ENGINEERED hMPV F PROTEIN IMMUNOGENS AND RELATED VACCINES

Abstract

The present invention provides engineered human metapneumovirus (hMPV) F protein trimer immunogens. These engineered proteins are stabilized via specific modifications introduced into both the F2 subunit and the F1 subunit of a wildtype hMPV soluble F sequence, e.g., shortened F2 subunit C -terminus and interprotomer disulfide bond in the F1 subunit. Also provided in the invention are nanoparticle vaccines that contain the engineered hMPV soluble F immunogens displayed on self-assembling nanoparticles. The invention also provides methods of using such vaccine compositions in various therapeutic applications, e.g., for preventing or treating hMPV infections.

Description

Attorney Docket No.: TSRI 2231.1PC ENGINEERED hMPV F PROTEIN IMMUNOGENS AND RELATED VACCINES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Numbers 63 / 614,729 (filed December 26, 2023; now pending). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which is submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml file is named “2231_1PC_Sequence Listing”, was created on December 11, 2024, and is 37 KB bytes in size. BACKGROUND OF THE INVENTION

[0003] Human metapneumovirus (hMPV), along with respiratory syncytial virus (RSV) and parainfluenza virus (PIV), belongs to the Paramyxoviridae family which are enveloped, non-segmented, negative-sense, single-stranded RNA viruses. RSV has been extensively studied since its discovery over 60 years ago, and hMPV was identified ~20 years ago with active ongoing research. RSV and hMPV pose a major burden on human health. RSV is a prevalent cause of acute lower respiratory infection (ALRI) in neonates and infants. RSV causes acute respiratory infection, accounting for ~66,000-200,000 deaths and 3.5 million hospitalizations worldwide in children under 5 years of age. Children infected with hMPV usually exhibit symptoms similar to those of RSV infection. Since discovery, hMPV has been detected in 4–16% of patients with ALRIs. Like RSV, hMPV causes disease primarily in children but can also infect adults and immunocompromised individuals

[0004] RSV and hMPV share similar genomic compositions with several key differences. The RSV genome encodes three envelope glycoproteins, the attachment (G) protein, fusion (F) protein, and small hydrophobic (SH) protein, and eight non- structural proteins (NS1, NS2, N, P, M, M2-1, M2-2, and L). The hMPV genome contains eight open reading frames (ORFs) encoding nucleoprotein (N), phosphoprotein(P), matrix protein (M), transcription enhancer protein (M2), and three envelope glycoproteins (G, F, and SH). For both RSV and hMPV, F and G are crucial for infectivity and pathogenesis and can be recognized by host neutralizing antibodies (NAbs). To enable cell entry, the RSV-F precursor, F0, is first cleaved by furin-like proteases at two sites to remove a 27-aa peptide (P27) and generate two subunits: a N- terminal F2 that is attached to a larger C-terminal F1 subunit by two disulfide bonds to form a heterodimeric protomer, three of which form a functional trimer. For hMPV, proteolytic cleavage at a single site by serine proteases transforms F0 into F2 and F1 subunits, which form a functional prefusion trimer. The metastable prefusion RSV-F and hMPV-F undergo irreversible refolding, during which the hydrophobic fusion peptides are ejected from the central cavity of the F trimer and insert into host cell membranes, to facilitate virus-host membrane fusion and rapid transition of F into a highly stable postfusion form.

[0005] In the last decade, significant advancements have been made in RSV and hMPV vaccine research. First, multiple antigenic sites (AS) on the RSV F protein have been identified that can be recognized by NAbs and non-NAbs. Importantly, co- crystallization of a potent human NAb D25 with RSV-F resulted in the first atomic structure of prefusion F trimer and revealed a novel antigenic site (Ø) near the apex. Second, an in-depth structural understanding has been achieved for the F protein at both prefusion and postfusion states for RSV and hMPV. Despite only ~30% sequence identity, RSV and hMPV share a high structural similarity for prefusion state, in which they have a “football” shaped trimer structure, and postfusion state, in which they have an elongated “stick” shaped trimer structure. Third, structure-based antigen design has played an essential role in the development of prefusion RSV-F vaccines. These studies have paved the way for two approved RSV vaccines, ABRYSVO (GlaxoKlineSmith, GSK) and AREXVY (Pfizer). For hMPV, similar design strategies (e.g., disulfide bond, proline, and cavity-filling substitutions) were recently used to stabilize prefusion F for vaccine development. However, these recently reported hMPV-F constructs contain mutations that can either disrupt trimer formation or introduce unintended intraprotomer disulfide bonds.

[0006] There is an unmet need in the art for better and more effective hMPV prefusion-closed hMPV-F antigens for vaccine development. The present invention is directed to this and other unmet needs in the art.SUMMARY OF THE INVENTION

[0007] In one aspect, the invention provides engineered or redesigned fusion (F) protein trimer immunogens of human metapneumovirus (hMPV). These engineered immunogens contain from N-terminus to C-terminus a modified F2 subunit and a modified F1 subunit. The modified F2 subunit contains an E80D substitution and a C- terminal truncation of about 8-12 amino acid residues. The modified F1 subunit contains an A185P substitution and an engineered disulfide bond via T127C / N153C substitutions. The noted amino acid numbering, based on hMPV isolate Arg / 2 / 02 (UniProt ID Q1A2Z0), is readily applicable to other hMPV subgroups or isolates due to identical protein structures and highly conserved sequences.

[0008] In some engineered hMPV F immunogens of the invention, the modified F2 subunit is covalently bonded to the modified F1 subunit via a peptide linker. In some of these embodiments, the employed peptide linker contain about 2-10 glycine residues. In some engineered hMPV F immunogens of the invention, C-terminal truncation in the modified F2 subunit contains a deletion of 10 C-terminal amino acid residues of the wildtype sequence. In some embodiments, the modified F2 subunit contains SEQ ID NO:4 or a conservatively modified variant thereof, and the modified F1 subunit contains SEQ ID NO:5 or a conservatively modified variant thereof. In some of these embodiments, the engineered hMPV F trimer immunogen contains the sequence set forth in SEQ ID NO:7.

[0009] In some embodiments, other than the above-noted mutations in the modified F2 subunit and the modified F1 subunit, the engineered hMPV F immunogens of the invention additionally contain a V155P substitution in the modified F1 subunit. As exemplification, one such engineered hMPV F immunogen contains the sequence set forth in SEQ ID NO:8. In some other embodiments, other than the above-noted mutations in the modified F2 subunit and the modified F1 subunit, the engineered hMPV F immunogens of the invention additionally contain an interprotomer disulfide bond formed by A120C and Q426C substitutions in the modified F1 subunit. As exemplification, one such engineered hMPV F immunogen contains the sequence set forth in SEQ ID NO:9. In still some other engineered hMPV F immunogens of the invention, the C-terminal truncation in the F2 subunit entails replacement of the C- terminal sequence DQLAREEQIENPRQSR (SEQ ID NO:12) with DGHGHP (SEQ IDNO:13), and the modified F1 subunit additionally contains A120V and Q426L substitutions. As exemplification, one such engineered hMPV F immunogen contains the sequence set forth in SEQ ID NO:10.

[0010] To promote trimer formation and stability, the engineered hMPV F immunogens of the invention can additionally contain a C-terminal trimerization motif. In some of these embodiments, the trimerization motif is a foldon motif, e.g., one containing the sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:6). In various embodiments, the trimerization motif is fused to the C-terminus of the modified F1 subunit via a peptide linker or an enzymatic site, e.g., an AS dipeptide. As exemplification, specific examples of engineered hMPV F trimer immunogens containing a C-terminal trimerization motif are set forth in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, the engineered hMPV F trimer immunogens of the invention can also include an N-terminal leader (signal peptide) sequence, with or without the C-terminal trimerization motif.

[0011] In a related aspect, the invention provides nanoparticle vaccines that contain an engineered hMPV F trimer immunogen described herein that is displayed on the surface of a self-assembling nanoparticle. In some preferred embodiments, the self- assembling nanoparticle contains a trimeric sequence, and the C-terminus of the engineered hMPV F trimer immunogen is fused to the N-terminus of a subunit sequence of the nanoparticle. In some of these embodiments, the employed self- assembling nanoparticle is a I3-01 variant, e.g., an I3-01 variant that contains SEQ ID NO:31 (I3-01v9b) or SEQ ID NO:32 (I3-01v9c).

[0012] In another aspect, the invention provides polynucleotide molecules that encode the engineered hMPV F trimer immunogens or the nanoparticle vaccines described herein. In another related aspect, the invention provides pharmaceutical compositions suitable for therapeutic applications of the engineered hMPV F trimer immunogens described herein. These compositions typically contain an engineered hMPV F trimer immunogen, a nanoparticle vaccine displaying the immunogen, or a polynucleotide encoding the immunogen or nanoparticle vaccine, and a pharmaceutically acceptable carrier. In still another aspect, the invention provides therapeutic methods for preventing or treating metapneumovirus infections in human subjects. These methods involve administering to the subjects a therapeutically effective amount of a pharmaceutical composition described herein.

[0013] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A-1E. Design and in vitro characterization of hMPV-F UFCM1series. SEC profile (left top), DSC profile (left bottom), representative 2D classification images (middle), and 3D reconstruction from nsEM analysis (right) for UFCM1(Fig. 1A), UFCM1-P2 (Fig.1B), UFCM1-P2-iSS (Fig.1C), and UFCM1-P2-F2C-VL (Fig.1D). All constructs were transiently expressed in 25 ml ExpiCHO cells and purified using an MPE8 antibody column and a Nickel column. The trimer (T) peak is marked on the profile. Fig.1E: SDS-PAGE analysis of four UFCM1trimers under reducing conditions. Prior to the analysis, UFCM1trimers were treated with 5 mM disuccinimidyl glutarate (DSG) for crosslinking F protomers.

