypeptide (e.g., a yeast transcription factor GCN4 polypeptide). In some cases, a multimerization domain can be obtained from a collagen XVIII polypeptide (e.g., a human collagen XVIII polypeptide).

In some cases, a multimerization domain can be a self-assembling multimerization domain. For example, when two or more (e.g., two, three, four, five, or more) polypeptides including a multimerization domain (e.g., two or more polypeptides each including a SARS- CoV-2 immunogen fused to a multimerization domain) are present, the two or more polypeptides can assemble (e.g., self-assemble) in vivo to form a multimer.

In some cases, a multimerization domain can be a trimerization domain. For example, when three polypeptides including a multimerization domain (e.g., three polypeptides each containing an immunogen such as a SARS-CoV-2 immunogen fused to a multimerization domain) are present, the three polypeptides can form a timer (e.g., a trimeric immunogen such as a trimeric SARS-CoV-2 immunogen).

Two or more (e.g., two, three, four, five, or more) polypeptides including a multimerization domain (e.g., polypeptides each containing an immunogen such as a SARS- CoV-2 immunogen fused to a multimerization domain) can form a multimeric polypeptide by any appropriate means. For example, two or more polypeptides including a multimerization domain can assemble into a multimeric polypeptide by hydrogen bonds, steric effects, hydrophobic effects, and/or one or more salt bridges.

A multimerization domain can include any appropriate sequence provided that two or more (e.g., two, three, four, five, or more) polypeptides including a multimerization domain (e g., polypeptides each containing an immunogen such as a SARS-CoV-2 immunogen fused to a multimerization domain) can assemble (e.g., self-assemble) to form a multimeric polypeptide. Examples of such multimerization domains include, without limitation, those set forth in Table 3.

Table 3. Exemplary multimerization domains.

In some cases, a multimerization domain can be as described elsewhere (see, e.g., Letarov et al., Biochemistry (Mose), 64(7):817-23 (1999), at, for example, Figure 1).

In some cases, a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) and a multimerization domain that can form a multimeric immunogen provided herein also can include a scaffold polypeptide. A scaffold polypeptide can be any appropriate scaffold polypeptide. A scaffold polypeptide can be a synthetic polypeptide or can be obtained from a naturally-occurring polypeptide. In some cases, a scaffold polypeptide can be obtained from a neutrophil-activating protein (NAP) polypeptide (e g., H. pylori NAP polypeptide). In some cases, a scaffold polypeptide can be obtained from a ferritin polypeptide (e.g., a hybrid ferritin polypeptide such as a hybrid H. /?j/orz-bullfrog ferritin polypeptide). In some cases, a scaffold polypeptide can be obtained from an encapsuling polypeptide (e.g., a Termotoga maritima encapsuling polypeptide). In some cases, two or more (e.g., two, three, four, five, six, seven, eight, nine, ten, 11, 12, or more) polypeptides including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain and a scaffold polypeptide can assemble (e.g., self-assemble) into a nanoparticle. For example, when two or more polypeptides including an immunogen fused to a multimerization domain and a scaffold polypeptide (e.g., two or more polypeptides each including an immunogen fused to a multimerization domain and a scaffold polypeptide) are present, the two or more polypeptides can assemble (e.g., self-assemble) to form a nanoparticle. In some cases, twelve polypeptides including an immunogen (e.g., a SARS- CoV-2 immunogen) fused to a multimerization domain and a scaffold polypeptide can assemble (e.g., self-assemble) to form a dodecameric nanoparticle.

Two or more (e.g., two, three, four, five, or more) scaffold polypeptides can form a nanoparticle by any appropriate means. For example, two or more polypeptides including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain and a scaffold polypeptide (e.g., two or more polypeptides each including an immunogen fused to a multimerization domain and a scaffold polypeptide) can assemble into a nanoparticle by hydrogen bonds, steric effects, hydrophobic effects, and/or one or more salt bridges.

A scaffold polypeptide can include any appropriate sequence provided that two or more (e.g., two, three, four, five, six, seven, eight, nine, ten, 11, 12, or more) polypeptides including a scaffold polypeptide (e.g., two or more polypeptides each including an immunogen fused to a multimerization domain and a scaffold polypeptide) can assemble into a nanoparticle. Examples of such scaffold polypeptides include, without limitation, those set forth in Table 4.

Table 4. Exemplary scaffold polypeptides. IVQLGHHPLVTLSEAIKLTRVKEETKTSFHSKDI FKEILEDYKYLEKEFEELSNTAE

KEGDKVTVTYADDQLAKLQKSIWMLQAHLA

MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEEFADMFDDLAER 36

IVQLGHHPLVTLTEALKLTRVKEETKTSFHSKDI FKEILEDYKYLEKEFKELSNTAE KEGDKVTVTYADDQLAKLQKSIWMLQAHLA

KT FE ILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEE IYEE FADMFDDLAERI 37

VQLGHHPLVTLSEAIKLTRVKEETKTSFHSKDI FKEILEDYKHLEKE FKELSNTAEK

EGDKVTVTYADDQLAKLQKSIWMLQAHLA

In some cases, a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain also can include one or more additional components. Examples of additional components that can be included in a polypeptide including an immunogen fused to a multimerization domain provided herein include, without limitation, leader sequences, epitope tags, and detectable markers.

When an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein includes a leader sequence, the leader sequence can be any appropriate leader sequence. Examples of leader sequences that can be included in aa immunogen fused to a multimerization domain provided herein provided herein include, without limitation, IgGic leader sequences (e.g., murine IgGK leader sequences), melittin signal sequence sequences, CD5 signal sequences (e.g., human CD5 signal sequences), and IL2 signal sequences (e.g., human IL2 signal sequences).

When an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein includes an epitope tag, the epitope tag can be any appropriate epitope tag. Examples of epitope tags that can be included in an immunogen fused to a multimerization domain provided herein provided herein include, without limitation, hemagglutinin (HA) tags, polyhistidine (HIS) tags, FLAG tags, and Strep tags.

When an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein includes a detectable marker, the detectable marker can be any appropriate detectable marker. Examples of detectable markers that can be included in an immunogen fused to a multimerization domain provided herein provided herein include, without limitation, bioluminescent polypeptides (e.g., luciferase polypeptides), and fluorescent polypeptides (e.g., green fluorescent polypeptides (GFPs)).

In some cases, any two components in a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be separated by a linker. In some cases, a linker can be a cleavable linker (e.g., a protease-sensitive sequence). Examples of linkers that can be used to separate two components in a polypeptide including an immunogen fused to a multimerization domain include, without limitation, GlySer linkers, gly linkers, proline-rich linker, and elastin-like linkers, and protease-sensitive sequences.

In some cases, a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can include a IgGK leader sequence, followed by a SARS-CoV-2 immunogen comprising, consisting essentially of, or consisting of an amino acid sequence set forth in SEQ ID NO: 13, followed by a multimerization domain comprising, consisting essentially of, or consisting of an amino acid sequence set forth in SEQ ID NO:21, followed by a NAP polypeptide. For example, a recombinant MeV vector including nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain can include a IgGK leader sequence, followed by SARS-CoV-2 immunogen comprising, consisting essentially of, or consisting of an amino acid sequence set forth in SEQ ID NO: 13, followed by a multimerization domain comprising, consisting essentially of, or consisting of an amino acid sequence set forth in SEQ ID NO:21, followed by a NAP polypeptide.

In some cases, a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can include a IgGK leader sequence, followed by SARS-CoV-2 immunogen comprising, consisting essentially of, or consisting of an amino acid sequence set forth in SEQ ID NO: 14, followed by a multimerization domain comprising, consisting essentially of, or consisting of an amino acid sequence set forth in SEQ ID NO:21, followed by a NAP polypeptide. For example, a nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain can include a IgGK leader sequence, followed by SARS-CoV-2 immunogen comprising, consisting essentially of, or consisting of an amino acid sequence set forth in SEQ ID NO: 14, followed by a multimerization domain comprising, consisting essentially of, or consisting of an amino acid sequence set forth in SEQ ID NO:21, followed by a NAP polypeptide.

Any appropriate method can be used to administer a multimeric immunogen provided herein to a mammal (e.g., a human). In some cases, one or more multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens) can be administered to a mammal (e.g., a human). In some cases, one or more polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain (e.g., a composition including one or more polypeptides each including an immunogen fused to a multimerization domain) can be administered to a mammal (e.g., a human). For example, one or more polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain (e.g., a composition including one or more polypeptides each including an immunogen fused to a multimerization domain) can be administered to a mammal (e.g., a human) such that two or more of the polypeptides can form a multimeric immunogen within the mammal. In some cases, nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be administered to a mammal (e.g., a human). For example, nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain (e.g., a composition including one or more polypeptides each including an immunogen fused to a multimerization domain) can be administered to a mammal (e.g., a human) such that the polypeptide is expressed and two or more of the polypeptides can form a multimeric immunogen within the mammal.

In cases where nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain is administered to a mammal (e.g., a human), the nucleic acid can be any appropriate nucleic acid. The term “nucleic acid” as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. A nucleic acid can be double-stranded or single-stranded. A single-stranded nucleic acid can be the sense strand or the antisense strand. In addition, a nucleic acid can be circular or linear.

In cases where nucleic acid encoding a polypeptide including an immunogen (e.g., a

SARS-CoV-2 immunogen) fused to a multimerization domain is administered to a mammal (e.g., a human), the nucleic acid can be in the form of a vector (e.g., a non-viral vector or a viral vector). When a vector including nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain is a non- viral vector, any appropriate non-viral vector can be used. In some cases, a non-viral vector can be an expression plasmid (e.g., a cDNA expression vector).

When a vector including nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, any appropriate viral vector can be used. In some cases, a viral vector can be a MeV (e.g., a recombinant MeV). A recombinant MeV vector including nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be any appropriate recombinant MeV vector. MeV (also referred to as MV) is a single- stranded, negative-sense, enveloped, non-segmented RNA virus of the genus Morbillivirus within the family Paramyxoviridae. The MeV genome encodes six main polypeptides: a nucleoprotein (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide, a fusion (F) polypeptide, a hemagglutinin (H) polypeptide, and an RNA dependent RNA polymerase (L) polypeptide, as well as the C and V non-structural proteins that serve as innate immunity antagonists. MV has a lipid membrane envelope, with which virion surface glycoproteins H and F are associated.

Nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be at any appropriate location within the genome of a recombinant MeV vector including nucleic acid encoding an immunogen fused to a multimerization domain. In some cases, nucleic acid encoding an immunogen (e.g., a SARS- CoV-2 immunogen) fused to a multimerization domain can be located between the MeV-P coding sequence of a recombinant MeV vector and the MeV-M coding sequence of a recombinant MeV vector.

