Viral vector-based vaccine for human metapneumovirus

Abstract

The present disclosure provides human metapneumovirus (hMPV) vaccines that include an hMPV F protein antigen, and methods of eliciting an immune response by administering the vaccines.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 284,407, filed November 30, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] CRADA Statement This invention was made in the fulfillment of a Cooperative Research and Development Agreement with the National Institutes of Health, an agency of the Department of Health and Human Services. The United States Government has certain rights in this invention. [Background technology]

[0003] Human metapneumovirus (hMPV) is a major cause of acute respiratory infections, especially in children, immunocompromised patients, and the elderly. Closely related to avian metapneumovirus subtype C, hMPV has been circulating for at least 65 years, and nearly all children are infected with hMPV by age 5. However, immunity is incomplete, and reinfection occurs throughout adulthood. Symptoms are similar to those of other respiratory viral infections, ranging from mild (e.g., cough, rhinorrhea, and fever) to severe (e.g., bronchiolitis and pneumonia).

[0004] Currently, despite the high disease burden, there are no licensed vaccines or therapeutics against hMPV. Thus, there is a need for an hMPV vaccine that induces a strong immune response for potent neutralization of hMPV infection. Summary of the Invention [Means for solving the problem]

[0005] In one aspect, a viral vector is provided that encodes a human metapneumovirus (hMPV) F polypeptide antigen that lacks a transmembrane domain, lacks a cytoplasmic tail, and includes a human rhinovirus 3C (HRV-3C) protease cleavage site.

[0006] In certain exemplary embodiments, the viral vector comprises a backbone derived from a parainfluenza virus (PIV).

[0007] In certain exemplary embodiments, the F polypeptide further comprises an F cleavage site mutation comprising the amino acid substitutions Q100R and S101R, which replace the glutamine at amino acid position 100 of SEQ ID NO:1 with arginine and the serine at amino acid position 101 of SEQ ID NO:1 with arginine.

[0008] In certain exemplary embodiments, the F polypeptide includes a signal peptide.

[0009] In certain exemplary embodiments, the F polypeptide optionally comprises at least one tag sequence that is a polyhistidine tag (e.g., a 6xHis tag, an 8xHis tag, etc.) and / or a Strep II tag.

[0010] In certain exemplary embodiments, the F polypeptide comprises a foldon domain.

[0011] In certain exemplary embodiments, the pre-fusion F polypeptide comprises an amino acid substitution that replaces the wild-type amino acid at position 160 of SEQ ID NO:1, and an amino acid substitution that replaces the wild-type amino acid at position 46 of SEQ ID NO:1.

[0012] In certain exemplary embodiments, the F polypeptide comprises an amino acid substitution substituting a threonine at amino acid position 160 of SEQ ID NO:1 and an amino acid substitution substituting an asparagine at amino acid position 46 of SEQ ID NO:1.

[0013] In certain exemplary embodiments, the pre-fusion F1 polypeptide comprises an amino acid substitution that replaces the amino acid at position 160 with phenylalanine, tryptophan, tyrosine, valine, alanine, isoleucine, or leucine. In certain exemplary embodiments, the pre-fusion F1 polypeptide comprises an amino acid substitution that replaces the amino acid at position 160 with phenylalanine.

[0014] In certain embodiments, the pre-fusion F2 polypeptide comprises a phenylalanine, tryptophan, tyrosine, valine, alanine, isoleucine, or leucine substitution at position 160 of SEQ ID NO:1, and / or a valine, alanine, isoleucine, leucine, phenylalanine, tyrosine, or proline substitution at position 46 of SEQ ID NO:1.

[0015] In certain exemplary embodiments, the pre-fusion F1 polypeptide comprises an amino acid substitution that replaces the amino acid at position 46 with valine, alanine, isoleucine, leucine, phenylalanine, tyrosine, or proline. In certain exemplary embodiments, the pre-fusion F1 polypeptide comprises an amino acid substitution that replaces the amino acid at position 46 with valine.

[0016] In certain exemplary embodiments, live attenuated viruses, including viral vectors, are provided.

[0017] In certain exemplary embodiments, a pharmaceutical composition comprising a viral vector is provided.