[0015] Figures 2A-2D. NsEM and antigenic characterization of hMPV-F UFCM1series. Fig.2A: The nsEM analysis of UFCM1-P2-iSS bound to NAb MPE8. The representative 2D classification images are shown on the top, while side and top views of the 3D reconstruction of the complex are shown on the bottom left and right, respectively. A 3.25 Å-resolution cryo-EM model of MPE8 scFv-bound v3B Δ12_D454C-V458C (PDB ID: 8F6X) is used for density fitting. Fig.2B: The nsEM analysis of UFCM1-P2-iSS bound to NAb 101F. The representative 2D classification images are shown on the left, while side and top views of the 3D reconstruction of the complex are shown on the right. A model of 101F Fab modeled on to a prefusion hMPV-F trimer (PDB ID: 5WB0) is used for density fitting. Fig.2C: ELISA analysis of the UFCM1series binding to 4 antibodies. Top: ELISA binding curves. Bottom: ELISA- derived EC50(µg / ml) values. Fig.2D: BLI analysis of the UFCM1series binding to 4 antibodies. Left: Octet binding curves. Right: The matrix of peak values at the highest antigen concentration. Sensorgrams were obtained from an Octet RED96 instrument using an antigen titration series of six concentrations (starting at 600 nM followed by two-fold dilutions).

[0016] Figures 3A-3D. Crystallographic analysis of hMPV-F UFCM1-P2-iSS. Fig. 3A: Crystal structure of UFCM1-P2-iSS, with a resolution of 6.0 Å, is superimposed onto that of 115-BV (PDB ID: 5WB0), which are shown as ribbons models within thegray trimer surface. Due to the limited resolution, structural details cannot be determined for the F2-F1liker, A344-S347, and V442-E457. A dotted line is added to show where the missing F2-F1like might be located. Fig.3B: Structural details of the intra-F2disulfide bond T127C-Q153C, the V155P (P2) mutation inserted into the β3 / β4 hairpin tip for destabilizing the postfusion state, and the interprotomer disulfide bond A120C-Q426C (iSS), which are shown in the top, middle, and bottom insets, respectively. The crystal structure of 115-BV is included for comparison. Fig.3C: Crystal structures of UFCM1-P2-iSS and DS-CavEs2 (PDB ID: 7SEJ) are superimposed and shown as ribbons models within the gray trimer surface. The extended α1 helix in DS-CavEs2 that will clash with an adjacent protomer in a prefusion-closed trimer is circled in a black dotted line box. The closed-up views of this region in DS-CavEs2 and UFCM1-P2-iSS are shown in the right insets. Fig.3D: Crystal structures of UFCM1-P2- iSS and v3B Δ12_D454C-V458C (PDB ID: 8F6X) are superimposed and shown as ribbons models within the gray trimer surface. The F2-F1linker region is circled in a dotted line box.

[0017] Figures 4A-4E. Antibody responses to rationally designed hMPV-F trimer vaccines in mice. Fig.4A: Schematic representation of the mouse immunization regimen for both hMPV-F vaccines (n = 10 mice / group). Fig.4B and Fig.4C: HMPV-F vaccine-induced binding antibody responses against hMPV-F UFCM1-P2-iSS(1TD0) and RSV-F sc9-10 DS-Cav1(1TD0). Fig.4D and Fig.4E: HMPV-F vaccine-induced neutralizing antibody responses against live hMPV-GFP and live RSV-A2-GFP. EC50values (were derived from the ELISA analysis of mouse serum against coating antigens, with geometric mean EC50values labeled on the plots. ID50titers were derived from the live RSV and hMPV neutralization assays, with geometric mean ID50values labeled on the plots. Of note, the ID50values were derived by setting the lower and upper constraints of % neutralization set at 0.0 and 100.0, respectively. The data was analyzed using one-way ANOVA, followed by post-hoc analysis using Tukey’s multiple comparison test for each time point. For significance, ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. DETAILED DESCRIPTION I Overview

[0018] Human metapneumovirus (hMPV) F protein belongs to the family of class I viral fusion proteins that mediate the fusion of viral envelope and cell membrane during infection. hMPV F is first synthesized as a polypeptide precursor, F0, and is then cleaved by an unknown enzyme to generate a heterodimer containing a F2 subunit and a F1 subunit, which are connected by disulfide bonds. The mature prefusion F protein forms a trimer of F2-F1 heterodimers. The prefusion conformation of hMPV F is meta- stable and undergoes conformational rearrangement to the postfusion state during the process of membrane fusion.

[0019] The present invention is derived in part from studies undertaken by the inventors to rationally design new, stable hMPV prefusion F trimers by minimizing F metastability. As detailed herein, the inventors first engineered a base construct, termed UFCM1, which contains a minimum set of mutations and retains hMPV-F in a prefusion conformation (albeit open). Several key mutations were then introduced into the base construct, including a well-positioned interprotomer disulfide bond, to effectively stabilize hMPV-F in a prefusion-closed trimer conformation. Additionally, the inventors tested the possibility of replacing this interprotomer disulfide bond with hydrophobic mutations. Furthermore, the structural integrity of the redesigned hMPV-F proteins, UFCM1-P2-iSS, was validated by negative-stain EM and a 6 Å-resolution crystal structure. In vivo studies confirmed that this redesigned hMPV-F protein could induce robust antibody responses in mice with potent NAb titers.

[0020] The invention accordingly provides engineered hMPV F trimer immunogens and vaccine compositions (including nanoparticle vaccines) in accordance with the engineering strategy described herein. Related polynucleotide sequences, expression vectors and pharmaceutical compositions are also provided in the invention. There are a number of technological advantages provided by the engineered hMPV immunogens described herein. The stabilization strategy described herein, with a minimum set of mutations, can effectively stabilize hMPV-F in a native-like, prefusion- closed trimer conformation, enabling the development of both trimer and nanoparticle vaccines. In contrast, previously reported hMPV-F designs all contain suboptimal mutations that either disrupt trimer formation or introduce unintended disulfide bonds that rearrange / destroy the local structure. The engineered hMPV F trimer proteins of the invention can be transiently expressed in, e.g., ExpiCHO cells. Since CHO is one of the principal mammalian cell lines used for industrial manufacture of proteintherapeutics and vaccines and ExpiCHO is a transient version of this CHO cell line, hMPV-F trimers and nanoparticles obtained from the ExpiCHO production are expected to have the same properties as those from industrial CHO production.

[0021] Unless otherwise specified herein, the hMPV vaccine immunogens of the invention, the encoding polynucleotides, expression vectors and host cells, as well as the related therapeutic applications, can all be generated or performed in accordance with the procedures exemplified herein or routinely practiced methods well known in the art. See, e.g., Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos.4,965,343, and 5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rded., 2000); Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol.57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998).

[0022] The following sections provide additional guidance for practicing the compositions and methods of the present invention. II. Definitions

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1sted., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar(Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rded., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1sted., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4thed., 2000). Further clarifications of some of these terms as they apply specifically to this invention are provided herein.

[0024] As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, "an Env-derived trimer" can refer to both single or plural Env-derived trimer molecules, and can be considered equivalent to the phrase "at least one Env-derived trimer."

[0025] As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and / or cellular type directed against that protein. Unless otherwise noted, the term “vaccine immunogen” is used interchangeably with “protein antigen” or “immunogen polypeptide”.

[0026] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For polypeptide sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0027] Epitope refers to an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and / or T cells respond. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

[0028] Effective amount of a vaccine or other agent that is sufficient to generate a desired response, such as reduce or eliminate a sign or symptom of a condition or disease, such as bronchiolitis or pneumonia. For instance, this can be the amount necessary to inhibit viral replication or to measurably alter outward symptoms of the viral infection. In general, this amount will be sufficient to measurably inhibit virus (for example, hMPV) replication or infectivity. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve in vitro inhibition of viral replication. In some embodiments, an "effective amount" is one that treats (including prophylaxis) one or more symptoms and / or underlying causes of any of a disorder or disease, for example to treat hMPV infection. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with bronchiolitis.

[0029] Unless otherwise noted, a fusion protein is a recombinant protein containing amino acid sequence from at least two unrelated proteins that have been joined together, via a peptide bond, to make a single protein. Thus, it does not encompass the naturally existing hMPV surface antigen that is termed fusion (F) protein as described herein. The unrelated amino acid sequences can be joined directly to each other or they can be joined using a linker sequence. As used herein, proteins are unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment(s) (e.g., inside a cell). For example, the amino acid sequences of bacterial enzymes such as B. stearothermophilus dihydrolipoyl acyltransferase (E2p) and the amino acid sequence of an engineered hMPV soluble F protein are not normally found joined together via a peptide bond.

[0030] Immunogen is a protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogen can lead to protective immunity and / or proactive immunity against a pathogen of interest.

[0031] Immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against virus expressing the immunogenic polypeptide, or induces a measurable B cell response (such as production of antibodies) against the immunogenic polypeptide.

[0032] Sequence identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0033] Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity / similarity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math.2:482, 1981; Needleman & Wunsch, J. Mol. Biol.48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237- 44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio.24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0034] The term "subject" refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. Preferably, the subject is human.

[0035] The term “treating” or “alleviating” includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., an hMPV infection), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0036] Vaccine refers to a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell or one or more cellular constituents. In some embodiments of the invention, vaccines or vaccine immunogens or vaccine compositions are expressed from fusion constructs and self-assemble into nanoparticles displaying an immunogen polypeptide or protein on the surface.