In some cases, a recombinant MeV vector including nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be a resurfaced MeV vector. The term “resurfaced” as used herein with reference to a MeV vector refers to an MeV vector having a modified H polypeptide and/or a modified F polypeptide. For example, a recombinant MeV vector can be resurfaced by modifying the nucleic acid encoding a H polypeptide within the genome of a MeV such that the MeV expresses a modified H polypeptide. In another example, a recombinant MeV vector can be resurfaced by modifying the nucleic acid encoding a F polypeptide within the genome of a MeV such that the MeV expresses a modified F polypeptide. In another example, a recombinant MeV vector can be resurfaced by modifying the nucleic acid encoding a H polypeptide within the genome of a MeV such that the MeV expresses a modified H polypeptide, and by modifying the nucleic acid encoding a F polypeptide within the genome of a MeV such that the MeV expresses a modified F polypeptide.

A modified H polypeptide expressed by a recombinant MeV vector including nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be any appropriate modified H polypeptide. In some cases, a modified H polypeptide can have one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) modifications (e.g., amino acid substitutions) relative to a WT MeV H polypeptide (e.g., a MeV vaccine strain H polypeptide). Examples of WT MeV H polypeptides include, without limitation, those set forth in the NCBI databases at, for example, accession no. AIY5560 (version AIY5560.1), accession no. AVA07189 (version AVA07189.1), and accession no. AFB35727 (version AFB35727.1). In some cases, a WT H polypeptide can have an amino acid sequence set forth in SEQ ID NO:3 (see, e.g., Example 3). For example, a modified H polypeptide can have one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) modifications (e.g., amino acid substitutions) relative to the amino acid sequence set forth in SEQ ID NO:3. Examples of modified H polypeptides that can be expressed by a recombinant MeV vector including nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain include, without limitation, H polypeptides having one or more of the following amino acid substitutions: H17S, D149N, A165T, S189P, G211S, E235G, N238D, S240N, L249P, V2801, N282K, G302R, E303G, Q311R, Q334H, A359T, K364N, R377Q, E379G, M378K, P397L, T420A, V421A, L423P, E471K, F476L, N481Y, G491D, H495R, D505T, R533G, V562T, D574A, K576R, 1594L, G603E, T609N, G613E, and T614A, as numbered in SEQ ID NO:3. In some cases, a modified H polypeptide can comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO:4 (see, e.g., Example 3).

A modified F polypeptide expressed by a recombinant MeV vector including nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be any appropriate modified F polypeptide. In some cases, a modified F polypeptide can have one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) modifications (e.g., amino acid substitutions) relative to a WT MeV F polypeptide e.g., a MeV vaccine strain F polypeptide). Examples of WT MeV F polypeptides include, without limitation, those set forth in the NCBI databases at, for example, accession no. AIY55563 (version Al Y55563.1), accession no. AXI82411 (version AXI82411.1), and accession no. BDB95832 (version BDB95832.1). In some cases, a modified F polypeptide can have one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) modifications (e.g., amino acid substitutions) relative to a WT canine distemper virus (CDV) F polypeptide. In some cases, a WT F polypeptide can have an amino acid sequence set forth in SEQ ID NO: 5 (see, e.g., Example 4). For example, a modified F polypeptide can have one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) modifications (e.g., amino acid substitutions) relative to the amino acid sequence set forth in SEQ ID NO: 5. In some cases, a modified F polypeptide can be a recombinant polypeptide. For example, a modified F polypeptide can include a CDV F polypeptide fused to a MeV F polypeptide signal peptide. In some cases, a modified F polypeptide can comprise, consist essentially of, or consist of an amino acid sequence set forth in SEQ ID NO:6 (see, e.g., Example 4). In some cases, a modified F polypeptide can be as described elsewhere (see, e.g., International Patent Application Publication No. WO 2018/212842 at, for example, page 17, lines 9-27).

In some cases, a recombinant MeV vector including nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain also can include nucleic acid encoding one or more additional components. Examples of additional components that can be included in recombinant MeV vector including nucleic acid encoding an immunogen fused to a multimerization domain provided herein include, without limitation, detectable markers.

When a recombinant MeV vector including nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain includes nucleic acid encoding a detectable marker, the detectable marker can be any appropriate detectable marker. Examples of detectable markers include, without limitation, luciferase polypeptides (e g., firefly luciferase polypeptides).

In some cases, a recombinant MeV vector described elsewhere (see, e.g., Munoz-Alia et al., Cell. Rep. Med., 2(4): 100225 (2021) at, for example, Figure 1; and International Patent Application Publication No. WO 2018/212842 at, for example, page 15, line 2 to page 18, line 2, Figure 9, and Figure 15) can be designed to include nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain as described herein.

This document also provides nucleic acid molecules that can encode a recombinant MeV vector provided herein (e.g., a MeV vector including nucleic acid encoding an immunogen (e.g., a SARS-CoV-2 immunogen) and nucleic acid encoding a multimerization domain).

This document also provides cells (e.g., cell lines) containing recombinant MeV vectors including nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain. In some cases, cells containing a recombinant MeV vector can be used to propagate the recombinant MeV vector (e.g., to establish a stock of the recombinant MeV vector). For example, a stock of the recombinant MeV vector can be produced by growth in mammalian cells. In some cases, a stock of the recombinant MeV vector can be aliquoted and frozen, and can be stored at -70°C to -80°C (e.g., at concentrations higher than the therapeutically effective dose). In some cases, a stock of the recombinant MeV vector can be stored in a stabilizing solution. Examples of stabilizing solutions include, without limitation, sugars (e.g., trehalose, dextrose, and glucose), amino acids, glycerol, gelatin, monosodium glutamate, Ca²⁺, and Mg²⁺. In some cases, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be formulated into a composition (e.g., a pharmaceutical composition such as a vaccine composition) for administration to a mammal. For example, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and corn oil. Suitable pharmaceutical formulations depend in part upon the use and the route of administration. Such forms should not prevent the composition or formulation from reaching target cells or from exerting its effect. For example, pharmacological compositions injected into the blood stream should be soluble. In some cases, a composition provided herein (e.g., a composition that includes multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS- CoV-2 immunogen) fused to a multimerization domain) can be a vaccine composition. For example, a composition containing multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be formulated into a composition together with one or more adjuvants. Examples of adjuvants that can be included within a vaccine composition provided herein include, without limitation, CpG oligonucleotide motifs, aluminum (e.g., aluminum salts such as aluminum sulfate, aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), monophosphoryl lipid A, aluminumphosphylate, MF59, AS03, AS04, alhydroxiquim-II, and Matrix-M™ In some cases, an adjuvant included within a vaccine composition provided herein can be a non-naturally occurring (e.g., artificial) adjuvant.

In cases where a vaccine composition provided herein includes multimeric SARS- CoV-2 immunogens, polypeptides each including a SARS-CoV-2 immunogen fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including a SARS-CoV- 2 immunogen fused to a multimerization domain, the vaccine composition can be a multivalent vaccine composition having the ability to increase immune responses against multiple members of the coronavirus family within a mammal (e.g., a human).

In some cases, a vaccine composition provided herein can have the ability to increase immune responses against a virus in the alphavirus genus. In some cases, a vaccine composition provided herein can have the ability to increase immune responses against a virus in the betacoronavirus genus (e.g., embecoviruses (previous lineage A), sarbecoviruses (previous lineage B), and merbecoviruses (previous lineage C)). For example, a vaccine composition provided herein can have the ability to increase immune responses against SARS-CoV-2, 229E, NL63, OC43, HKU1, Middle East Respiratory Syndrome (MERS)- CoV, Severe Acute Respiratory Syndrome (SARS)-CoV, or any combination thereof. In some cases, a vaccine composition provided herein can be used as a multivalent vaccine composition having the ability to increase immune responses against one or more lineages, clades, or strains of SARS-CoV-2. For example, a vaccine composition provided herein can have the ability to increase immune responses against B.1.17 (alpha), Bl.351 (beta), Pl (gamma), B.1.617.2 (delta), B.1.1.529 (omicron), B.1.526 (iota), B.1.617.1 (kappa), C.37 (lamda), B.1.621 (mu), B.1.427/B.1.429 (epsilon), P2 (zeta), or any combination thereof.

This document also provides methods for increasing an immune response against a particular immunogen within a mammal (e.g., a human). In some cases, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be administered to a mammal (e.g., human) to increase an immune response against the immunogen within the mammal. For example, multimeric SARS-CoV-2 immunogens, polypeptides each including a SARS-CoV- 2 immunogen fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain can be administered to a mammal (e.g., human) to increase an immune response against a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) within the mammal. For example, multimeric SARS-CoV-2 immunogens, polypeptides each including a SARS-CoV-2 immunogen fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain can be administered to a mammal (e.g., a human) having or at risk of developing a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) to treat that mammal.

Any appropriate mammal can be administered multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain to treat that mammal. Examples of mammals that can be administered multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein include, without limitation, humans, non-human primates (e.g., monkeys or apes), horses, dogs, cats, bovine species, pigs, sheep, mice, rats, hamsters, bats, guinea pigs, cotton rats, and ferrets. In some cases, a human identified as having or as being at risk of developing a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) can be administered multimeric SARS-CoV-2 immunogens, polypeptides each including a SARS- CoV-2 immunogen fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain provided herein to treat that human. For example, a human identified as having been exposed to a coronavirus can be administered multimeric SARS-CoV-2 immunogens, polypeptides each including a SARS-CoV-2 immunogen fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain provided herein to treat that human.

In some cases, the methods described herein can include identifying a mammal (e.g., a human) as needing an increase in an immune response against an infection (e.g., a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19)). For example, humans identified as having been in recent (e.g., within one to two weeks) contact with one or more humans having or suspected of having a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) can be identified as needing an increase in an immune response against a coronavirus such as SARS-CoV-2 and can be administered a vaccine composition including multimeric SARS-CoV-2 immunogens, polypeptides each including a SARS-CoV- 2 immunogen fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain provided herein.