[0018] In certain exemplary embodiments, the live-attenuated virus or pharmaceutical composition comprises a vaccine.

[0019] In certain exemplary embodiments, the method of inducing an immune response against hMPV and / or HPIV3 or protecting a subject from hMPV and / or HPIV3 infection comprises administering a vaccine to the subject.

[0020] In certain exemplary embodiments, the vaccine is co-administered with an adjuvant. In certain exemplary embodiments, the vaccine is administered in combination with an additional vaccine. In certain exemplary embodiments, the additional vaccine is a respiratory syncytial virus (RSV) vaccine or an influenza vaccine.

[0021] In certain exemplary embodiments, the subject is a human, hi certain exemplary embodiments, the human subject is an infant, a young child, or an elderly person.

[0022] In certain exemplary embodiments, the vaccine increases serum concentrations of neutralizing antibodies and the subject has pre-existing hMPV immunity.

[0023] In certain exemplary embodiments, vaccines are provided for use in eliciting an immune response against hMPV and HPIV3 or protecting a subject from hMPV and HPIV3 infection, comprising administering a viral vector, live attenuated virus, or vaccine to a subject.

[0024] In certain exemplary embodiments, the viral vectors, live attenuated viruses, or vaccines are for use in the manufacture of a medicament for inducing an immune response against hMPV and HPIV3, or for protecting a subject from hMPV and HPIV3 infection.

[0025] In certain exemplary embodiments, there is provided a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of a viral vector, a live attenuated virus, or a vaccine.

[0026] In certain exemplary embodiments, methods are provided for preventing hMPV infection and HPIV3 infection or alleviating one or more symptoms of hMPV infection and HPIV3 infection, comprising administering a prophylactically effective amount of a viral vector, live attenuated virus, or vaccine to a subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally.

[0027] In certain exemplary embodiments, the use of a viral vector, live attenuated virus, or vaccine is provided for the manufacture of a medicament for use in treating a subject in need of treatment.

[0028] In certain exemplary embodiments, the viral vector, live attenuated virus, or vaccine is for use in treating a subject in need of treatment.

[0029] In certain exemplary embodiments, a kit is provided that includes a container that contains a single-use or multi-use dose of a viral vector, a live-attenuated virus, or a vaccine, optionally where the container is a vial or a pre-filled syringe or injector.

[0030] In certain exemplary embodiments, the viral vector, live attenuated virus, or vaccine comprises an hMPV F nucleic acid sequence having at least 95% identity to SEQ ID NO:8, or comprises SEQ ID NO:8.

[0031] In another aspect, a viral vector is provided encoding a human metapneumovirus (hMPV) F polypeptide antigen, the F polypeptide lacking a transmembrane domain, lacking a cytoplasmic tail, and comprising an F0 cleavage site mutation comprising amino acid substitutions Q100R and S101R, which substitute arginine for glutamine at amino acid position 100 of SEQ ID NO:1 and arginine for serine at amino acid position 101 of SEQ ID NO:1; a human rhinovirus 3C (HRV-3C) protease cleavage site; a heterologous signal peptide; a polyhistidine tag (e.g., a 6xHis tag, 8xHis tag, etc.) and / or a Strep II tag; and a foldon domain.

[0032] In another aspect, a viral vector is provided encoding a human metapneumovirus (hMPV) F polypeptide antigen, the F polypeptide lacking a transmembrane domain, lacking a cytoplasmic tail, and comprising an amino acid substitution substituting a threonine at amino acid position 160 of SEQ ID NO:1 and an amino acid substitution substituting an asparagine at amino acid position 46 of SEQ ID NO:1.

[0033] In certain exemplary embodiments, the F1 polypeptide comprises an amino acid substitution that replaces the threonine at amino acid position 160 with phenylalanine, tryptophan, tyrosine, valine, alanine, isoleucine, or leucine. In certain exemplary embodiments, the F1 polypeptide comprises an amino acid substitution T160F that replaces the threonine at amino acid position 160 with phenylalanine.

[0034] In certain exemplary embodiments, the F polypeptide comprises an amino acid substitution that replaces the asparagine at amino acid position 46 with valine, alanine, isoleucine, leucine, phenylalanine, tyrosine, or proline. In certain exemplary embodiments, the F polypeptide comprises an amino acid substitution N46V that replaces the asparagine at amino acid position 46 with valine.