[0037] Virus-like particle (VLP) refers to a non-replicating, viral shell, derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, for example, Baker et al. (1991) Biophys. J.60:1445-1456; and Hagensee et al. (1994) J. Virol.68:4503-4505.For example, VLPs can be isolated by density gradient centrifugation and / or identified by characteristic density banding. Alternatively, cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.

[0038] A self-assembling nanoparticle refers to a ball-shape protein shell with a diameter of tens of nanometers and well-defined surface geometry that is formed by identical copies of a non-viral protein capable of automatically assembling into a nanoparticle with a similar appearance to VLPs. A notable example of self-assembling nanoparticles is engineered protein I3-01 (Hsia et al., Nature 535, 136-139, 2016) and variants derived therefrom, including I3-01v9b and I3-01v9c exemplified herein. Other examples include ferritin (FR), which is conserved across species and forms a 24-mer, as well as B. stearothermophilus dihydrolipoyl acyltransferase (E2p), Aquifex aeolicus lumazine synthase (LS), and Thermotoga maritima encapsulin, which all form 60-mers. Self-assembling nanoparticles can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for nanoparticle production, detection, and characterization can be conducted using the same techniques developed for VLPs. III. hMPV fusion (F) glycoproteins

[0039] The invention provides novel engineered immunogenic proteins and vaccine compositions that contain a modified soluble F glycoprotein sequence of a human metapneumovirus. Human metapneumovirus (hMPV) is a negative-sense single-stranded enveloped RNA virus in the family Pneumoviridae. There are two circulating genotypes of hMPV (A and B), which are further divided into four subgroups, A1, A2, B1, and B2, based on the sequence variability of the surface proteins. The hMPV F protein plays an indispensable role in viral infection. It belongs to the family of class I viral fusion proteins that mediate the fusion of viral envelope and cell membrane during infection. The hMPV F protein is highly conserved among hMPV subgroups, and also shares similar structural topology and approximately 30% amino acid sequence homology with the respiratory syncytial virus (RSV) F protein.

[0040] The hMPV F protein is homotrimeric. It is first synthesized as a polypeptide precursor, F0, which contains from the N-terminus to the C-terminus: the leader sequence, the F2 subunit (domain), the ectodomain portion of the F1 subunitwhich includes at its N-terminus a hydrophobic fusion peptide (FP) and two heptad repeat regions (HRA and HRB), a single-pass transmembrane domain (TM), and a c- terminal cytoplasmic tail. The F0 precursor is cleaved by an unknown enzyme to generate a F1-F2 heterodimer connected by disulfide bonds, which form the mature trimeric prefusion structure. The prefusion conformation of hMPV F is meta-stable and undergoes conformational rearrangement to the postfusion state during the process of membrane fusion. As a reference, the full length hMPV F sequence is illustrated with the wildtype F protein sequence of hMPV isolate Arg / 2 / 02, SEQ ID NO:1 (GenBank Accession ABD27846.1; UniProt ID Q1A2Z0; Galiano et al., J. Med. Virol.78, 631- 637, 2006). It contains 530 amino acid residues, including N-terminal leader (signal peptide) MSWKVVIIFSLLITPQHG (SEQ ID NO:15), F2 subunit (L19-R102), ectodomain of F1 subunit (F103-L481) including fusion peptide F103-T127, and the transmembrane domain and intracellular C-terminus (S482-V530). The engineered hMPV F trimer immunogens of the invention is described by referring to the amino acid numbering of this exemplified hMPV F protein sequence. Due to their highly conserved sequences and identical domain structures among the F proteins of different hMPV subgroups and isolates, the modifications to the exemplified sequence in the engineered hMPV F trimer immunogens of the invention can be readily applied to the other hMPV subgroups and isolates.

[0041] Wildtype hMPV F sequence (SEQ ID NO:1) MSWKVVIIFSLLITPQHG LKESYLEESCST ITEGYLSVLR TGWYTNVFTL EVGDVENLTCADGPSLIKTE LDLTKSALRE LRTVSADQLA REEQIENPRQ SR FVLGAIAL GVATAAAVTA GVAIAKTIRL ESEVTAIKNA LKKTNEAVST LGNGVRVLAT AVRELKDFVS KNLTRAINKN KCDIADLKMA VSFSQFNRRF LNVVRQFSDN AGITPAISLD LMTDAELARA VSNMPTSAGQ IKLMLENRAM VRRKGFGILI GVYGSSVIYM VQLPIFGVID TPCWIVKAAP SCSEKKGNYA CLLREDQGWY CQNAGSTVYY PNEKDCETRG DHVFCDTAAG INVAEQSKEC NINISTTNYP CKVSTGRHPI SMVALSPLGA LVACYKGVSC SIGSNRVGII KQLNKGCSYI TNQDADTVTI DNTVYQLSKV EGEQHVIKGR PVSSSFDPVK FPEDQFNVAL DQVFESIENS QALVDQSNRIL SSAEKGNTG FIIVIILTAV LGSTMILVSV FIIIKKTKKP TGAPPELSGV IV. Engineered hMPV fusion protein (F) trimer immunogens

[0042] The invention provides engineered (redesigned or modified) hMPV soluble F sequences that can be employed for generating vaccine compositions. The engineered soluble hMPV F trimer immunogens or proteins of the invention are stabilized by introducing modifications into the wildtype hMPV soluble F sequences. Specifically,the engineered hMPV F trimer immunogens contain a modified F2 subunit and a modified F1 subunit ectodomain, each containing one or more mutations or modifications relative to the wildtype F2 subunit and the wildtype F1 subunit sequences, respectively. A reference wildtype hMPV soluble F sequence, based on hMPV Arg / 2 / 02 isolate, is shown in SEQ ID NO:2. Unless otherwise noted, amino acid numbering of the various sequence modifications in the engineered hMPV trimer immunogens of the invention is based on the wildtype F sequence of this prototype hMPV isolate.

[0043] Wildtype soluble hMPV F sequence (GenBank Accession ABD27846; SEQ ID NO:2): MSWKVVIIFSLLITPQHG LKESYLEESCST ITEGYLSVLR TGWYTNVFTL EVGDVENLTCADGPSLIKTE LDLTKSALRE LRTVSADQLA REEQIENPRQ SR FVLGAIAL GVATAAAVTA GVAIAKTIRL ESEVTAIKNA LKKTNEAVST KNLTRAINKN

[0044] In this reference or prototype wildtype hMPV soluble F sequence, the N- terminal leader sequence (SEQ ID NO:15) is shown in italicized font, the F2 subunit sequence (SEQ ID NO:16) is underlined, and the ectodomain of the F1 subunit sequence (SEQ ID NO:17) is dotted underlined. It is noted that while the N-terminal leader sequence (signal peptide) is required for recombinant expression of the hMPV F protein, it is not a necessary component of the engineered hMPV F trimer immunogens of the invention. The engineered F trimer proteins do not require the N-terminal leader for their biochemical activities or immunogenic properties. Thus, the reference wildtype hMPV soluble F sequence for engineering the hMPV F trimer immunogens of the invention can also be the N-terminal leader deleted portion of SEQ ID NO:2, which is shown as SEQ ID NO:3 below.

[0045] N-terminal leader deleted wildtype soluble hMPV F sequence (GenBank Accession ABD27846; SEQ ID NO:3): LKESYLEESCST ITEGYLSVLR TGWYTNVFTL EVGDVENLTCADGPSLIKTE LDLTKSALRE LRTVSADQLA REEQIENPRQ SR FVLGAIAL GVATAAAVTA GVAIAKTIRL ESEVTAIKNA LKKTNEAVST LGNGVRVLAT AVRELKDFVSKNLTRAINKN KCDIADLKMA VSFSQFNRRF LNVVRQFSDN AGITPAISLD

[0046] Typically, the modified F2 subunit in the engineered hMPV F trimer immunogens of the invention contains an E80D substitution and also a shortened C- terminus relative to the wildtype F2 subunit sequence, and the modified F1 subunit contains an A185P substitution and an engineered disulfide bond via T127C / N153C substitutions relative to the wildtype F1 subunit ectodomain sequence. In these engineered hMPV F immunogens, the truncation at the C-terminus of the F2 subunit can be a deletion of about 8 to about 12 amino acid residues. As an exemplification, 10 N-terminal residues at the C-terminus, E93-R102 (EQIENPRQSR; SEQ ID NO:21) is deleted. In some embodiments, a short peptide linker is inserted between the modified F2 subunit C-terminus and the modified F1 subunit ectodomain. In various embodiments, the peptide linker can contain from about 2 to about 10 residues (e.g., Gly and / or Ser residues). As exemplification, a GGGGGG (SEQ ID NO:22) peptide linker is employed in some of the engineered F trimer immunogens herein. One specific engineered hMPV F trimer immunogen exemplified herein is hMPV UMCMI(SEQ ID NO:7). It contains modified F2 subunit sequence (SEQ ID NO:4) and modified F1 subunit ectodomain sequence (SEQ ID NO:5) connected by a (G)6(SEQ ID NO:22) linker.