In some cases, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein can be used to increase an immune response against a coronavirus such as SARS- CoV-2 within a mammal (e.g., a human). For example, a vaccine composition including multimeric SARS-CoV-2 immunogens, polypeptides each including a SARS-CoV-2 immunogen fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein can be administered to a mammal (e.g., a human) in need thereof (e.g., a mammal needing an increase in an immune response against a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) such as a mammal having or at risk of developing COVID-19) to increase an immune response (e.g., an increased antibody response and/or an increased T cell response) against a coronavirus such as SARS-CoV-2. An immune response against a coronavirus such as SARS-CoV-2 within a mammal (e.g., a human) can be a humoral antibody response and/or a cellular immune response. When an immune response against a coronavirus such as SARS-CoV-2 within a mammal (e.g., a human) is cellular immune response, the immune response can be a T helper type 1 (TH1) cell-mediated response and/or a TH2 cell-mediated response. When an immune response against a coronavirus such as SARS-CoV-2 within a mammal (e.g., a human) is cellular immune response, the immune response can involve any appropriate T cells (e.g., CD4⁺ T cells and CD8⁺ T cells).

In some cases, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein can be used to delay or prevent the development of one or more symptoms of an infection (e.g., a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19)) within a mammal at risk of developing that infection. For example, multimeric SARS-CoV-2 immunogens, polypeptides each including a SARS-CoV-2 immunogen fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including a SARS-CoV- 2 immunogen fused to a multimerization domain provided herein can be administered to a mammal (e.g., a human) in need thereof (e.g., a mammal needing an increase in an immune response against a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) such as a mammal having or at risk of developing COVID-19) to delay or prevent the development of one or more symptoms of a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) in the mammal. Symptoms of a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) include, without limitation, fever, chills, cough, shortness of breath, difficulty breathing, fatigue, muscle aches, body aches, headache, loss of taste, loss of smell, sore throat, congestion, runny nose, nausea, vomiting, diarrhea, and persistent pain or pressure in the chest. For example, the materials and methods described herein can be used to delay the onset of one or more symptoms of a coronavirus infection (e g., a SARS-CoV-2 infection such as COVID-19) within a mammal at risk of developing a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

In some cases, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein can be used to reduce the duration and/or the severity of one or more symptoms of an infection (e.g., a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) present within a mammal having a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID- 19)). For example, multimeric SARS-CoV-2 immunogens, polypeptides each including a SARS-CoV-2 immunogen fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain provided herein can be administered to a mammal (e.g., a human) in need thereof (e.g., a mammal needing an increase in an immune response against a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) such as a mammal having or at risk of developing CO VID-19) to reduce the duration and/or the severity of one or more symptoms of a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) within the mammal. Symptoms of a coronavirus infection (e.g., a SARS-CoV-2 infection such as CO VID-19) include, without limitation, fever, chills, cough, shortness of breath, difficulty breathing, fatigue, muscle aches, body aches, headache, loss of taste, loss of smell, sore throat, congestion, runny nose, nausea, vomiting, diarrhea, and persistent pain or pressure in the chest. For example, the methods and materials described herein can be used to reduce the duration and/or the severity of one or more symptoms of one or more symptoms of a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) present within a mammal having a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID-19) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

When administering a composition (e.g., a vaccine composition) provided herein to a mammal (e.g., a human), any appropriate route of administration can be used. For example, a composition (e.g., a vaccine composition) provided herein can be administered to a mammal (e.g., a human) intranasally (e.g., via an intranasal spray), intravenously (e.g., via an intravenous injection or infusion), subcutaneously (e.g., via a subcutaneous injection), intraperitoneally (e.g., via an intraperitoneal injection), orally, via inhalation, or intramuscularly (e.g., via intramuscular injection). In some cases, the route and/or mode of administration of a composition (e.g., a vaccine composition) provided herein can be adjusted for the mammal being treated.

In some cases, an effective amount of multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV- 2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be an amount that increases an immune response against an infection (e.g., a SARS-CoV-2 infection such as COVID-19) within the mammal (e.g., a human) without producing significant toxicity to the mammal. For example, an effective amount of a composition (e.g., a vaccine composition) provided herein can be from about 2 pg to about 480 pg (e.g., from about 2 pg to about 450 pg, from about 2 pg to about 400 pg, from about 2 pg to about 350 pg, from about 2 pg to about 300 pg, from about 2 pg to about 250 pg, from about 2 pg to about 200 pg, from about 2 pg to about 150 pg, from about 2 pg to about 100 pg, from about 2 pg to about 50 pg, from about 50 pg to about 480 pg, from about 100 pg to about 480 pg, from about 150 pg to about 480 pg, from about 200 pg to about 480 pg, from about 250 pg to about 480 pg, from about 300 pg to about 480 pg, from about 350 pg to about 480 pg, from about 400 pg to about 480 pg, from about 50 pg to about

450 pg, from about 100 pg to about 400 pg, from about 150 pg to about 350 pg, from about

200 pg to about 300 pg, from about 50 pg to about 150 pg, from about 100 pg to about 200 pg, from about 150 pg to about 250 pg, from about 200 pg to about 300 pg, from about 250 pg to about 350 pg, from about 300 pg to about 400 pg, or from about 350 gg to about 450 gg) of multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens). For example, an effective amount of a composition (e.g., a vaccine composition) provided herein can be from about 1 pg to about 200 gg (from about 1 gg to about 175 gg, from about 1 gg to about 150 gg, from about 1 gg to about 125 gg, from about 1 gg to about 100 gg, from about 1 gg to about 75 gg, from about 1 gg to about 50 gg, from about 1 gg to about 25 gg, from about 25 gg to about 200 gg, from about 50 gg to about 200 gg, from about 75 gg to about 200 gg, from about 100 gg to about 200 gg, from about 125 gg to about 200 gg, from about 150 gg to about 200 gg, from about 175 gg to about 200 gg, from about 25 gg to about 175 gg, from about 50 gg to about 150 gg, from about 25 gg to about 75 gg, from about 50 gg to about 100 gg, from about 75 gg to about 125 gg, from about 100 gg to about 150 gg, or from about 125 gg to about 175 gg) nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain. In some cases, an effective amount of a composition (e.g., a vaccine composition) provided herein can be from about 1x10⁵ plaque- forming units (pfu) to about 2x10⁶ pfu of recombinant MeV vector including nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the severity of an infection (e.g., a SARS-CoV-2 virus infection such as COVID-19) when treating a mammal having such an infection, the route of administration, the age and general health condition of the mammal, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents (e.g., antiviral agents such as remdesivir (e.g., VEKLURY®), galidesivir, and/or favipiravir (e.g., AVIGAN®)), and the judgment of the treating physician may require an increase or decrease in the actual effective amount of a recombinant MeV vector that is administered.

In some cases, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain can be administered to a mammal once (e.g., in a single administration).

In some cases, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein can be administered to a mammal several times (e.g., as several administrations). For example, a mammal (e.g., a human) in need thereof (e.g., a mammal needing an increase in an immune response against an infection (e.g., a coronavirus infection (e.g., a SARS-CoV-2 infection such as COVID- 19)) can be administered a first dose of multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein, and can be administered a boost (e.g., a second dose) from about 3 weeks to about 28 weeks (e.g., from about 3 weeks to about 24 weeks, from about 3 weeks to about 20 weeks, from about 3 weeks to about 15 weeks, from about 3 weeks to about 12 weeks, from about 3 weeks to about 8 weeks, from about 3 weeks to about 4 weeks, from about 4 weeks to about 28 weeks, from about 8 weeks to about 28 weeks, from about 12 weeks to about 28 weeks, from about 15 weeks to about 28 weeks, from about 20 weeks to about 28 weeks, from about 24 weeks to about 28 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 18 weeks, from about 4 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 18 weeks, or from about 18 weeks to about 24 weeks) after the administration of the first dose. In some cases, additional subsequent doses (e.g., subsequent boosters) can be administered.

In some cases, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein can be administered as a heterologous boost to a mammal (e.g., a human) that previously received a vaccine targeting a SARS-CoV-2 immunogen (e.g., a COVID-19 vaccine). For example, multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein can be administered as a heterologous vaccination boost to a human who previously received a COVID-19 vaccine (e.g., a Pfizer® vaccine such as Comirnaty®, an AstraZeneca® vaccine such as Vaxzevria®, a Moderna® vaccine such as Spikevax®, a Novavax® vaccine such as Nuvaxovid®, and/or Sputnik).

This document also provides kits containing multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens), polypeptides each including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain, and/or nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain provided herein. In some cases, a kit provided herein can include multimeric immunogens (e.g., multimeric SARS-CoV-2 immunogens). In some cases, a kit provided herein can include polypeptides each including an immunogen (e.g., a SARS-CoV- 2 immunogen) fused to a multimerization domain. In some cases, a kit provided herein can include nucleic acid encoding a polypeptide including an immunogen (e.g., a SARS-CoV-2 immunogen) fused to a multimerization domain.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: Neutralizing Antibodies Against SARS-CoF-2

This Example describes the generation of multimeric SARS-CoV-2 immunogens that can induce a neutralizing antibody response against SARS-CoV-2 variants. For example, a resurfaced MeV vector (e.g., MeV-MR) encoding a stabilized and multimeric SARS-CoV-2 spike glycoprotein can be used a vaccine composition to induce a neutralizing antibody response against SARS-CoV-2 variants. Materials and Methods

Cells and Viruses

BHK cells (ATCC), were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (GE Healthcare Life) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 units/mL of penicillin, and 100 pg/mL of streptomycin (ThermoFisher). Vero African green monkey kidney cells expressing a membrane-anchored single-chain variable fragment (scFv) specific for a hexahistidine peptide (6x HIS-tag) were cultured in DMEM-5% FBS. Cells were incubated at 37°C in 5% CO2 with saturating humidity. The Indiana strain-based Vesicular Stomatitis Virus (VSV) expressed SARS-CoV- 2 spike in place of VSV-G and was trans-complemented with VSV-G. The recombinant measles virus (rMeV) based on the Moraten vaccine strain and expressing firefly luciferase was as described elsewhere (Munoz-Alia MA, et aL, Cell Rep Med 2021; 2(4): 100225). SARS-COV-2 virus stocks were grown on TMPRSS2 overexpressing Vero-E6 cells were maintained DMEM- 10 FBS, 100 unit/mL of penicillin, 100 pg/mL of streptomycin, 1% NEAA, 3 pg/mL of puromycin, and 100 pg/mL of normocin. USA-WA1/2020 virus was obtained from BEI Resources (NR-52281), and omicron BAI virus (hCoV-19/USA/NY- MSHSPSP-PV44476/2021, GISAID: EPI_ISL_7908052) was obtained from the Mount Sinai Pathogen Surveillance Program.