[0035] In certain embodiments, the hMPV polypeptide comprises a substitution at position 160 of SEQ ID NO:1 and a substitution at position 46 of SEQ ID NO:1, where the substitutions are "stabilizing substitutions" that stabilize the tertiary and / or quaternary structure of the hMPV polypeptide. Stabilizing substitutions include, but are not limited to, hydrophobic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, and methionine); hydrophilic amino acids (e.g., cysteine, serine, threonine, asparagine, and glutamine); amino acids that form disulfide bonds (e.g., cysteine); amino acids that form hydrogen bonds (e.g., tryptophan, histidine, tyrosine, and phenylalanine); charged amino acids (e.g., aspartic acid, glutamic acid, arginine, lysine, and histidine), and the like.

[0036] In certain exemplary embodiments, the F2 polypeptide comprises at least 95% sequence identity to SEQ ID NO:7.

[0037] In certain exemplary embodiments, the F polypeptide further comprises an F cleavage site mutation comprising the amino acid substitutions Q100R and S101R, which replace the glutamine at amino acid position 100 of SEQ ID NO:1 with arginine and the serine at amino acid position 101 of SEQ ID NO:1 with arginine.

[0038] In certain exemplary embodiments, the F polypeptide includes a signal peptide.

[0039] In certain exemplary embodiments, the F polypeptide optionally comprises at least one tag sequence that is a polyhistidine tag (e.g., a 6xHis tag, an 8xHis tag, etc.) and / or a Strep II tag.

[0040] In certain exemplary embodiments, the F polypeptide comprises a foldon domain.

[0041] In certain exemplary embodiments, live attenuated viruses, including viral vectors, are provided.

[0042] In certain exemplary embodiments, a pharmaceutical composition comprising a viral vector is provided.

[0043] In certain exemplary embodiments, the live-attenuated virus or pharmaceutical composition comprises a vaccine.

[0044] In certain exemplary embodiments, the method of inducing an immune response against hMPV and / or HPIV3 or protecting a subject from hMPV and / or HPIV3 infection comprises administering a vaccine to the subject.

[0045] In certain exemplary embodiments, the vaccine is co-administered with an adjuvant. In certain exemplary embodiments, the vaccine is administered in combination with an additional vaccine. In certain exemplary embodiments, the additional vaccine is a respiratory syncytial virus (RSV) vaccine or an influenza vaccine.

[0046] In certain exemplary embodiments, the subject is a human, hi certain exemplary embodiments, the human subject is an infant, a young child, or an elderly person.

[0047] In certain exemplary embodiments, the vaccine increases serum concentrations of neutralizing antibodies and the subject has pre-existing hMPV immunity.

[0048] In certain exemplary embodiments, vaccines are provided for use in eliciting an immune response against hMPV and HPIV3 or protecting a subject from hMPV and HPIV3 infection, comprising administering a viral vector, live attenuated virus, or vaccine to a subject.

[0049] In certain exemplary embodiments, the viral vectors, live attenuated viruses, or vaccines are for use in the manufacture of a medicament for inducing an immune response against hMPV and HPIV3, or for protecting a subject from hMPV and HPIV3 infection.

[0050] In certain exemplary embodiments, there is provided a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of a viral vector, a live attenuated virus, or a vaccine.

[0051] In certain exemplary embodiments, methods are provided for preventing hMPV infection and HPIV3 infection or alleviating one or more symptoms of hMPV infection and HPIV3 infection, comprising administering a prophylactically effective amount of a viral vector, live attenuated virus, or vaccine to a subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally.

[0052] In certain exemplary embodiments, the use of a viral vector, live attenuated virus, or vaccine is provided for the manufacture of a medicament for use in treating a subject in need of treatment.

[0053] In certain exemplary embodiments, the viral vector, live attenuated virus, or vaccine is for use in treating a subject in need of treatment.

[0054] In certain exemplary embodiments, a kit is provided that includes a container that contains a single-use or multi-use dose of a viral vector, a live-attenuated virus, or a vaccine, optionally where the container is a vial or a pre-filled syringe or injector.