[0047] Modified F2 in UMCMI(SEQ ID NO:4): containing E80D substitution and a shortened C terminus. LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLARE

[0048] Modified F1 in UMCMI(SEQ ID NO:5): containing A185P substitution and an engineered disulfide bond T127C / N153C. FVLGAIALGVATAAAVTAGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGVR VLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSD NAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGS TVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPI SMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RIL

[0049] UMCMI: without N-terminal leader and C-terminal foldon and His tag (SEQ ID NO:7) LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLARE-GGGGGG- FVLGAIALGVATAAAVTAGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGVR VLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSD NAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGS TVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPI SMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTV YQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RIL

[0050] Other than the above-noted modifications, the modified F2 subunit sequence and the modified F1 subunit ectodomain sequence in some engineered hMPV soluble F proteins of the invention can contain one or more additional mutations in comparison to their respective wildtype counterpart sequences. In some of these embodiments, the engineered hMPV F trimer immunogens of the invention can additionally contain a V155P substitution in the F1 subunit. An exemplary engineered hMPV F trimer immunogen containing such modifications is shown in SEQ ID NO:8. In some embodiments, the engineered hMPV F trimer immunogens further contain an engineered interprotomer disulfide bond, which functions to maintain a prefusion- closed trimer. As exemplification, the engineered interprotomer disulfide bond can be formed by A120C and Q426C substitutions in the modified F1 subunit. An exemplary engineered hMPV F trimer immunogen containing such modifications is shown in SEQ ID NO:9. It is noted that the formation of this interprotomer disulfide bond is due to the Cβ-Cβ distance for A120 and Q426 in two adjacent F1 subunits. In contrast, in the constructs containing the T127C / N153C substitutions as described above, the Cβ-Cβ distance for T127 and N153 within the same F1 subunit necessitates the formation of an intraprotomer disulfide bond.

[0051] In still some other embodiments, instead of introducing an interprotomer covalent disulfide bond, an interprotomer non-covalent hydrophobic interaction can be engineered in the modified F1 subunit. As exemplification, the hydrophobic interaction can be engineered by replacing each of A120 and Q426 with a hydrophobic residue.For example, these two positions can have A120V and Q426L substitutions as exemplified herein. In some of these embodiments, the shortened F2 subunit C- terminus can be further optimized by removing one or more charged residues. As exemplification, the shortened F2 C-terminal fragment D87-E92, DQLARE (SEQ ID NO:19), can be replaced with a non-charged sequence fragment DGHGHP (SEQ ID NO:20). An exemplary engineered hMPV F trimer immunogen containing such modifications is shown in SEQ ID NO:10. In this exemplified embodiment, the modifications in the F2 subunit motif fragment (10-aa C-terminal truncation plus the further optimization) are equivalent to replacing the C-terminal residues DQLAREEQIENPRQSR (SEQ ID NO:18) with a shorter and optimized sequence motif DGHGHP (SEQ ID NO:20).

[0052] UMCMI-P2: without N-terminal leader sequence and C-terminal foldon and His tag (SEQ ID NO:8). LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLARE-GGGGGG- FVLGAIALGVATAAAVTAGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGPR VLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSD NAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGS TVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPI SMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTV YQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RIL

[0053] UFCM1-P2-iSS: without N-terminal leader sequence and C-terminal foldon and His tag (SEQ ID NO:9). LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLARE-GGGGGG- FVLGAIALGVATAAAVTCGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGPR VLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSD NAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGS TVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPI SMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTV YCLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RIL

[0054] UFCM1-P2-F2C-VL: without N-terminal leader sequence and C-terminal foldon and His tag (SEQ ID NO:10).LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADGHGHP-GGGGGG- FVLGAIALGVATAAAVTVGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGPR VLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSD NAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGS TVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPI SMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTV YLLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RIL

[0055] Other than the modified F2 and F1 subunits, some engineered hMPV F trimer immunogens of the invention can additionally contain a trimerization motif at the C-terminus. This motif functions to further stabilize the trimer and also to increase the trimer ratio within the total protein yield. Suitable trimerization motifs for the invention include, e.g., T4 fibritin foldon (PDB ID: 4NCV) and viral capsid protein SHP (PDB: 1TD0). T4 fibritin (foldon) is well known in the art, and constitutes the C-terminal 30 amino acid residues of the trimeric protein fibritin from bacteriophage T4, and functions in promoting folding and trimerization of fibritin. See, e.g., Papanikolopoulou et al., J. Biol. Chem.279: 8991-8998, 2004; and Guthe et al., J. Mol. Biol.337: 905- 915, 2004. Similarly, the SHP protein and its used as a functional trimerization motis are also well known in the art. See, e.g., Dreier et al., Proc Natl Acad Sci USA 110: E869–E877, 2013; and Hanzelmann et al., Structure 24: 140–147, 2016. In some exemplified embodiments, the trimerization motif in the engineered hMPV F immunogens contains a foldon sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:6). Exemplary engineered hMPV F trimer immunogens containing such a trimerization motif are shown in SEQ ID NOs:11-14. In some other embodiments, the employed trimerization motif can contain a sequence that is a conservatively modified variant or substantially identical (e.g., at least 90%, 95% or 99% identical) sequence of a known foldon sequence or SHP protein sequence. In various embodiments, the trimerization motif can be inserted at the C-terminus of the F1 subunit ectodomain via a restriction site or a short peptide linker, e.g., an GS or AS dipeptide as exemplified herein. In some embodiments, the hMPV F trimer immunogens of the invention can also contain a N-terminal leader sequence to facilitate their recombinant expressions. For example, the N-terminal leader of a wildtype hMPV F protein (e.g., SEQ ID NO:15) can be employed in these embodiments. In some embodiments, an His6-tag canbe further added to the C-terminus of the trimerization motif to facilitate protein purification, e.g., by using a Nickel column.

[0056] UMCMI: has C-terminal “AS” restriction site + foldon (SEQ ID NO:11) LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLARE-GGGGGG- FVLGAIALGVATAAAVTAGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGVR VLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSD NAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGS TVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPI SMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTV YQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RIL-ASGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0057] UMCMI-P2: has C-terminal “AS” site + foldon (SEQ ID NO:12) LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLARE-GGGGGG- FVLGAIALGVATAAAVTAGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGPR VLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSD NAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGS TVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPI SMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTV YQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RIL-ASGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0058] UFCM1-P2-iSS: has C-terminal “AS” site + foldon (SEQ ID NO:13) LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLARE-GGGGGG- FVLGAIALGVATAAAVTCGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGPR VLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSD NAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGS TVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPI SMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTV YCLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RILASGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0059] UFCM1-P2-F2C-VL: has C-terminal “AS” site + foldon (SEQ ID NO:14) LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADGHGHP-GGGGGG- FVLGAIALGVATAAAVTVGVAIAKCIRLESEVTAIKNALKKTNEAVSTLGCGPR VLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSD NAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPI SMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTV YLLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSN RIL-ASGYIPEAPRDGQAYVRKDGEWVLLSTFL

[0060] The engineered hMPV F trimer immunogens of the invention are exemplified with the reference wildtype hMPV soluble F protein sequence shown in SEQ ID NO:2 or the wildtype soluble sequence without the N-terminal leader (signal peptide) (SEQ ID NO:3) from hMPV Arg / 2 / 02 isolate. Due to identical domain structures and highly conserved sequence among different hMPV strains, engineered or redesigned soluble F immunogens derived from other known hMPV strains or isolates can also be generated in accordance with the redesign strategy described herein. In addition to the hMPV F sequence exemplified herein (GenBank Accession No. ABD27846), there are many other known hMPV homolog F protein sequences that have been described in the literature. See, e.g., GenBank Accession Nos. ABD27847 - ABD27856 (Galiano et al., J. Med. Virol.78, 631-637, 2006); GenBank Accession No. ABM67072 (Mok et al., J. Virol.81 (24), 13710-13722; 2007); GenBank Accession No. AAU25820 (Ishiguro et al., Clin. Diagn. Lab. Immunol.12, 202-205, 2005); GenBank Accession Nos. AGW43131 - AGW43223 (Papenburg et al., J. Clin. Virol. 58, 541-547, 2013); GenBank Accession Nos. AEZ52343 - AEZ52366 (Klemenc et al., J. Clin. Virol.54, 371-375, 2012); and GenBank Accession Nos. AVV63136 and AVV63127. Any of these and other known hMPV F protein sequences can be readily employed to generate engineered hMPV trimer immunogens in accordance with the strategy described herein. V. Nanoparticle displayed vaccine compositions

[0061] The invention provides vaccine compositions that contain a heterologous scaffold that display the stabilized hMPV soluble F proteins or immunogens described herein. Any heterologous scaffold can be used to present the engineered soluble F proteins or immunogens in the construction of the vaccines of the invention. This includes a virus-like particle (VLP) such as bacteriophage QβVLP and nanoparticles. Various nanoparticle platforms can be employed in generating the vaccine compositions of the invention. In general, the nanoparticles employed in the invention need to be formed by multiple copies of a single subunit. The nanoparticles aretypically ball-like shaped, and / or have rotational symetry (e.g., with 3-fold and 5-fold axis), e.g., with an icosahedral structure exemplified herein. Additionally or alternatively, the amino-terminus of the particle subunit has to be exposed and in close proximity to the 3-fold axis, and the spacing of three amino-termini has to closely match the spacing of the carboxyol-termini of the displayed trimeric stabilized soluble F protein.