Constructs and virus rescue

The codon-optimized gene encoding Wuhan-Hu- 1 (GenBank MN908947.3) was used as the basis for all the SARS-CoV-2 spike constructs. The beta variant of the SARS-CoV-2 spike (L18F, D80A, D215G, del242/243, R246I, K417N, E484K, N501Y, A701V) was synthesized in two fragments (GENEWIZ), and was cloned into a pcDNA3.1+ expression vector (ThermoFisher Scientific) using an InFusion HD kit (Takara). All the other variants were obtained from InvivoGene (Toulouse, France). Amino acid substitutions and deletions were introduced into the SARS-CoV-2 spike (See FIG. 2A) using standard molecular biology techniques and confirmed by Sanger sequencing (GENEWIZ). When indicated, the constructs also incorporated a C-terminal thrombin cleavage site LEVLFQGP (SEQ ID NO: 38), a “FoldON” sequence GYPPEAPRDGQAYVRKDEWVLLSTFLG (SEQ ID N0:21), and the neutrophil-activating protein of H. pylori (Genebank accession no. WP_000846461) at the extreme C-terminus of the construct.

All SARS-CoV-2 spike constructs were inserted directly by InFusion cloning into the Mlu/Aatll sites of the vector pSMART LC MeVvac2 (eGFP)P encoding MeV-HA8/CDV-F. The inserts were modified at the STOP codon to ensure compliance with the paramyxovirus rule of six. Rescue of rMeV was carried out on co-transfected BHK cells.

Virus infections and multi-step growth curves

Viruses were propagated by infecting Vero cells at an MOI of 0.03 in VP productionserum free medium (ThermoFisher Scientific) supplemented with L-glutamine (ThermoFisher). Virus titers were determined on Vero cells pre-seeded on a 96-well plate at 10,000 cells/well and infected with serial ten-fold dilution in Opti-MEM I reduced-serum medium (ThermoFisher). After a 90 minute absorption-period, cells were replenished with viral growth medium (DMEM + 5% FBS). Titer was determined 2-3 days post-infection by a naked-eye using a microscope and calculated as plaque forming units. For virus growth analysis, Vero cells were pre-seeded on a 6-well plate at 400,000 cells/well and infected at an MOI of 0.03. After absorption, the inoculum was removed, cells were washed thrice with Dulbecco’s phosphate-buffered saline (DPBS) (Mediatech), and the medium was replaced with 1 rnL of VP-SFM. At various time point after infection, the cell culture fluid and cell lysates were harvested, and virus titers were determined as described above.

Next-Generation Sequencing

RNA from virus stocks was extracted with QIAam Viral RNA mini (QIAgen), and one-step cDNA synthesis was next done with SuperScript IV RT Viral cDNA (ThermoFisher Scientific) using the primer pairs shown in Table 5.

Table 5. Primers used for RT-qPCR analysis

DNA fragments were gel-purified using QIAquick gel extraction kit (QIAgen), and amplicon sequencing was performed by the CCIB DNA Core Facility at Massachusetts General Hospital (Cambridge, MA). Illumina compatible adapters with unique barcodes were ligated onto each sample during library construction. Libraries were pooled in equimolar concentrations for multiplexed sequencing on the Illumina MiSeq platform with 2x150 run parameters. Upon completion of the NGS run, data were analyzed, demultiplexed, and subsequently entered into an automated de novo assembly pipeline, UltraCycler vl.O.

Western blot

Cells grown on a 6-well plate were infected with various rMeV at MOI 0.03. At 36 - 48 hours post-infection, supernatant was collected and filtered through a 0.45 pm size pore membrane. In the meantime, cells were lysed in M-PER mammalian protein extraction reagent (ThermoFisher Scientific) supplemented with halt protease inhibitor cocktail (ThermoFisher Scientific). Protein content was determined using Pierce Coomassie Plus assay Kit (ThermoFisher) and or 3 pg of cell lysate or ~20 μL of supernatant was separated on a pre-cast 12% or 4-12% Bis-Tris polyacrylamide gel before being transfer to PDVF membrane using an iBlot2 dry blotting system (Thermofisher Scientific). The blot was then probed with anti-SARS-CoV-2- spike RBD (GeneTex) or anti-SARS-CoV-2 spike (GeneTex), anti-MeV nucleocapsid (LsBio), and anti-HA peroxidase (MilliporeSigma) and developed with KwikQuant Western blot detection kit using a KwikQuant Imager (Kindle Bioscience LLC). Expression of purification of antibodies

VH and VH sequences were synthesized as gBlock fragments (GENEWIZ), and were cloned into the vectors pFUSEss-CHig-hGl and pFUSE2-CLIg-hK (InvivoGen, San Diego, CA, USA) using an InFusion HD kit. Recombinant antibodies were produced by plasmid cotransfection using the Expi293 expression system kit (ThermoFisher). The culture supernatant was collected and loaded at 4 mL/minute on a 5 mL HiTrap Protein G column (Cytiva) equilibrated with 10 mM phosphate, pH 7 using a Bio-Rad NGC FPLC system. The media was tittered to pH 7 with 1 M monosodium phosphate before loading. Antibody was eluted with 100 mM glycine, pH 2.7 and collected in tubes containing 1 M dibasic sodium phosphate to neutralize the pH. The eluate was concentrated to <4 mL with a 4 mL 50 KDa MWCO Amicon ultra centrifugal filter, and buffer exchanged on a 10 mL Zeba desalting column (ThermoFisher) equilibrated in PBS. Final antibody concentration was determined using the protein extinction coefficient for IgG.

Generation of pseudovirus particles displaying SARS-CoV-2 spike and pseudovirus neutralization assay

Single-round pseudotyped lentivirus particles were produced by co-transfection of HEK293 T-cells with plasmids pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI), HDM-Hgpm2 (BEI), HDM-tatlb (BEI), pRC-CMV-Revlb (BEI), and SARS-CoV-2 spike plasmid. Virus containing supernatants were harvested 72 hours post-transfection and filtered using a 0.45 pm syringe filters, aliquoted, and stored at -80°C until further use. For neutralization assays, virus was diluted to yield ~ 50,000 relative light units (RLU)/well and incubated for 1 hour at 37°C with 2-fold dilutions of heat-inactivated serum. Cells were then infected in quadruplicate and lysed 72 hours later using the Bio-Gio luciferase assay system (PROMEGA) to measure luciferase activity. The percentage of neutralization was calculated based on the relative luminescence units (RU) of virus-only control. ECso titers were calculated using a log (agonist) versus normalized response (variable slope) nonlinear function in Prism 9 for macOS (GraphPrism). Alternatively, IMMUNO-CRON and IMUNO-CoV v2.0 (Imanis Life Sciences), which use a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins, were used to determined pseudovirus neutralizing antibodies.

Measles virus neutralization assay

A luciferase-based neutralization assay was used. In brief, 2-fold serial solutions serum samples were mixed with an equal volume rMeV-Fluc and incubated for 1 hour at 37°C. The virus-serum mix was subsequently added to Vero cells for 48 hours before adding 50 nmoles of D-Luciferin (GoldBio) for measurement of luminescence. The percentage of neutralization was calculated based on the RLU of virus-only control, and subsequently analyze in Prism 9 to calculate the ECso using non-sigmoidal dose-response. ECso values were converted to mIU/mL by using the third international standard for anti-measles serum (National Institute for Biological Standards and Control).

Mice immunizations

Male and female eight- to 19-week-old mice deficient for type I IFN receptor (FNAR ‘) and transgenically expressing human CD4644 were vaccinated intraperitoneally with 1x10⁵ to 2x10⁶ pfu of recombinant viruses or purified SARS-CoV-2 spike protein adjuvant with aluminum hydroxide (Alhydrogel adjuvant 2%) (InvivoGen). A prime-boost vaccination regimen was used, and serum samples were collected before the vaccination booster and at the end of the study. At this point, mice were terminated and splenocytes were harvested for study of the cellular immune responses. All serum samples were heat inactivated for 30 minutes at 56°C before assessed the humoral immune responses.

Passive serum transfer

129S1 mice (Strain #:002448) and transgenic K18-hACE2 mice (Strain #:034860) were purchased from Jackson Laboratories and housed in a temperature-controlled vivarium with a 12 hour day/night regime with water and food ad libitum. For passive immunization, 150 μL of pooled serum was passively transferred by intraperitoneal injection 2 hours before infection. The mice were infected intra-nasally with 10⁴ pfu/L virus diluted in PBS, given in 75mg/kg; xylazine 7.5 mg/kg). Mice were monitored daily and body weights were recorded. On day 3 or 5 post-infection, mice were euthanized via intraperitoneal injection of sodium pentobarbital (292.50 mg/kg). Lungs and nasal turbinates were isolated aseptically in 500 pL of PBS and homogenized for further use. Homogenates from lung and nasal turbinates were titrated for determining the virus load by plaque assay on Vero-TMPRSS2 cells.

Recombinant SARS-CoV-2 antigens

Recombinant SARS-CoV-2 proteins produced in a baculovirus system were commercially obtained from Sino Biologicals: S1+S2 ectodomain, SI, RBD, S2, and nucleocapsid. Trimeric SARS-CoV-2 spike and spike-ferritine proteins (SARS-CoV-2S6p3 and SARS-CoV-2S6p312, respectively) were produced by transient expression ofExpi293F cells (ThermoFisher). Clarified supernatants were purified by affinity chromatography using an anti-HA affinity matrix (Millipore Sigma) pre-equilibrated with 20 mM Tris, 0.1 M NaCl, and 0.1 mM EDTA, pH 7.5 (equilibration buffer). The column was washed with equilibration buffer containing 0.05% Tween 20 and then eluted with 1 mg/mL of HA synthetic peptide (ThermoFisher) per manufacturer’s instructions. Fractions containing the eluted proteins were combined, concentrated, and dialyzed against Dulbecco’s PBS (Cat# 25-508, Genesee Scientific) using Pierce protein concentrator, 10K MWCO (ThermoFisher). The HA matrix was regenerated with 20 V of 0.1 M glycine, pH 2.0 (Santa Cruz Biotechnology), and reequilibrated before proceeding with the next purification round. Protein concentration was determined using Pierce 660 protein assay kit (ThermoFisher). SARS-CoV-2S6p312 was purified after SARS-CoV-2S6p3 and stored at -80°C until used.