[0055] In certain exemplary embodiments, which are viral vectors, live attenuated viruses, or vaccines, the vector comprises an hMPV F nucleic acid sequence having at least 95% identity to SEQ ID NO:8, or comprises SEQ ID NO:8.

[0056] In another aspect, a viral vector is provided encoding an antigenic human metapneumovirus (hMPV) pre-fusion F polypeptide, the pre-fusion F polypeptide lacking a transmembrane domain, lacking a cytoplasmic tail, and comprising: an amino acid substitution T160F, which replaces the threonine at amino acid position 160 of SEQ ID NO:1 with a phenylalanine, and an amino acid substitution N46V, which replaces the asparagine at amino acid position 46 of SEQ ID NO:1 with a valine; an F0 cleavage site mutation comprising amino acid substitutions Q100R and S101R, which replace the glutamine at amino acid position 100 of SEQ ID NO:1 with an arginine and the serine at amino acid position 101 of SEQ ID NO:1 with an arginine; a human rhinovirus 3C (HRV-3C) protease cleavage site; a signal peptide; a polyhistidine tag (e.g., a 6xHis tag, an 8xHis tag, etc.) and / or a Strep II tag; and a foldon domain.

[0057] In certain exemplary embodiments, hMPV F is derived from A strain hMPV.

[0058] In certain exemplary embodiments, the hMPV F is an hMPV of the A1 or A2 subtype.

[0059] In certain exemplary embodiments, the pre-fusion F polypeptide comprises at least 95% sequence identity to or comprises SEQ ID NO:3.

[0060] In certain exemplary embodiments, the F2 polypeptide comprises at least 95% sequence identity to SEQ ID NO:7.

[0061] In certain exemplary embodiments, the F polypeptide is a pre-fusion F polypeptide.

[0062] In certain exemplary embodiments, the viral vector backbone is derived from a parainfluenza virus (PIV).

[0063] In certain exemplary embodiments, the F polypeptide comprises the amino acid substitution T160F, which replaces the threonine at amino acid position 160 with a phenylalanine, and the amino acid substitution N46V, which replaces the asparagine at amino acid position 46 with a valine.

[0064] In certain exemplary embodiments, the F polypeptide comprises SEQ ID NO:7.

[0065] In certain exemplary embodiments, the viral vector comprises a nucleic acid molecule having at least 95% sequence identity to SEQ ID NO: 8. In certain exemplary embodiments, the nucleic acid molecule comprises SEQ ID NO:8.

[0066] In certain exemplary embodiments, the hMPV F polypeptide is a pre-fusion F polypeptide.

[0067] In certain exemplary embodiments, the viral vector backbone is a chimeric bovine / human parainfluenza 3 virus (rB / HPIV3) vector backbone.

[0068] In certain exemplary embodiments, the viral vector backbone is a human parainfluenza virus type 3 (HPIV3) vector backbone.

[0069] In certain exemplary embodiments, live attenuated viruses, including viral vectors, are provided.

[0070] In certain exemplary embodiments, a pharmaceutical composition comprising a viral vector is provided.

[0071] In certain exemplary embodiments, the live-attenuated virus or pharmaceutical composition comprises a vaccine.

[0072] In certain exemplary embodiments, the method of inducing an immune response against hMPV and / or HPIV3 or protecting a subject from hMPV and / or HPIV3 infection comprises administering a vaccine to the subject.

[0073] In certain exemplary embodiments, the vaccine is co-administered with an adjuvant. In certain exemplary embodiments, the vaccine is administered in combination with an additional vaccine. In certain exemplary embodiments, the additional vaccine is a respiratory syncytial virus (RSV) vaccine or an influenza vaccine.

[0074] In certain exemplary embodiments, the subject is a human, hi certain exemplary embodiments, the human subject is an infant, a young child, or an elderly person.

[0075] In certain exemplary embodiments, the vaccine increases serum concentrations of neutralizing antibodies and the subject has pre-existing hMPV immunity.

[0076] In certain exemplary embodiments, vaccines are provided for use in eliciting an immune response against hMPV and HPIV3 or protecting a subject from hMPV and HPIV3 infection, comprising administering a viral vector, live attenuated virus, or vaccine to a subject.