[0062] In various embodiments, the employed self-assembling naoparticles have a diameter of about 25nm or less (usually assembled from 12, 24, or 60 sububits) and 3- fold axes on the particle surface. Such nanoparticles provide suitable particle platforms to produce multivalent vaccines. For example, the hMPV F trimer immunogen protein or polypeptide can be presented on self-assembling nanoparticles such as I3-01 or derivatives. I3-01 is an engineered protein that can self-assemble into hyperstable nanoparticles. See, e.g., Hsia et al., Nature 535, 136-139, 2016. Database search reveals that I3-01 is engineered from a bacterial enzyme with a known crystal structure (PDB ID: 1VLW). In some preferred embodiments, the hMPV F trimer immunogens can be displayed with I3-01 variant scaffolds such as I3-01v9b (SEQ ID NO:31) and I3-01v9c (seq id no:32). Other examples of nanoparticles suitable for the invention include nanoparticles derived from ferritin (FR) or E2p. Well known and routinely used in the art, ferritin is a globular protein found in all animals, bacteria, and plants. As is well known in the art, it acts primarily to control the rate and location of polynuclear Fe(III)2O3formation through the transportation of hydrated iron ions and protons to and from a mineralized core. The globular form of ferritin is made up of monomeric subunit proteins (also referred to as monomeric ferritin subunits), which are polypeptides having a molecule weight of approximately 17-20 kDa. E2p is a redesigned variant of dihydrolipoyl acyltransferase from Bacillus stearothermophilus that has been shown to self-assemble into thermostable 60-meric nanoparticle. See, e.g., He et al., Nat. Commun.7:12041, 2016. Sequences of the subunits of these proteins are known in the art. See, e.g., WO2017 / 192434. More detailed information on the structural and functional properties of the various nanoparticle scaffolds, as well as their use in presenting trimeric protein immunogens, is provided in the art. See, e.g., WO2017 / 192434, WO2019 / 089817 and WO2019 / 241483. In various embodiments, the hMPV vaccine compositions of the invention can employ any of these knownnanoparticles, as well as their conservatively modified variants or variants with substantially identical (e.g., at least 90%, 95% or 99% identical) sequences.

[0063] I3-01v9b (for trimeric antigen display; with the 1stresidue mutated to G) (SEQ ID NO:31) GAEKMIKEI-GSGS-EELQKKM- EELFKKHKIVAVLRANSVEEAKMKALAVFVGGVHLIEITFTVPDADTVIKELSF LKELGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDAEITVFCLEKGVFYMPGV MTPTELVKAMKLGHNILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDN VCEWFKAGVLAVGVGSALVKGTIAEVAAKAAAFVEKIRGCTE

[0064] I3-01v9c (for trimeric antigen display; with the 1stresidue mutated to G) (SEQ ID NO:32) GAEKMIKEI-GPPS-EELQKKM- EELFKKHKIVAVLRANSVEEAKMKALAVFVGGVHLIEITFTVPDADTVIKELSF LKELGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDAEITVFCLEKGVFYMPGV MTPTELVKAMKLGHNILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDN VCEWFKAGVLAVGVGSALVKGTIAEVAAKAAAFVEKIRGCTE

[0065] In addition to the nanoparticle sequences noted above, many other nanoparticles or VLPs known in the art may also be used in the practice of the invention. These include, e.g., Aquifex aeolicus lumazine synthase, Thermotoga Maritima encapsulin, Myxococcus xanthus encapsulin, bacteriophage Qbeta virus particle, Flock House Virus (FHV) particle, ORSAY virus particle, and infectious bursal disease virus (IBDV) particle.

[0066] Other than the displayed soluble F immunogen, the nanoparticle vaccine compositions of the invention can include additional motifs for better biological or pharmaceutical properties. The additional structural components can function to facilitate the immunogen display on the surface of the nanoparticles, to enhance the stability of the displayed immunogens, and / or to improve yield and purity of the self- assembled protein vaccines. In these embodiments, one or more linkers (linker sequences, motifs or moieties) can be used to connect the various structural components in the constructs. One example of the additional structural components is a trimerization motif such as foldon as noted above. In some embodiments, the coding sequence of a polypeptide fragment or motif that serves as an active site for chemical conjugation can be inserted into the construct at an appropriate position. In some other embodiments, additional structural components such as a CD4+T-helper epitope or a CD8+T-cell epitope can also be inserted into the nanoparticle construct at anappropriate position. These include, e.g., the PADRE T-helper epitope as exemplified herein.

[0067] In still some other embodiments, the nanoparticle vaccines of the invention can contain a locking domain that stabilizes the nanoparticle. The locking domain coding sequence can be fused directly or indirectly to the C-terminus of the nanoparticle subunit coding sequence. The locking domain stabilizes the nanoparticles from the inside so that the nanoparticles presenting the hMPV soluble F immunogen polypeptide can remain intact during manufacture, vaccine formulation, and immunization. The nanoparticle vaccine immunogens thus constructed have significantly enhanced stability. In general, the locking domain suitable for the invention is a protein subunit that can naturally form a dimer with another protein subunit in solution through non-covalent interactions at the interface. In some preferred embodiments, the two protein subunits can be identical in sequence and form a homodimer. In some other embodiments, the two protein subunits can be different proteins, or two different domains of a single protein derived through engineering, that can form a heterodimer in solution through non-covalent interactions at the interface. Typically, the locking domain is covalently fused to the nanoparticle subunit to which the immunogen polypeptide is linked. Examples of specific locking domains and guidance on the use of a locking domain (e.g., LD7 or LD4 as noted below) in the construction of nanoparticle displayed trimeric immunogens can be found in the art, e.g., WO2019 / 241483. Two specific locking domains suitable for use in the nanoparticle vaccines of the invention are LD4 (SEQ ID NO:29) and LD7 (SEQ ID NO:30).

[0068] Locking domain LD4 (SEQ ID NO:29): FSEEQKKALDLAFYFDRRLTPEWRRYLSQRLGLNEEQIERWFRRKEQQIGWSH PQFEK

[0069] Locking domain LD7 (SEQ ID NO:30): SPAVDIGDRLDELEKALEALSAEDGHDDVGQRLESLLRRWNSRRAD

[0070] Nanparticles displaying any of the engineered hMPV F protein trimer immunogens described herein can be constructed by fusing the immunogen polypeptide or subunit of multimeric immunogen protein (e.g., a trimer immunogen) to the subunit sequence of the nanoparticle (e.g., subunit sequence of E2p, I3-01v9b or I3-01v9c), as well as the other optional or alternative components described herein (e.g., a lockingdomain or a trimerization motif). To construct the nanoparticle displayed fusion vaccine immunogens of the invention, one or more linker motifs or moieties may be employed to facilitate connection and maintain structural integrity of the different components. Typically, the linker motifs contain short peptide sequences. In various embodiments, the linkers or linker motifs can be any flexible peptides that connect two protein domains or motifs without interfering with their functions. For example, any of these linkers used in the constructs can be GC-rich peptides with a sequence of (GaSb)n, wherein a is an integer of about 1-5, b is an integer of about 0-2, and n is an integer of about 1-5. In some embodiments, the employed linkers comprise a sequence GSGS (SEQ ID NO:27) or GSGSGSGS (SEQ ID NO:28). Detailed procedures for recombinant production of the vaccine compositions of the invention can be based on the protocols described herein and / or other methods that have been described in the art, e.g., He et al., Nat. Comm.7, 12041, 2016; Kong et al., Nat. Comm.7, 12040, 2016; He et al., Sci Adv.4(11):eaau6769, 2018; WO2017 / 192434; WO2019 / 089817 and WO2019 / 241483. VI. Polynucleotides and expression constructs

[0071] The engineered hMPV soluble F proteins and the related vaccine compositions of the invention are typically produced by first generating expression constructs (i.e., expression vectors) that contain operably linked coding sequences of the various structural components described herein. Accordingly, in some related aspects, the invention provides substantially purified polynucleotides (DNA or RNA) that encode the nanoparticle displayed hMPV soluble F immunogens as described herein, as well as expression vectors that harbor such polynucleotides (e.g., CMV vectors) and host cells for producing the vaccine immunogens (e.g., ExpiCHO cell line exemplified herein). The fusion polypeptides encoded by the polynucleotides or expressed from the vectors are also included in the invention. As described herein, such polypeptides will self-assemble into nanoparticle vaccines that display the immunogen polypeptides or proteins on its surface.

[0072] The polynucleotides and related vectors can be readily generated with standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfecting, transient gene expression and obtaining stable transfected cell lines are described in the art, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res.19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0073] The selection of a particular vector depends upon the intended use of the fusion polypeptides. For example, the selected vector must be capable of driving expression of the fusion polypeptide in the desired cell type, whether that cell type be prokaryotic or eukaryotic. Many vectors contain sequences allowing both prokaryotic vector replication and eukaryotic expression of operably linked gene sequences. Vectors useful for the invention may be autonomously replicating, that is, the vector exists extrachromosomally and its replication is not necessarily directly linked to the replication of the host cell's genome. Alternatively, the replication of the vector may be linked to the replication of the host's chromosomal DNA, for example, the vector may be integrated into the chromosome of the host cell as achieved by retroviral vectors and in stably transfected cell lines. Both viral-based and nonviral expression vectors can be used to produce the immunogens in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet.15:345, 1997). Useful viral vectors include vectors based on lentiviruses or other retroviruses, adenoviruses, adenoassociated viruses, Cytomegalovirus, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol.49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0074] Depending on the specific vector used for expressing the fusion polypeptide, various known cells or cell lines can be employed in the practice of the invention. The host cell can be any cell into which recombinant vectors carrying a fusion of the invention may be introduced and wherein the vectors are permitted to drive the expression of the fusion polypeptide is useful for the invention. It may beprokaryotic, such as any of a number of bacterial strains, or may be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells including, for example, rodent, simian or human cells. Cells expressing the fusion polypeptides of the invention may be primary cultured cells or may be an established cell line. Thus, in addition to the cell lines exemplified herein (e.g., CHO cells), a number of other host cell lines capable well known in the art may also be used in the practice of the invention. These include, e.g., various Cos cell lines, HeLa cells, Sf9 cells, HEK293, AtT20, BV2, and N18 cells, myeloma cell lines, transformed B-cells and hybridomas.