Antigen binding ELISA

IgG binding to SARS-CoV-2 or MeV antigens were measured by ELISA using clear flat -bottom immuno nonsterile 96-well plates (ThermoFisher Scientific) coated overnight at 4°C with 100 ng of recombinant SARS-CoV-2 proteins or Ipg of MeV bulk antigen (Institut Virion\Serion GmbH, Wurzburg, Germany) in 50 mM carbonate-bicarbonate buffer, pH 9.6. Plates were washed and blocked with 2% bovine serum albumin (BSA) in PBS for 2 hours at room temperature (RT). Plates were washed again and incubated with serial dilutions of mouse sera and incubated for 1 hour at 37°C. Plates were washed three times with PBS with 0.05% Tween 20 and then incubated for 1 hour at RT with horse radish peroxidase (HRP)- conjugated anti-mouse IgG (1 :5,000) (ThermoFisher Scientific), IgGl (1 :5,000) (Jackson ImmunoResearch), or IgG2a (1 :5,000) (Jackson ImmunoResearch) secondary antibody. After final wash, plates were developed using 50 μL of 1-Step Ultra TMB (3, 3’, 5,5’- tetramethylbenzidine; ThermoFisher Scientific) and the reaction stopped with an equal volume of 2 M sulfuric acid before the optical density (OD) was read at 405 nm using an Infinite M200Pro microplate reader (Tecan). The endpoint titers of serum IgG responses were determined as the dilution that emitted an optimal density exceeding average of OD values plus three standard deviations of pooled negative serum samples. Alternatively, anti- SARS-CoV-2 binding IgG was reported as pg/mL based on a standard curve carried out using a SARS-CoV-2 spike neutralizing antibody (Sino Biological).

T-cell responses to viral antigens

Interferon (IFN)-y ELISPOT assays were carried out on mouse splenocytes to assess T-cell responses against measles virus and SARS-CoV-2 peptides. Briefly, 5* 10⁵ isolated splenocytes were co-cultured with different stimuli in 200 μL of RPMI-10% FBS complete media for 48 hours on TFN-y-coated plates (R&D systems). 15-mer overlapping peptides from SARS-CoV-2 spike glycoprotein (JPT peptides) and MeV- nucleoprotein (Genscript) were used to stimulate splenocytes at 5 pg/mL. As a positive control, PMA/ionomycin cell stimulation cocktail (Biolegend) was used at 2.5 pl/mL, and as negative control, splenocytes were stimulated with equivalent DMSO concentration (0.8%). At 48 hours post incubation, plates were developed in accordance with manufacturer instructions. Developed IFN-y spots were counted with automated ELISPOT reader (CTL Analyzers LLC, USA). Each spot represented a single reactive IFN-y-secreting T-cell.

Detection of TH1/Th2 cytokines using ex vivo stimulation of splenocytes with antigen peptides

Frozen splenocytes were thawed and incubated with 50 μg/mL of DNase 1 (Roche), for 5 minutes at 37°C. Cells were then washed twice and re-suspended with RPML1640 media with 10% (vol./vol.) of heat-inactivated fetal bovine serum. Splenocytes (le6/well in 96 well plate) were stimulated for 24 hours with 15-mer overlapping peptides from SARS- CoV-2 spike glycoprotein (JPT Peptide Technologies) or VSV-N (Genscript) at a concentration 2.5 pg/mL. Supernatants were collected, centrifuged at 1,800 RPM for 5 minutes, and stored at -80°C until analysis. Supernatants were then analyzed for the expression of IFN-y, IL-6, IL-18, GM-CSF, IL-10, IL12p70, IL-13, IL-2, IL-4, TNF-a, and IL-5 cytokines using the mouse cytokine 11-plex antibody bead kit (Thl/Th2 Cytokine 11- Plex Mouse ProcartaPlex™ Panel, ThermoFisher). Preparation of samples, along with kit standards, detection antibody and streptavidin-PE, were carried out per manufacturer’s instructions. Cytokine bead fluorescence intensity was measured using the Luminex 200 system (Luminex Corp.), and data was quantitated with xPONENT® Software.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 9.1.0 version for Mac OS 10.15.7. Significant differences among groups were determined using either a two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test or a two-tailed T- test.

Results

Immunogenicity of SARS-CoV-2 Spike antigens in IFNAR~'~ -CD46Ge mice.

Since the SARS-CoV-2 spike is a long 1273 aa protein, this study evaluated the antigenic properties of the SARS-CoV-2 spike protein and three different subunits: (1) the full-length spike ectodomain (S1+S2, amino acids 16 to 1213), (2) the SI domain (amino acids 16 to 685), (3) the S2 domain (amino acids 868 to 685) and (4), the receptor binding domain (RBD, amino acids 319 to 541). Type-I interferon, human CD46 transgenic mice (IFNAR -CD46Ge) were immunized twice at 3 -week intervals with 5 pg of recombinant proteins adjuvanted with aluminum hydroxide gel (Alum) via the intraperitoneal route. Serum samples were then collected on days 21 (before boost) and 42 and analyzed for binding antibodies by ELISA using various spike proteins and domains. After a first immunization, binding antibodies were low to absent, but they were significantly increased after a second dose (FIGS. 1A and IB). The exception was antisera generated with SI -RBD, which exhibited no binding to S1+S2 (FIG. 1A). When analyzed more in detail, both the full- length spike ectodomain (S1+S2) and the S2 subunit elicited comparably strong IgG binding antibodies not only to the S1+S2 but also to the S2 (FIG. IB). On the contrary, both the Sl- RBD and the SI domains generated antibodies that were specific to the Sl-RBD (FIG. IB). These results indicated that the RBD is immunodominant in SI domain, however, most of epitopes laid within the S2 subunit. Alternatively, the lower immunogenicity of the SI and Sl-RBD could be related to loss of structural epitopes in the truncated soluble forms.

Neutralizing antibody (nAb) responses against SARS-COV-2 were next measured using lentiviral pseudotype assay. Some neutralization activity was observed in anti-sera generated by the full-length S1-S2 ectodomain. However, the nAb titers were low and only observed in three out of five animals (FIG. 1C). No neutralizing activity was detected in animals immunized with the other three immunogens. This was also true for antisera generated with the S2 domain, although it contained IgG binding antibodies of similar magnitude to those found after immunization with S1+S2. Thus, antibodies elicited by the soluble and purified full-length spike target predominantly non-neutralizing epitopes.

To assess the ability of the various spike domains to induce a T-cell response, splenocytes from immunized animals were collected three weeks after the booster and analyzed by ELISPOT for by antigen-specific IFN- y. While a similar basic reactivity to unspecific T-cell stimulation with was observed across different groups, no reactivity was observed when splenocytes were stimulated ex vivo with two different pools of SARS-CoV-2 spike peptides (FIG. ID). Taken together, the full-length SARS-CoV-2 spike was the only antigen able to elicit an immune response which exclusively engaged the humoral arm of the immune response but was narrow in the neutralizing activity.

Multivalent display of SARS-CoV-2 spike proteins.

To determine whether the high ratio of binding to neutralizing antibodies for the soluble purified protein related to the lack of a quaternary assembly of the prefusion trimer, a self-trimerizing T4 fibritin motif (FoldON) was included to the full-length spike ectodomain in conjugation with prefusion stabilizing mutations. Other modifications included genetic fusion at the C-terminus of the SARS-CoV-2 spike and of the H. pylori NAP (FIG. 2A). NAP is a 27 nm wide dodecameric protein with four 3-fold axes, a feature that could allow multivalent display of immunogens on the exterior surface. Next, both trimeric and full- length spike ectodomain (S1+S2, herein termed SARS-CoV-2S6p3) and SARS-CoV-2Sp3- NAP (herein termed SARs-CoV-2S6p312) were recombinantly expressed using mammalian cells to ensure proper folding and glycosylation pattern of the proteins. SDS-PAGE analysis followed with Coomassie blue staining of the purified SARS-CoV-2S6p3 and SARS-CoV- 2S6p312 revealed an apparent molecular weight of 180 kDa and 210 kDa, respectively, under reducing condition, which suggest proper genetic fusion of NAP (FIG. 2B, left panel). This analysis also revealed that the preparations were highly pure. Further native gel electrophoresis demonstrated that both SARS-CoV-2 spikes preferentially assembled as mature trimers (three -270 kDa units, FIG. 2B, right panel).

To compare the immunogenicity of the spike proteins, 5-10 IFNAR ' -CD46Ge mice were vaccinated with alum-adj uvanted formulations containing 1 pg or 5 pg of SARS-CoV-2 spike. Serum samples were then collected at week 3 and neutralizing antibodies were determined by VSV-SARS-CoV-2-S pseudoviruses (FIG. 2C). Mirroring the data presented above, not all the animals vaccinated with a prefusion, trimeric SARS-CoV-2 (SARS-CoV- 2S6p3) elicited neutralizing antibody (7/10 for the 1 pg dose and 8/10 for the 5 pg dose) and those that did, had low geometric mean titer (GMT); i.e., 66 and 144. In contrast, all animals vaccinated with a homologous spike fused to NAP (SARS-CoV-2S6p312) seroconverted and elicited a GMT one order of magnitude higher; i.e., 1143 and 1159 for the 1 pg and 5 pg dose, respectively. These data strongly suggested that the SARS-CoV-2 was poorly immunogenic but that a multivalent display of SARS-CoV-2 on a self-assembling nanoparticle scaffold markedly improved its immunogenicity.

Generation and Characterization of rMeV expressing soluble SARS-CoV-2 spike

To examine the immunogenicity of various length SARS-CoV-2 spike proteins when expressed in Moraten Resurfaced measles virus (MeV-MR). A panel of rMeV-MR encoding unmodified or modified versions of the spike with different truncations were cloned between the MeV-P and MeV-M coding sequences of the rMeV-MR. Among the modifications into the spike, the native signal sequence for the murine IgG K leader sequence was replaced followed by a hemagglutinin (HA) tag and included for comparison two sets of prefusion- stabilized forms, the S-2P construct 57 and the so-called HexaPro (S-6P,) and genetic fusion of NAP was included with or without the presence of the FoldON domain.

Altogether, six different constructs were designed (FIG. 3 A): (i) WT leader sequence with S deletion of the cytoplasmic tail (Cov-SACT); (ii) WT leader sequence with S deletion of the cytoplasmic tail in addition to alteration of the furin cleavage site and six prolinestabilizing amino acid substitutions (Cov-S6ACT); (iii) IgG K leader sequence with spike deletion of the transmembrane region, reflecting the soluble ectodomain, as well as the cytoplasmic tail, alteration of the furin cleavage site and two proline-stabilizing amino acid substitutions (CoV-S2p); (iv) IgG K leader sequence with spike deletion of the transmembrane region and cytoplasmic tail, with mutation of the furin cleavage site and two proline-stabilizing mutations, fused to NAP (CoV-S2pl2); (v) IgG K leader sequence with spike deletion of the transmembrane region and cytoplasmic tail, with alteration of the furin cleavage site and two proline-stabilizing amino acid substitutions, and a FoldON trimerization motif followed by NAP (CoV-S2p312); (vi) IgG K leader sequence with spike deletion of the transmembrane region and cytoplasmic tail, with alteration of the furin cleavage site and six proline-stabilizing amino acid substitutions, and a FoldON trimerization motif followed by NAP (CoV-S6p312).

All rMeVs were rescued and propagated on Vero cells to produce virus stock, reaching comparable titers (~ 10⁶ pfu/mL). Next, virus integrity was assessed by full-genome Next-Generation Sequencing (NGS). Unlike the case for the MeV coding-sequences, some amino acid changes were noted on the spike. Namely, the C0V-S6ACT construct encoded 15 not-engineered amino acid changes in addition to an early stop termination due to a single point mutation. Similarly, a single A890V amino acid substation was present on the CoV- S6p312 construct. No amino acid changes were observed in any of the other constructs. These results suggest that a non- fusogenic version of the spike were subjected to selection pressure when displayed onto the MeV coat.

Finally, the expression of the spike protein was analyzed by western blot analysis of Vero cells infected with rMeVs (FIG. 3B). Similar MeV-N antigenic material was immunodetected for all the rMeV, suggesting similar kinetic growth of the viruses. When cells were infected with rMeV expressing SARS-CoV-2 spike with prefusion stabilizing amino acid changes (MR-S2p or S6p), the full-length spike proteins were detected with antibodies to the SARS-CoV-2 spike and to the N-terminus HA-tag. Among these constructs and in the absence of C-terminal NAP, recombinant spike was mostly soluble expressed and secreted into the culture medium (MR-CoV-S2p). When NAP was genetically added to the C-terminus of the spike, the spike was detected both in the culture medium and the cell pellet, and an additional band >250 kDa was detected. In cells infected with the MR- CoV- SACT, both full-length and cleaved spike were detected exclusively with the SARS-CoV-2 spike antibody, and they were detected predominantly in the cell lysate. Compared to the soluble and prefusion stabilized constructs where low band intensity was noted when using an anti-SARS-Co-V-2 spike antibody, a prominent signal was observed for SACT. These results indicated that the rMeV expressed various recombinant spike proteins and that both oligomerization status and epitope accessibility varies among them.

Immunogenicity of MeV expressing SARS-CoV-spike antigens.

To evaluate the immunogenicity of the different vaccine candidates, IxlO⁵ plaqueforming units of various viruses were used to vaccinate 8 to 12-weeks-old IFNAR ^/_- CD46Ge mice on days 0 and 21. Serum samples were then collected at days 21 (before boost) and 41 to assess the presence of S- and MeV-specific IgG antibodies by ELISA. As negative control for vaccination, an isogenic MeV-MR encoding an irrelevant antigen or a VSV G protein pseudotyped VSV expressing SARS2 spike (VSV(+G)SARS2) was used.

End-point titers of sera from animals vaccinated with rMeV constructs developed binding antibodies to MeV antigens that were detected 3 weeks after the first vaccination, and that increased by more than one-log after a second dose with the homologous virus, indicating vaccine uptake in all animals (FIG. 4A). Even though IgG antibodies specific to MeV were detected in animals that received MeV-MR, seroconversion to S ARS2 was not observed in all groups even after two doses. Specific IgG antibodies to SARS2 spike were detected in 100% of animals vaccinated once with rMeV expressing a trimeric and stabilized SARS2 spike, CoV-S2p312 and CoV-S6p312, and twice in those animals that received rMeV-MR-CoV-S2pl2 and VSV(+G)SARS2 (FIG. 4B). The neutralizing activity of the antibodies was next measured using SARS2 spike-pseudovirus. Sera from animals vaccinated with the trimeric and stabilized SARS2 spike (CoV-S2p312 and CoV-S6p312) contained pseudovirus-neutralizing antibodies after one dose, whereas two doses of VSV(+G)SARS2 were required to detect neutralizing activity in some of the vaccinated animals (FIG. 4C). An additional experiment showed that increasing the virus inoculum for the vaccination studies from IxlO⁵ to 5xl0⁵ or 2xl0⁶ boosted binding antibodies to SARS2 spike unrelated to the age of the animals. However, pseudovirus-neutralization activity was mainly unaffected (FIG. 9). Among animals vaccinated with trimeric and stabilized SARS2 construct, the biggest difference was observed in their pseudovirus-neutralizing activity, where one dose of the rMeV-MR-CoV-S6p312 was superior than two doses of rMeV-CoV- S2p312.

Cell-mediated immunity was assessed on day 41 by ELISPOT analysis. All animals that had been vaccinated with any rMeV showed a strong IFN- y -producing T-cell response (FIG. 4D). Similarly, splenocytes of all animals that received viral vectors expressing SARS2 constructs showed a reactivity to SARS2 peptides, even though some animals previously failed to mount an SARS2-specific IgG response. When two pools of SARS2 spike were used to stimulate the splenocytes, a higher frequency of IFN- y -producing T-cells for peptides spanning the S2 subunit (aa 633 to 1258) was observed. These results demonstrated that trimerization of the SARS2 spike protein improves induction of SARS2 IgG antibodies and that prefusion stabilizing mutations augment neutralizing activity.

Skewing of the humoral immune response

Th2-skewed immune responses have been observed in severe SARS-CoV-2 patients. To examine T-cell polarization, the two IgG subclasses of SARS-CoV-2 spike-specific antibodies were determined by ELISA. As a control for a Th2-skewed response, serum from mice immunized twice with alum-adj uvanted SARS-CoV-2 spike protein were used. In these mice, a significant (p<0.05) difference in IgGl and IgG2a subclasses were observed, with IgGl being greater than IgG2a (FIG. 5A). In contrast, mice vaccinated with MeV-MR-CoV- S6p312 elicited comparable antibody titers of IgGl and IgG2a after one dose. After two doses, a significant predominance of IgG2a was observed, indicative of a Th 1 -skewed response. To confirm the Thl/Th2 balance, splenocytes of vaccinated animals were treated with dimethyl sulfoxide (DMSO) or a SARS-CoV-2 peptide pool, and cytokine secretion was quantified in the cell culture supernatant with a ProcartaPlex multiplex panel. The results showed a strong polarity towards Thl by the production of IL-ip, IL-2, IL- 12, TNF-a, and IFN-y. Th2 cytokines (IL-6, IL-5, IL-4, and IL- 13) were not detected. Collectively, both humoral and cellular responses revealed a Thl -biased immunity elicited by MR-SARS-CoV- 2S6p312.

Neutralizing antibody response against SARS-CoV-2 variants

The study assessed whether this favorable Th-1 type immune response elicited in MeV-vaccinated animals would neutralize SARS-CoV-2 variants. To evaluate whether the omicron variant was resistant to neutralizing antibody responses elicited in mice vaccinated with MeV-MR-CoV-S6p312, antibody-neutralization was performed with pseudoviruses expressing the omicron BA.l variant SARS-CoV-spike containing the lineage-defining amino acid changes: A67V, deletion (A)H69-V70, T95I, G142D, AV143-Y145, AN211, L212I, ins214EPE, T547K, D614G, H655Y, N679K, P681H, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H,N764K, D796Y, N856K, Q954H, N969K, L981F. Moreover, additional pseudoviruses harboring spike proteins of other variants were also generated.

Sera from mice vaccinated with a single vaccine dose of MR-SARS-CoV-2S6p312 similarly neutralized (p>0.05) pseudoviruses harboring spikes from epsilon, delta, beta, kappa, alpha and gamma. However, a partial or complete loss of neutralization was seen for pseudoviruses harboring spikes from gamma, lambda, omicron-BA. l, mu, and iota.

Immunogenicity of heterologous booster with omicron-based MR-SARS-CoV-2S6p312

To begin to address whether a homologous wt MR-SARS-CoV-2S6p312 or a heterologous omicron BA.l-matched MR-SARS-CoV-2S6p312 vaccine candidate could broaden neutralizing responses against omicron variant, two cohorts of 15-17-weeks old IFNAR -CD46Ge mice sequentially vaccinated at weeks 0 and 10 with two doses of wt or one dose of wt and another dose of the BA.1-matched MR-SARS-CoV-2S6p312 vaccine candidate. The study used 10-week between vaccination doses to ensure the presence of affinity-mature, class-switched memory B cells and long-lived plasma cells. The study collected blood samples to measure VSV-SARs-CoV-2-S pseudoviruses neutralizing antibodies at peak levels (week 3), before boosting (week 10), and 3 weeks thereafter (3- weeks post-boosting). Comparing the 3-week to 10 week-serum samples, a statistically significant reduction in Wuhan-neutralization activity was observed (3 -fold, p<0.0005) in wt vaccinated mice (FIG. 6A). Confirming the previous results, omicron-neutralizing antibodies were low to absent in these animals (FIG. 6B). A homologous wt booster shot significantly augmented Wuhan-neutralizing antibodies (p<0,0005), reaching levels comparable to week 3 post first dose (FIG. 6A, left panel). Although omicron-neutralizing antibodies were now detected in all the animals, GMT of neutralization were low (i.e., (1/dilution) + SEM of 72.8 ± 11.0; FIG. 6B, left panel). On the other hand, an omicron-based booster shot augmented antibody titers not only against omicron variant but also rescued antibody titers against Wuhan-pseudoviruses (FIG. 6A and 6B, right panels). The neutralization titers of Wuhan- pseudovirus were equivalent (p>0.05) between animals receiving wt or omicron-based boosters. Together, these data strongly suggested that omicron could be considered a SARS- CoV-2 serotype and that an omicron-based booster can restore not only neutralizing antibody titers against the homotypic virus, but also historical SARS-CoV-2 variants.

Protection against SARS-CoV-2 by passive transfer of serum

To determine the protective efficacy of homologous and heterologous boost, a passive antibody transfer was conducted followed by challenge with SARS-CoV-2 virus. Sera was pooled from the IFNAR ' -CD46Ge mice boosted with wt or omicron-based MR-SARS- CoV-2S6p312 and 150 μL was administered into the peritoneum of K18-hACE mice, which express hACE2 under an epithelial cytokeratin promoter. As mock-vaccination control, serum from IFNAR -CD46Ge mice vaccinated twice with a MeV-MR empty vector. Animals were then challenged 2 hours later by the intranasal route with 10⁴ pfu of: 1) USA- WA1/2020 SARS-CoV-2 virus, or 2) omicron BAI virus. Mice were monitored for sign of clinical disease following infection, including daily weight changes. On day 5 postinfection, mice were euthanized and lung tissues and nasal turbinates were collected to determine virus titers by plaque assay. For mice challenged with USA-WA1/2020, those that were pretreated with vaccination serum showed no sights of weight loss, contrary to the sham group where weight loss was observed starting at 4 days post infection (dpi) (FIG. 6C). Although a substantial replication occurred in the lungs of passively immunized animals, serum from animals receiving both vaccinations demonstrated similar reduced lung viral titers (~ 16-fold). Only animals that received serum from wt MR-SARS-CoV-2S6p312 animals showed a reduction in nasal viral titers (175-fold) compared to the empty MeV-MR control group.