[0075] The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. The fusion polypeptide-expressing vectors may be introduced to the selected host cells by any of a number of suitable methods known to those skilled in the art. For the introduction of fusion polypeptide-encoding vectors to mammalian cells, the method used will depend upon the form of the vector. For plasmid vectors, DNA encoding the fusion polypeptide sequences may be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection (“lipofection”), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation. These methods are detailed, for example, in Brent et al., supra. Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture. For example, LipofectAMINE™ (Life Technologies) or LipoTaxi™ (Stratagene) kits are available. Other companies offering reagents and methods for lipofection include Bio-Rad Laboratories, CLONTECH, Glen Research, Life Technologies, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

[0076] For long-term, high-yield production of recombinant fusion polypeptides, stable expression is preferred. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the fusion polypeptide- encoding sequences controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers. The selectable marker in the recombinant vector confers resistance to the selection and allows cells to stably integrate the vector into theirchromosomes. Commonly used selectable markers include neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J. Mol. Biol., 150:1, 1981); and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30: 147, 1984). Through appropriate selections, the transfected cells can contain integrated copies of the fusion polypeptide encoding sequence. VII. Pharmaceutical compositions and therapeutic applications

[0077] In another aspect, the invention provides pharmaceutical compositions and related therapeutic methods of using the engineered hMPV F trimer immunogens and nanoparticle vaccine compositions as described herein. In some embodiments, the soluble hMPV F trimer immunogens and related vaccines can be used for preventing infections. Some embodiments of the invention relate to use of pharmaceutical compositions containing the engineered hMPV F trimer proteins for treating hMPV infections.

[0078] In the practice of the various therapeutic methods of the invention, the subjects in need of prevention or treatment of a disease or condition (e.g., hMPV infection) is administered with an engineered hMPV F trimer immunogen, a nanoparticle vaccine containing the same, or an encoding polynucleotide described herein. Typically, a nanoparticle vaccine, an immunogen protein or an encoding polynucleotide disclosed herein is included in a pharmaceutical composition. The pharmaceutical composition can be either a therapeutic formulation or a prophylactic formulation. Typically, the composition can additionally include one or more pharmaceutically acceptable vehicles and, optionally, other therapeutic ingredients (for example, antiviral drugs). Various pharmaceutically acceptable additives can also be used in the compositions.

[0079] Some of the pharmaceutical compositions of the invention are vaccine compositions. For vaccine compositions, appropriate adjuvants can be additionally included. Examples of suitable adjuvants include, e.g., aluminum hydroxide, lecithin, Freund's adjuvant, MPLTMand IL-12. In some embodiments, the vaccine compositions or nanoparticle immunogens disclosed herein can be formulated as a controlled-release or time-release formulation. This can be achieved in a composition that contains a slow release polymer or via a microencapsulated delivery system or bioadhesive gel. The various pharmaceutical compositions can be prepared in accordance with standardprocedures well known in the art. See, e.g., Remington’s Pharmaceutical Sciences, 19thEd., Mack Publishing Company, Easton, Pa., 1995; Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978); U.S. Pat. Nos.4,652,441 and 4,917,893; U.S. Pat. Nos.4,677,191 and 4,728,721; and U.S. Pat. No.4,675,189.

[0080] The pharmaceutical compositions of the invention can be readily employed in a variety of therapeutic or prophylactic applications, e.g., for treating hMPV infection or bronchiolitis, or eliciting an immune response to hMPV in a human subject. In various embodiments, the vaccine compositions can be used for treating or preventing infections caused by hMPV. In some embodiments, an hMPV nanoparticle vaccine composition can be administered to a subject to induce an immune response to hMPV, e.g., to induce production of broadly neutralizing antibodies to the virus. For subjects at risk of developing an hMPV infection, a vaccine composition of the invention can be administered to provide prophylactic protection against the viral infection. Depending on the specific subject and conditions, pharmaceutical compositions of the invention can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. In general, the pharmaceutical composition is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate a selected disease or condition or one or more symptom(s) thereof. For therapeutic applications, the compositions should contain a therapeutically effective amount of the hMPV immunogen or nanoparticle containing the immunogen as described herein. For prophylactic applications, the compositions should contain a prophylactically effective amount of the immunogen or nanoparticle. The appropriate amount of the immunogen or nanoparticle can be determined based on the specific disease or condition to be treated or prevented, severity, age of the subject, and other personal attributes of the specific subject (e.g., the general state of the subject's health and the robustness of the subject's immune system). Determination of effective dosages is additionally guided with animal model studies followed up by human clinical trials, and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject.

[0081] For prophylactic applications, the immunogenic composition is provided in advance of any symptom, for example in advance of infection. The prophylactic administration of the immunogenic compositions serves to prevent or ameliorate any subsequent infection. Thus, in some embodiments, a subject to be treated is one who has, or is at risk for developing, an hMPV infection, for example because of exposure or the possibility of exposure to an hMPV. Following administration of a therapeutically effective amount of the disclosed therapeutic compositions, the subject can be monitored for an hMPV infection, symptoms associated with an hMPV infection, or both.

[0082] For therapeutic applications, the immunogenic composition is provided at or after the onset of a symptom of disease or infection, for example after development of a symptom of hMPV infection or after diagnosis of the infection. The immunogenic composition can thus be provided prior to the anticipated exposure to the virus so as to attenuate the anticipated severity, duration or extent of an infection and / or associated disease symptoms, after exposure or suspected exposure to the virus, or after the actual initiation of an infection. The pharmaceutical composition of the invention can be combined with other agents known in the art for treating or preventing hMPV infections.

[0083] The pharmaceutical compositions or nanoparticle vaccine compositions containing the novel hMPV F immunogens as described herein can be provided as components of a kit. Optionally, such a kit includes additional components including packaging, instructions and various other reagents, such as buffers, substrates, antibodies or ligands, such as control antibodies or ligands, and detection reagents. An optional instruction sheet can be additionally provided in the kits. EXAMPLES

[0084] The following examples are offered to illustrate, but not to limit the present invention. Example 1 Previous hMPV-F designs and our rational design of the UFCM1series constructs

[0085] Multiple designs have been proposed to stabilize prefusion hMPV-F , including an early construct 115-BV (Battles et al., Nat Comm 2017, 8, 1528), DS-Cav1Es2 (Hsieh et al., Nat Comm 2022, 131299), and V3B ∆12_D454C-V458C (Ou et al., PLoS Pathog 2023, 19, e1011584). However, these hMPV-F designs appeared to either be unstable or contain suboptimal mutations. For example, although 115-BV led to the first crystal structure of prefusion hMPV-F trimer, it is not stable enough to retain the prefusion conformation in the solution phase. In DS-Cav1Es2, a disulfide bond (T365C / V463C) was introduced between β14 and α10 that would destabilize the C- terminal stalk essential to hMPV-F trimerization (Hsieh et al., Nat Comm 2022, 13 1299). As a result, the cleaved DS-CavEs2 hMPV-F was determined as a prefusion monomer. Kwong and colleagues evaluated various disulfide bonds, proline mutations, and cleavage site linkers, arriving at a construct containing a short F2-F1 linker and three disulfide bonds (Stewart-Jones et al., PNAS 2022, 118, e2106196118; Ou et al., PLoS Pathog 2023, 19, e1011584). In their construct design, the D454C / V458C mutation was initially introduced as an interprotomer disulfide bond, but high- resolution cryo-EM revealed the formation of an intra-F1 disulfide bond that altered the local structure around the trimer base.

[0086] Here, we followed a similar minimalist approach to rationally design uncleaved, prefusion-closed (UFC) trimers for hMPV-F. To this end, we developed a base construct, termed UFCM1, which places a G6 linker between the shortened F2 C- terminus (F2-E92) and hydrophobic fusion peptide (F1-F103), in addition to A185P, E80D, and disulfide bond (T127C / N153C) mutations, as well as a C-terminal His6 tag to facilitate Nickel purification. We then hypothesized that a second proline mutation (V155P, or termed P2) in the HR1N-equivalent β3-β4 hairpin region can destabilize the postfusion state, producing a UFCM1-P2 construct. We further hypothesized that a single interprotomer disulfide bond (A120C / Q426C, or termed iSS) is sufficient to maintain a prefusion-closed trimer, leading to a UFCM1-P2-iSS construct. Lastly, we created a UFCM1-P2-F2C-VL construct, in which the shortened F2 C-terminus (residues 87-92) was modified to remove buried charges and the interprotomer disulfide bond mutation (iSS) was replaced with a hydrophobic contact (A120V / Q426L). This UFCM1- P2-F2C-VL construct was designed to examine the possibility of using a non-covalent interaction across the protomer-protomer interface to replace the interprotomer disulfide bond (A120C / Q426C). The sequences of the four tested constructs are shown below. A summary of the structural features of these redesigned hMPV soluble Fconstructs, including the mutations relative to the wildtype sequence, is provided in Table 1.