For mice challenge with BA.1 virus, the study did not observe any body weight loss, and viral titers in the lungs and in the nasal turbinates were —100-fold lower than those previously detected with USA-WA1/2020. Although SARS-CoV-2 virus was recovered in the lungs of all vaccinated mice after the challenge, no infectious virus was detected in the nasal turbinates. Mice vaccinated with omicron-based MR-SARS-CoV-2S6p312 vector showed a ~4-fold reduction in lung viral titers compared to wt and mock-vaccinated animals. Thus, protection against BA.l was improved in animals boosted with a BAl-based booster vaccine.

Effect of pre-existing measles immunity on the immunogenicity of MeV-MR vectored CO VID 19 vaccine candidate

To test whether the observed pseudovirus-neutralizing responses could be hampered by measles pre-immunity, IFN AR -CD46Gc mice were vaccinated in the presence or absence of MeV-specific IgG. For this, 400 mIU of MeV neutralizing antibodies (nAb) were administered three hours prior to vaccination with either the MeV-MR-CoV-S6p312 or the MeV Moraten vaccine, which was used for vaccination control. Three weeks later, animals were boosted following another passive administration of MeV-nAbs (FIG. 7A). The neutralizing antibody response was assessed against the MeV Moraten vaccine or SARS2- pseudoviruses as well as against T-cell immunity. The MeV neutralizing antibodies were not detected at any time point in mice vaccinated with MeV-MR-CoV-S6p312. In contrast, naive animals vaccinated with the homologous Moraten virus developed mean MeV neutralization titers of 6,194 mIU/mL. However, when animals were passively immunized, the production of MeV nAbs was reduced at 136 mIU/mL (FIG. 7B). Data obtained at the same time point were also collected to analyze the immune response against the SARS-CoV-2 spike protein generated in response to MR-CoV-S6p312. Similar levels of pseudovirus nAbs were present in naive animals and animals with preexisting anti-MeV antibodies. Pseudovirus nAbs were not detected after vaccination with the MeV Moraten vaccine (FIG. 7C). ELISPOT assays performed three weeks after the second dose revealed no significant differences (p>0.05) in the number of SARS-CoV-2 spikespecific IFNy-producing cells in the animals vaccinated in die presence or absence of preexisting anii-MeV antibodies. However, a significant decrease in the number of MeV-N- specific IFN-y-producing cells was observed (FIG. 7D). In conclusion, pre-existing MeV nAbs do not hamper the immunogenicity of the MeV-MR-vectored vaccine, since titers were comparable to those observed in naive animals. Collectively, these results suggest that a MeV/SARS-CoV-2 vaccine candidate based on a remodeled MeV can be used as an effective strategy to elicit long-lasting nAb responses against SARS-CoV-2 virus in a measles-immune human population.

Example 2: Exemplary SARS-CoV-2 Polypeptides LYLSSHRGVITDNQANWAVPTTRTDDKLQKGTCFQQACKGKIQALCENLEWAPLKD SRIP S YGVL S VNL SL AAEPKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNL ALGVINTLKWIPRLKVSPYLFTVPIEEADEDCRAPTYLPAEVTGDVKLSSNLVILPGQD LQYVLATYDTSGVEHAVVYYVYSPGGSFSYVYPFRLPIKGTPIELQVECFTWAQRLW CRHFCVLADSESGGHLTHSGMVGMEVSCTVNREDEANRR (SEQ ID N0:4)

Example 4: Exemplary MeV F Polypeptides

WT MeV F polypeptide

MSIMGLKVNVSAIFMAVLLTLQTPTGQIHWGNLSKIGVVGIGSASYKVMTRSSHQSL VIKLMPNITLLNNCTRVEIAEYRRLLRTVLEPIRDALNAMTQNIRPVQSVASSRRHKRF AGVVLAGAALGVATAAQITAGIALHQSMLNSQAIDNLRASLETTNQAIETIRQAGQE MIL AVQGVQD YINNELIP SMNQL S CDLIGQKLGLKLLRYYTEIL SLFGP SLRDPI S AEI S IQALSYALGGDINKVLEKLGYSGGDLLGILESGGIKARITHVDTESYFIVLSIAYPTLSE IKGVIVHRLEGVS YNIGS QEWYTTVPKYVATQGYLISNFDE S S CTFMPEGTVC SQNAL YPMSPLLQECLRGYTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTIINQD PDKILTYIAADHCPVVEVNGVTIQVGSRRYPDAVYLHRIDLGPPISLERLDVGTNLGN AIAKLEDAKELLESSDQILRSMKGLSSTSIVYILIAVCLGGLIGIPALICCCRGRCNKKG EQVGMSRPGLKPDLTGTSKSYVRSL (SEQ ID NO: 5)

Modified F polypeptide

MSIMGLKVNVSAIFMAVLLTLQTPTGQIHWNNLSTIGIIGTDSVHYKIMTRPSHQYLVI KLMPNVSLIENCTK AELGEYEKLLNS VLEPINQ ALTLMTKNVKPLQ SLGS GRRQRRF AGVVLAGVALGVATAAQITAGIALHQSNLNAQAIQSLRTSLEQSNKAIEEIREATQETV lAVQGVQDYVNNELVPAMQHMSCELVGQRLGLRLLRYYTELLSIFGPSLRDPISAEISI QALIYALGGEIHKILEKLGYSGSDMIAILESRGIKTKITHVDLPGKFIILSISYPTLSEVK GVIVHRLEAVSYNIGSQEWYTTVPRYIATNGYLISNFDESSCVFVSESA1CSQNSLYPM SPFLQQCIRGDTSSCARTLVSGTMGNKFILSKGNIVANCASILCKCYSTSTIINQSPDKL LTFIASDTCPLVEIDGATIQVGGRQYPDMVYEDKVALGPAISLDRLDVGTNLGNALKK LDDAKVLIDSSNQILETVRRSSFNFGSLLSVPILSCTALALLLLIYCCKRRYQQTLKQH TKVDPAFKPDLTGTSKSYVRSL

(SEQ ID N0:6)

Example 5: Treating SARS-CoV-2

A human identified as needing an increase in an immune response against a SARS- CoV-2 infection (e.g., COVID-19) such as a human having or at risk of developing COVID- 19 is administered one or more multimeric SARS-CoV-2 immunogens provided herein.

In some cases, multimeric SARS-CoV-2 immunogens assemble (e.g., self-assemble) into a nanoparticle in vivo.

The multimeric SARS-CoV-2 immunogens can induce an immune response against SARS-CoV-2 to prevent the development of one or more symptoms of COVID-19.

Example 6: Treating SARS-CoV-2

A human identified as needing an increase in an immune response against a SARS- CoV-2 infection (e.g., COVID-19) such as a human having or at risk of developing COVID- 19 is administered two or more (e.g., two, three, four, five, or more) polypeptides each containing a SARS-CoV-2 immunogen fiised to a multimerization domain, such that the two or more polypeptides can assemble (e.g., self-assemble) in vivo to form a multimeric SARS- CoV-2 immunogen.

In some cases, polypeptides each containing a SARS-CoV-2 immunogen fused to a multimerization domain also include a scaffold polypeptide, such that the multimeric SARS- CoV-2 immunogens assemble (e.g., self-assemble) into a nanoparticle in vivo.

Example 7: Treating SARS-CoV-2

A human identified as needing an increase in an immune response against a SARS- CoV-2 infection (e.g., COVID-19) such as a human having or at risk of developing COVID- 19 is administered recombinant MeV vectors provided herein (e.g., MeV vectors including nucleic acid encoding a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain) and/or nucleic acid molecules that can encode a recombinant MeV vector provided herein. The recombinant MeV vectors infect cells such that the infected cells express a polypeptide including a SARS-CoV-2 immunogen fused to a multimerization domain, and such that two or more (e.g., two, three, four, or more) of the polypeptides including a SARS-CoV-2 immunogen fused to a multimerization domain form a multimeric SARS-CoV-2 immunogen in vivo. The multimeric SARS-CoV-2 immunogens can induce an immune response against SARS-CoV-2 to reduce the severity of or eliminate one or more symptoms of COVID- 19. OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide comprising (1) an immunogen, (2) a multimerization domain, and (3) a scaffold polypeptide, wherein two or more of said polypeptides multimerize in vivo within a mammal administered said two or more of said polypeptides to form a multimeric immunogen, wherein two or more of said multimeric immunogens assemble in vivo to form a nanoparticle, and wherein said mammal produces an immune response against the immunogen.

2. The polypeptide of claim 1, wherein said mammal is a human.

3. The polypeptide of any one of claims 1-2, wherein said immunogen comprises a coronavirus amino acid sequence.

4. The polypeptide of claim 3, wherein said coronavirus amino acid sequence comprises at least a portion of a coronavirus spike (S) polypeptide.

5. The polypeptide of claim 4, wherein said portion of said coronavirus S polypeptide is at least 232 amino acids in length.

6. The polypeptide of any one of claims 3-5, wherein said coronavirus amino acid sequence is a full-length coronavirus S polypeptide.

7. The polypeptide of any one of claims 3-5, wherein said coronavirus amino acid sequence is a coronavirus S polypeptide comprising a modified furin cleavage site, wherein said modified furin cleavage site is resistant to proteolytic cleavage.

8. The polypeptide of claim 7, wherein said modified coronavirus S polypeptide fragment comprises two or more proline substitutions selection from the group consisting of F817P, A892P, A899P, K986P, V987P, and A942P.

9. The polypeptide of claim 8, wherein said modified coronavirus S polypeptide fragment comprises proline substitutions at K986P and V987P.

10. The polypeptide of claim 8, wherein said modified coronavirus S polypeptide fragment comprises proline substitutions at F817P, A892P, A899P, and A942P.

11. The polypeptide of claim 8, wherein said modified coronavirus S polypeptide fragment comprises proline substitutions at F817P, A892P, A899P, K986P, V987P, and A942P.

12. The polypeptide of any one of claims 3-7, wherein said coronavirus amino acid sequence comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 14.

13. The polypeptide of any one of claims 3-12, wherein said coronavirus is a betacoronavirus.

14. The polypeptide of claim 13, wherein said betacoronavirus is SARS-CoV-2.

15. The polypeptide of any one of claims 1-14, wherein said multimerization domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO 21.