[0087] hMPV-F UFCM1complete sequence (SEQ ID NO:23): including N-terminal leader, A185P & E80D mutations, shortened F2 C terminus DQLARE (SEQ ID NO:19) and inserted G6 linker, AS enzymatic site and foldon trimerization motif (SEQ ID NO:6), and GS linker and His6 tag. MSWKVVIIFSLLITPQHG- LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLAREGGGGGGFVLGAIALGVATAAAVTAGVAIAKCIRL ESEVTAIKNALKKTNEAVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCD IPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPS CSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIN VAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNR VGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKF PEDQFNVALDQVFESIENSQALVDQSNRIL- ASGYIPEAPRDGQAYVRKDGEWVLLSTFLGSHHHHHH

[0088] hMPV-F UFCM1-P2 complete sequence (SEQ ID NO:24): hMPV-F UFCM1(SEQ ID NO:23) plus a V155P mutation. MSWKVVIIFSLLITPQHG- LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLAREGGGGGGFVLGAIALGVATAAAVTAGVAIAKCIRL ESEVTAIKNALKKTNEAVSTLGCGPRVLATAVRELKDFVSKNLTRAINKNKCDI PDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPS CSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIN VAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNR VGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKF PEDQFNVALDQVFESIENSQALVDQSNRIL- ASGYIPEAPRDGQAYVRKDGEWVLLSTFLGSHHHHHH

[0089] hMPV-F UFCM1-P2-iSS complete sequence (SEQ ID NO:25): SEQ ID NO:24 plus an engineered interprotomer disulfide bond A120C-Q426C. MSWKVVIIFSLLITPQHG- LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADQLAREGGGGGGFVLGAIALGVATAAAVTCGVAIAKCIRL ESEVTAIKNALKKTNEAVSTLGCGPRVLATAVRELKDFVSKNLTRAINKNKCDI PDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTS AGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPS CSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIN VAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNR VGIIKQLNKGCSYITNQDADTVTIDNTVYCLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRIL- ASGYIPEAPRDGQAYVRKDGEWVLLSTFLGSHHHHHH

[0090] hMPV-F UFCM1-P2-F2C-VL complete sequence (SEQ ID NO:26): SEQ ID NO:24 plus replacement of the shortened F2 C-terminus DQLARE (SEQ ID NO:19) with DGHGHP (SEQ ID NO:20), and also A120V / Q426L substitutions. MSWKVVIIFSLLITPQHG- LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDL TKSALRDLRTVSADGHGHPGGGGGGFVLGAIALGVATAAAVTVGVAIAKCIR LESEVTAIKNALKKTNEAVSTLGCGPRVLATAVRELKDFVSKNLTRAINKNKC DIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPT SAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAP SCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGIN VAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNR VGIIKQLNKGCSYITNQDADTVTIDNTVYLLSKVEGEQHVIKGRPVSSSFDPVKF PEDQFNVALDQVFESIENSQALVDQSNRIL- ASGYIPEAPRDGQAYVRKDGEWVLLSTFLGSHHHHHHTable 1. Mutations in redesigned hMPV-F constructs with respect to hMPV strain Argentinean / 02 / 2002 (UniProt Q1A2Z0)a.ConstructFusion peptide and furinIntraprotomer Interprotomer Proline cleavage sitedisulfide bond disulfide bondmutationOthersa. Previous hMPV-F designs 115-BV Cleaved A185P DS-Cav1Es2bCleaved, Q100R, S101R A185P L219K, V231I, E453QUFCM1Uncleaved, E93-R102truncation, G6 linkerT127C-N153C A185P E80DaMutations in the previously reported hMPV-F designs, our design constructs that were used to examine hMPV-F metastability, and in the constructs for which crystal structures were successfully determined are listed in this table.bDS-Cav1Es2 was developed using a different hMPV strain, Tennessee / 03 / 2002 (UniProt H6X1Z0), as the backbone, but its F sequence is identical to that of the strain Argentinean / 02 / 2002 (UniProt Q1A2Z0).cv3B Δ12_D454C-V458C was developed using a different hMPV strain, Tennessee / 04 / 1999 (UniProt G3KCK8), as the backbone. Therefore, the mutations listed in “Others” mostly reflect the sequence difference between the two strains.dConstructs used in the crystallographic analysis.Example 2 Basic in vitro and structural characterization of hMPV-F UFCM1 series

[0091] Four hMPV-F designs, UFCM1, UFCM1-P2, UFCM1-P2-iSS, and UFCM1-P2- F2C-VL, were validated using an approach that combines biochemical, biophysical, and structural methods. Specifically, size exclusion chromatography (SEC), differential scanning calorimetry (DSC), and negative-stain electron microscopy (nsEM) were combined to evaluate various hMPV-F constructs. All constructs were transiently expressed in 25ml ExpiCHO cells and purified using two different methods. While a Nickel column was used to capture all F species, an antibody column was generated using NAb MPE8 to target the prefusion F in hMPV-F purification. For UFCM1, Nickel purification yielded a trimer peak at ~11.9 ml in the SEC profile that was 5-fold higher than that from an MPE8 column (Figure 1A, left top), as measured by the ultraviolet absorbance at 280 nm (UV280). Both purification methods showed acceptable purity. DSC produced a thermogram with overlapping peaks, with Tm1and Tm2determined at 53.0 ⁰C and 59.6 ⁰C, respectively (Figure 1A, left bottom). The SEC-purified trimer fractions were analyzed by nsEM, which did not identify any 2D classes representing prefusion-closed trimers (Figure 1A, right). Meanwhile, no postfusion molecules were found in the EM micrographs. For UFCM1-P2, the P2 mutation between β3 and β4 notably increased the hMPV-F yield, as evident from the SEC profile following Nickel purification (Figure 1B, left top). DSC generated similar thermal parameters with ~1 °C higher Tm1and Tonsetvalues (Figure 1B, left bottom). All 2D classes obtained from nsEM corresponded to prefusion hMPV-F monomers with similar shape to prefusion RSV-F monomers (Figure 1B, right). UFCM1-P2-iSS showed reduced yield after Nickel and MPE8 purification, but with an increased ratio of prefusion-closed trimers within the total hMPV-F protein (Figure 1C, left top). Further, DSC demonstrated a single narrow peak with a Tmof 72 ⁰C and a Tonsetof 58.3 ⁰C, which were significantly higher, ~12-19 ⁰C and ~13-14 ⁰C, respectively, than those of the two constructs lacking the iSS mutation (Figure 1C, left bottom). Remarkably, almost all 2D classes in the nsEM analysis represented prefusion-closed trimers (Figure 1C, middle), which was further confirmed by 3D reconstruction and structural fitting (PDB ID: 5WB0) (Figure 1C, right). The last construct, UFCM1-P2-F2C-VL, showed a low trimer yield after MPE8 purification although a Nickel column produced a similar SEC profile to UFCM1and UFCM1-P2 (Figure 1D, left top). The DSC thermogram contained two peaks: while Tm1was comparable to those of UFCM1and UFCM1-P2, Tm2increased to 83.6 ⁰C (Figure 1D, left bottom). Interestingly, the nsEM analysis of SEC-purified trimer fractions indicated the presence of prefusion-closed trimers, partially open trimers, and misfolded hMPV-F (Figure 1D, middle). The 3D reconstruction revealed a tightened trimer apex and a widening around the base, potentially corresponding to an unstable intermediate state (Figure 1D, right). In reducing SDS-PAGE analysis, the cross-linked hMPV-F produced monomer, dimer, and trimer bands on the gel for all four constructs except UFCM1-P2-iSS, which displayed a single trimer band (Figure 1E). In summary, both UFCM1and UFCM1-P2 can produce prefusion hMPV-F but they are open trimers, whereas UFCM1-P2-iSS shows outstanding thermostability and represents 100% prefusion-closed trimers in solution. The attempt to replace iSS with a non-covalent interaction in UFCM1-P2-F2C-VL was less successful, indicating that more in-depth analysis of hMPV-F metastability may be required. Example 3 NsEM analysis of UFCM1-P2-iSS / NAb complexes and antigenic profiling of the UFCM1 series

[0092] To further characterize UFCM1-P2-iSS, we performed nsEM analysis of purified protein in complex with Fabs of MPE8 and 101F. The 3D reconstruction showed three MPE8 Fabs binding laterally to site III of a prefusion-closed trimer (Figure 2A). The 3.25 Å-resolution cryoEM model (EMDB-28891) of a recently reported hMPV-F design, v3B Δ12_D454C-V458C, bound to three single-chain variable fragments (scFv) of MPE8 could be fitted into the EM density with an excellent match. The nsEM analysis indicated stronger 101F binding to UFCM1-P2-iSS, with more 2D classes showing two to three 101F Fabs binding to the hMPV-F trimer (Figure 2B, left). Indeed, a 3D reconstruction with more structural details was obtained for the 101F complex (Figure 2B, right). Structural fitting of an hMPV-F / 101F model revealed an upward angle of approach for 101F, which targets the exposed site IV epitope. Together, our results indicate that UFCM1-P2-iSS can preserve important epitopes on the prefusion-stabilized hMPV-F trimer ready for NAb recognition. Antigenicity of the four UFCM1constructs were evaluated by ELISA and BLI using four NAbs with known complex structures, MPE8, 101F, DS7, and 458. In ELISA (Figure 2C), UFCM1-P2-iSS bound preferably to MPE8 with a 3.5-5.7-fold higher EC50than other UFCM1constructs, consistent with the fact that MPE8 interacts with twoprotomers of a prefusion-closed trimer. In contrast, UFCM1-P2-iSS exhibited the lowest affinity for DS7, with a 5.0-8.3-fold difference in EC50compared to other UFCM1constructs (Figure 2C). Further analysis of the DS7 complex structure revealed that its binding requires the displacement of β22, which only occurs in monomers or open trimers. All four hMPV-F constructs exhibited similar binding to 101F and 458 (Figure 2C). BLI demonstrated consistent patterns compared to ELISA, with UFCM1-P2-iSS showing the highest MPE8-binding signal (Figure 2D). Example 4 Crystallographic characterization of the hMPV-F UFCM1-P2-iSS trimer

[0093] We obtained a 6 Å-resolution structure for the ExpiCHO-expressed, Nickel / SEC-purified UFCM1-P2-iSS using similar crystallization conditions to the first crystal structure of a prefusion hMPV-F construct, 115-BV. The UFCM1-P2-iSS structure was superimposed onto the 115-BV structure (PDB ID: 5WB0) for comparison (Figure 3A). In the symmetric unit, UFCM1-P2-iSS adopted the same form as 115-BV, enabling the trimer structure to be built in a similar manner. UFCM1-P2-iSS yielded a Cα-RMSD of 1.22 Å with respect to 115BV at the protomer level. The three key elements of the UFCM1-P2-iSS design were compared to 115-BV, which has by far the most complete F structure and minimum mutations (Figure 3B). The T127C / Q153C mutation was found critical to maintaining prefusion hMPV-F, with a Cβ-Cβ distance of 4.2 Å in the DS-CavEs2 structure. This disulfide bond had a Cβ-Cβ distance of 3.4 Å in UFCM1-P2-iSS, compared to a Cβ-Cβ distance of 4.9 Å between T127 and Q153 in 115-BV (Figure 3B, top). The V155P mutation widened the β3-β4 turn in UFCM1- P2-iSS, which would likely facilitate disulfide bond formation at position 153 to position 127 and destabilize the transition to the postfusion F conformation (Figure 3B, middle). The interprotomer disulfide bond (C120-C426) showed a Cβ-Cβ distance of 4.7 Å, thus locking hMPV-F in a prefusion-closed trimer conformation (Figure 3B, bottom). Doe to the limited resolution, the structure could not be resolved for the F2-F1 linkage, part of α8 (A344-S347), and part of α9-α10-β23 (V442-E457). Notwithstanding, our crystal structure validated the structural integrity of UFCM1-P2- iSS.