16. The polypeptide of any one of claims 1-15, wherein said immunogen further comprises an N-terminal leader sequence.

17. The polypeptide of claim 16, wherein said leader sequence is an IgG K leader sequence.

18. The polypeptide of any one of claims 1-17, wherein three of said polypeptide form a trimeric immunogen within said mammal.

19. The polypeptide of any one of claims 1-18, wherein said scaffold polypeptide is a C- terminal scaffold polypeptide.

20. The polypeptide of any one of claims 1-19, wherein said scaffold polypeptide is a neutrophil-activating protein (NAP) polypeptide.

21. The polypeptide of claim 20, wherein said NAP polypeptide is a Helycobacter pylori NAP polypeptide.

22. The polypeptide of any one of claims 1-21, wherein a polypeptide linker is located between said multimerization domain and said scaffold polypeptide.

23. The polypeptide of claim 22, wherein said polypeptide linker is a GlySer linker.

24. The polypeptide of any one of claims 1-23, wherein said nanoparticle comprises 12 of said multimeric immunogens.

25. A multimeric immunogen comprising two or more polypeptides, each polypeptide comprising (1) an immunogen, (2) a multimerization domain, and (3) a scaffold polypeptide, wherein two or more of said multimeric immunogens assemble in vivo within a mammal administered said two or more of said multimeric immunogens to form a nanoparticle, and wherein said mammal produces an immune response against the immunogen.

26. The multimeric immunogen of claim 25, wherein said mammal is a human.

27. The multimeric immunogen of any one of claims 25-26, wherein said immunogen comprises a coronavirus amino acid sequence.

28. The multimeric immunogen of claim 27, wherein said coronavirus amino acid sequence comprises at least a portion of a coronavirus S polypeptide.

29. The multimeric immunogen of claim 28, wherein said portion of said coronavirus S polypeptide is at least 232 amino acids in length.

30. The multimeric immunogen of any one of claims 27-29, wherein said coronavirus amino acid sequence is a full-length coronavirus S polypeptide.

31. The multimeric immunogen of any one of claims 27-30, wherein said coronavirus amino acid sequence is a coronavirus S polypeptide comprising a modified furin cleavage site, wherein said modified furin cleavage site is resistant to proteolytic cleavage.

32. The multimeric immunogen of claim 31, wherein said modified coronavirus S polypeptide fragment comprises two or more proline substitutions selection from the group consisting of F817P, A892P, A899P, K986P, V987P, and A942P.

33. The multimeric immunogen of claim 32, wherein said modified coronavirus S polypeptide fragment comprises proline substitutions at K986P and V987P.

34. The multimeric immunogen of claim 32, wherein said modified coronavirus S polypeptide fragment comprises proline substitutions at F817P, A892P, A899P, and A942P.

35. The multimeric immunogen of claim 32, wherein said modified coronavirus S polypeptide fragment comprises proline substitutions at F817P, A892P, A899P, K986P, V987P, and A942P

36. The multimeric immunogen of any one of claims 27-31, wherein said coronavirus amino acid sequence comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 14.

37. The multimeric immunogen of any one of claims 27-36, wherein said coronavirus is a betacoronavirus.

38. The multimeric immunogen of claim 37, wherein said betacoronavirus is SARS-CoV- 2.

39. The multimeric immunogen of any one of claims 25-38, wherein said multimerization domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:21.

40. The multimeric immunogen of any one of claims 25-39, wherein said immunogen further comprises an N-terminal leader sequence.

41. The multimeric immunogen of claim 40, wherein said leader sequence is an IgG K leader sequence.

42. The multimeric immunogen of any one of claims 25-41, wherein three of said immunogens form a trimeric complex within said mammal.

43. The multimeric immunogen of any one of claims 25-41, wherein said scaffold polypeptide is a C-terminal scaffold polypeptide.

44. The multimeric immunogen of any one of claims 25-43, wherein said scaffold polypeptide is a NAP polypeptide.

45. The multimeric immunogen of claim 44, wherein said NAP polypeptide is a H. pylori NAP polypeptide.

46. The multimeric immunogen of any one of claims 25-45, wherein a polypeptide linker is located between said multimerization domain and said scaffold polypeptide.

47. The multimeric immunogen of claim 46, wherein said polypeptide linker is a GlySer linker.

48. The multimeric immunogen of any one of claims 25-47, wherein said nanoparticle comprises 12 of said multimeric immunogens.

49. A nucleic acid encoding a polypeptide comprising (1) an immunogen, (2) a multimerization domain, and (3) a scaffold polypeptide, wherein cells within a mammal administered said nucleic acid express said polypeptide, wherein two or more of said polypeptide multimerize in vivo to form a multimeric immunogen, wherein two or more of said multimeric immunogen assemble in vivo to form a nanoparticle, and wherein said mammal produces an immune response against a coronavirus.

50. The nucleic acid of claim 49, wherein said mammal is a human.

51. The nucleic acid of any one of claims 49-50, wherein said immunogen comprises a coronavirus amino acid sequence.

52. The nucleic acid of claim 51, wherein said coronavirus amino acid sequence comprises at least a portion of a coronavirus S polypeptide.

53. The nucleic acid of claim 52, wherein said portion of said coronavirus S polypeptide is at least 232 amino acids in length.

54. The nucleic acid of any one of claims 51-53, wherein said coronavirus amino acid sequence is a full-length coronavirus S polypeptide.

55. The nucleic acid of any one of claims 51-54, wherein said coronavirus amino acid sequence is a coronavirus S polypeptide comprising a modified furin cleavage site, wherein said modified furin cleavage site is resistant to proteolytic cleavage.

56. The nucleic acid of claim 55, wherein said modified coronavirus S polypeptide fragment comprises two or more proline substitutions selection from the group consisting of F817P, A892P, A899P, K986P, V987P, and A942P.

57. The nucleic acid of claim 56, wherein said modified coronavirus S polypeptide fragment comprises proline substitutions at K986P and V987P.

58. The nucleic acid of claim 56, wherein said modified coronavirus S polypeptide fragment comprises proline substitutions at F817P, A892P, A899P, and A942P.

59. The nucleic acid of claim 56, wherein said modified coronavirus S polypeptide fragment comprises proline substitutions at F817P, A892P, A899P, K986P, V987P, and A942P.

60. The nucleic acid of any one of claims 51-55, wherein said coronavirus amino acid sequence comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 13 or the amino acid sequence set forth in SEQ ID NO: 14.

61. The nucleic acid of any one of claims 51-60, wherein said coronavirus is a betacoronavirus.

62. The nucleic acid of claim 61, wherein said betacoronavirus is SARS-CoV-2.

63. The nucleic acid of any one of claims 49-62, wherein said multimerization domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:21.

64. The nucleic acid of any one of claims 49-63, wherein said immunogen further comprises an N-terminal leader sequence.

65. The nucleic acid of claim 64, wherein said leader sequence is an IgG K leader sequence.

66. The nucleic acid of any one of claims 49-65, wherein said scaffold polypeptide is a C- terminal scaffold polypeptide.

67. The nucleic acid of any one of claims 49-66, wherein said scaffold polypeptide is a NAP polypeptide.

68. The nucleic acid of claim 67, wherein said NAP polypeptide is a H. pylori NAP polypeptide.

69. The nucleic acid of any one of claims 49-68, wherein a polypeptide linker is located between said multimerization domain and said scaffold polypeptide.

70. The nucleic acid of claim 69, wherein said polypeptide linker is a GlySer linker.

71. The nucleic acid of any one of claims 49-70, wherein said nanoparticle comprises 12 of said multimeric immunogens.

72. The nucleic acid of any one of claims 49-71, wherein the nucleic acid is in form of a viral vector.

73. The nucleic acid of claim 72, wherein the viral vector is a recombinant MeV vector.

74. The nucleic acid of claim 73, wherein said recombinant MeV comprises a modified H polypeptide.

75. The nucleic acid of claim 74, wherein said modified H polypeptide comprises, consists essentially of, or consists of an amino acid sequence set forth in SEQ ID NO:4.

76. The nucleic acid of claim 73, wherein said recombinant MeV comprises a modified F polypeptide.

77. The nucleic acid of claim 76, wherein said modified F polypeptide comprises, consists essentially of, or consists of an amino acid sequence set forth in SEQ ID NO:6.

78. A composition comprising the polypeptide of any one of claims 1-24, the multimeric immunogen of any one of claims 25-48, or the nucleic acid of any one of claims 49-77.

79. The compositions of claim 52, wherein said composition comprises an aluminum hydroxide adjuvant.

80. A method for inducing an immune response against a coronavirus in a mammal, wherein said method comprises administering the polypeptide of any one of claims 1-24, the multimeric immunogen of any one of claims 25-48, or the nucleic acid of any one of claims 49-77, or the composition of any one of claims 78-79 to said mammal under conditions wherein said nanoparticle in said mammal leads to induction of said immune response.

81. The method of claim 80, wherein said mammal is a human.

82. The method of any one of claims 80-81, wherein said coronavirus is a betacoronavirus.

83. The method of claim 82, wherein said betacoronavirus is SARS-CoV-2.

84. The method of claim 83, wherein said SARS-CoV-2 is selected from the group consisting of a B.1.17 (alpha), a Bl .351 (beta), a Pl (gamma), a B.1.617.2 (delta), a B.l.1.529 (omicron), a B.1.526 (iota), a B.1.617.1 (kappa), a C.37 (lamda), a B.1.621 (mu), a B.1.427/B.1.429 (epsilon), P2 (zeta), and any combination thereof.

85. The method of any one of claims 80-84, wherein said administering comprises a single administration.

86. The method of any one of claims 80-85, wherein said administering is a nasal administration.

87. The method of any one of claims 80-86, wherein said immune response is an IgG antibody response.

88. The method of any one of claims 80-86, wherein said immune response is an IgA antibody response.

89. The method of any one of claims 80-86, wherein said immune response is a Thl cell- mediated response.

90. The method of any one of claims 80-86, wherein said immune response is a Th2 cell- mediated response.

91. The use of a composition comprising the polypeptide of any one of claims 1-24, the multimeric immunogen of any one of claims 25-48, or the nucleic acid of any one of claims 49-77 to induce an immune response against a coronavirus in a mammal.

92. The polypeptide of any one of claims 1-24, the multimeric immunogen of any one of claims 25-48, or the nucleic acid of any one of claims 49-77 for use in the preparation of a medicament for inducing an immune response against a coronavirus in a mammal.

93. The polypeptide of any one of claims 1-24, the multimeric immunogen of any one of claims 25-48, or the nucleic acid of any one of claims 49-77for use in inducing an immune response against a coronavirus in a mammal.