[0094] The UFCM1-P2-iSS structure was then compared to two leading prefusion hMPV-F designs: DS-CavEs2 and v3B Δ12_D454C-V458C. Structural superpositionof UFCM1-P2-iSS and DS-CavEs2 (PDB ID: 7SEJ) revealed major differences in the stalk and α1 helix. Compared to a well-formed α10 helix in UFCM1-P2-iSS, DS- CavEs2 showed an incomplete α10 helix, due to the intra-F1 disulfide bond (T365C / V463C) between β14 and α10, which destabilizes the C-terminal trimeric stalk (Figure 3C, left). This may also explain why DS-CavEs2 was determined as a monomer with an extended α1 helix that would clash with the adjacent protomer in a prefusion- closed trimer (Figure 3C, right). Nonetheless, two cryo-EM structures showed a trimeric DS-CavEs2 in complex with NAbs that interact with two protomers at the trimer interface, although the α10 helix was partially unstructured. UFCM1-P2-iSS was then structurally superimposed onto v3B Δ12_D454C-V458C (PDB ID: 8F6X) (Figure 3D). While both designs showed similar cleavage site linker structures, the trimer base (β23 and α10) in v3B Δ12_D454C-V458C adopted a non-native conformation due to the unintended intraprotomer disulfide bond (D454C / V458C). In summary, our crystal structure validated the UFCM1-P2-iSS design and allowed for structural comparison with previously reported hMPV-F designs. Example 5 Antibody responses induced by rationally designed hMPV-F trimer vaccines in mice

[0095] The immunogenicity of three hMPV-F design constructs, UFCM1-P2, UFCM1-P2-iSS, and UFCM1-P2-F2C-VL, was assessed in mice (Figure 4A). The hMPV- F-specific binding antibody responses were measured by ELISA using a UFCM1-P2- iSS(1TD0) probe for all time points. In the UFCM1-P2-iSS(1TD0) construct, the original foldon motif is replaced with the 1TD0 motif to avoid recognition of foldon in the UFCM1-P2-iSS immunogen by antibodies in mouse sera. The EC50titers were calculated and plotted longitudinally for comparison (Figure 4B). Overall, all groups demonstrated strong binding antibody responses with EC50values of 9153 or greater after two vaccine doses. The UFCM1-P2 group reached the highest EC50titer at week 5, comparable to the UFCM1-P2-iSS group and 2.3-fold higher than the UFCM1-P2-F2C- VL group. Interestingly, the UFCM1-P2-F2C-VL group yielded higher EC50values than the other two groups at week 8. The cross-reactive antibody responses were assessed using week 11 serum samples against an RSV-F sc9-10 DS-Cav11TD0 probe (Figure 4C). The sc9-10 DS-Cav1 construct is a disulfide-locked RSV-F trimer (Joyce et al.,Nat Struct Mol Biol 2016, 23: 811-820). In the sc9-10 DS-Cav11TD0 probe, the original foldon motif is replaced with 1TD0 to avoid serum binding to foldon used in the hMPV-F trimer immunogen. All three groups showed negligible signals except some non-specific signals observed for UFCM1-P2. Live hMPV neutralization assays were used to assess serum NAb responses elicited by various hMPV-F constructs (Figure 4D). As expected, none of three hMPV-F trimers elicited NAb responses against autologous hMPV at week 2. Importantly, the UFCM1-P2-iSS group showed the most potent NAb titers, with the ID50titers of 33911, 37129 and 46408, which were 3.9-, 3.0-, and 2.1-fold higher than those elicited by UFCM1-P2 at weeks 5, 8 and 11, respectively. The results confirmed the importance of our rationally designed mutations for producing a stabilized, prefusion-closed hMPV-F trimer, which in turn induced a potent NAb response against hMPV. We next evaluated the cross-neutralizing activity of hMPV-F-induced mouse sera (Figure 4E). The live RSV assays detected no serum neutralization against RSV. Altogether, our results indicate that UFCM1-P2-iSS, as a fully closed prefusion hMPV-F trimer, can induce a potent NAb response in mice. Our study also suggests that such potent NAb response can be generated after only two immunizations. ***

[0096] The invention thus has been disclosed broadly and illustrated in reference to representative embodiments described above. It is understood that various modifications can be made to the present invention without departing from the spirit and scope thereof.

[0097] It is further noted that all publications, sequence accession numbers, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Claims

WHAT IS CLAIMED IS:

1. An engineered human metapneumovirus (hMPV) fusion protein (F) trimer immunogen, comprising from N-terminus to C-terminus (a) a modified F2 subunit and (b) a modified F1 subunit; where the modified F2 subunit comprises an E80D substitution and a C-terminal truncation of about 8-12 amino acid residues; and wherein the modified F1 subunit comprises an A185P substitution and an engineered disulfide bond via T127C / N153C substitutions; and wherein the amino acid numbering is based on hMPV F protein sequence with UniProt ID Q1A2Z0 (SEQ ID NO:1).

2. The engineered hMPV F trimer immunogen of claim 1, wherein the modified F2 subunit is covalently bonded to the modified F1 subunit via a peptide linker.

3. The engineered hMPV F trimer immunogen of any one of claims 1 to 2, wherein the peptide linker is (G)n, wherein n is an integer of about 2-10.

4. The engineered hMPV F trimer immunogen of any one of claims 1 to 3, wherein the modified F2 subunit comprises a truncation of 10 amino acid residues at the C-terminus.

5. The engineered hMPV F trimer immunogen of any one of claims 1 to 4, wherein the modified F2 subunit comprises SEQ ID NO:4 or a conservatively modified variant thereof, and the modified F1 subunit comprises SEQ ID NO:5 or a conservatively modified variant thereof.

6. The engineered hMPV F trimer immunogen of claim 1, comprising SEQ ID NO:

7.

7. The engineered hMPV F trimer immunogen of claim 2, wherein the modified F1 subunit further comprises a V155P substitution.

8. The engineered hMPV F trimer immunogen of claim 7, comprising SEQ ID NO:

8.

9. The engineered hMPV F trimer immunogen of claim 7, further comprising an interprotomer disulfide bond formed by A120C and Q426C substitutions in the modified F1 subunit.

10. The engineered hMPV F trimer immunogen of claim 9, comprising SEQ ID NO:

9.

11. The engineered hMPV F trimer immunogen of claim 7, wherein the C-terminal truncation in the F2 subunit comprises replacement of the C-terminal sequence DQLAREEQIENPRQSR (SEQ ID NO:12) with DGHGHP (SEQ ID NO:13), and wherein the modified F1 subunit further comprises A120V and Q426L substitutions.

12. The engineered hMPV F trimer immunogen of claim 11, comprising SEQ ID NO:

10.

13. The engineered hMPV F trimer immunogen of any one of claims 1- 12, further comprising a C-terminal trimerization motif.

14. The engineered hMPV F trimer immunogen of claim 13, wherein the trimerization motif comprises GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:6).

15. The engineered hMPV F trimer immunogen of claim 13, wherein the trimerization motif is linked to the C-terminus of the modified F1 subunit via an AS dipeptide.

16. The engineered hMPV F trimer immunogen of claim 13, comprising SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:

14.

17. The engineered hMPV F trimer immunogen of any one of claims 1- 16, further comprising an N-terminal leader (signal peptide) sequence.

18. A nanoparticle vaccine, comprising the engineered hMPV F trimer immunogen of any one of claims 1-17 that is displayed on the surface of a self- assembling nanoparticle.

19. The nanoparticle vaccine of claim 18, wherein the self-assembling nanoparticle comprises a trimeric sequence, and wherein C-terminus of the engineered hMPV F trimer immunogen is fused to N-terminus of subunit sequence of the nanoparticle.

20. The nanoparticle vaccine of claim 18, wherein the self-assembling nanoparticle is a I3-01 variant.

21. The nanoparticle vaccine of claim 18, wherein the I3-01 variant comprises SEQ ID NO:31 (I3-01v9b) or SEQ ID NO:32 (I3-01v9c).

22. A polynucleotide that encodes the engineered hMPV F trimer immunogen of any one of claims 1-17 or the nanoparticle vaccine of any one of claims 18-21.

23. A pharmaceutical composition, comprising engineered hMPV F trimer immunogen of claim 1, the nanoparticle vaccine of claim 18, or the polynucleotide of claim 22, and a pharmaceutically acceptable carrier.

24. A method of preventing or treating a metapneumovirus infection in a human subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 23.

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