[0077] In certain exemplary embodiments, the viral vectors, live attenuated viruses, or vaccines are for use in the manufacture of a medicament for inducing an immune response against hMPV and HPIV3, or for protecting a subject from hMPV and HPIV3 infection.

[0078] In certain exemplary embodiments, there is provided a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of a viral vector, a live attenuated virus, or a vaccine.

[0079] In certain exemplary embodiments, methods are provided for preventing hMPV infection and HPIV3 infection or alleviating one or more symptoms of hMPV infection and HPIV3 infection, comprising administering a prophylactically effective amount of a viral vector, live attenuated virus, or vaccine to a subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally.

[0080] In certain exemplary embodiments, the use of a viral vector, live attenuated virus, or vaccine is provided for the manufacture of a medicament for use in treating a subject in need of treatment.

[0081] In certain exemplary embodiments, the viral vector, live attenuated virus, or vaccine is for use in treating a subject in need of treatment.

[0082] In certain exemplary embodiments, a kit is provided that includes a container that contains a single-use or multi-use dose of a viral vector, a live-attenuated virus, or a vaccine, optionally where the container is a vial or a pre-filled syringe or injector.

[0083] In certain exemplary embodiments, which are viral vectors, live attenuated viruses, or vaccines, the vector comprises an hMPV F nucleic acid sequence having at least 95% identity to SEQ ID NO:8, or comprises SEQ ID NO:8.

[0084] The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of exemplary embodiments taken in conjunction with the accompanying drawings. This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0085] [Figure 1] FIG. 1 shows a schematic of the design of a recombinant bovine human parainfluenza virus 3 (B / HPIV3) expressing the human metapneumovirus (HMPV) fusion (F) protein from an added gene. The B / HPIV3 backbone contains the N, P, M, and L genes (labeled) from bovine parainfluenza virus 3 (BPIV3) and the F and HN (labeled) from human parainfluenza virus 3 (HPIV3). Gene start and gene end transcription signals are shown as light and dark grey boxes flanking each ORF, respectively. Eight different HMPV (CAN97-83) F versions were inserted into the AscI site between the N and P genes to obtain eight virus constructs. The HMPV F inserts were designed to be transcribed as separate mRNAs starting at the P gene start and ending at the N gene end. [Diagram 2] FIG. 1 shows design considerations for a panel of 21 candidate hMPV pre-fusion F antigens, shown as two exemplary constructs, D185P and T160F_N46V. Construct D185P is used as a benchmark reference for measuring the activity of various novel constructs. "(mut)" = shaded "ENPRRRR" amino acid sequence and shaded "P", shaded "V" and shaded "F" single amino acids; "Linker" = bold, underlined "GGGGS", "GRS" and "G" amino acid sequences; "foldon" = underlined "GYIPEAPRDGQAYVRKDGEWVLLSTFL" amino acid sequence; "8xHIS" = shaded "HHHHHHHH" amino acid sequence; "StrepII" = shaded "SAWSHPQFEK" sequence. [Diagram 3] FIG. 1 shows mouse IgG antibody titers measured on days 0, 21, and 35 against four hMPV pre-fusion F antigen protein constructs (data points listed from left to right at each time point are as follows): (1) A2-F D185P, (2) A2-F T160F_N46V, (3) A2-F K138F, (4) A2-F G366F_K362F, and controls: hMPV (5) A1-F pre-F lot 1, (6) A1-F pre-F lot 2, (7) A1-F post-F, and (8) B2pre-F. [Figure 4] Figure 1 shows mouse hMPV microneutralizing antibody titers measured on days 21 and 35 against four hMPV pre-fusion F antigen protein constructs: (1) A2-F D185P, (2) A2-F T160F_N46V, (3) A2-F K138F, (4) A2-F G366F_K362F and controls: hMPV (1) A1 pre-F lot 1, (2) A1 pre-F lot 2, (3) A1 postF, and (4) B2 pre-F. [Diagram 5] FIG. 1 shows SEC-MALS results for the reference A1 proteins, A1-A185P and A1-postF and the A2 protein antigen candidates, A2-T160F_N46V and A2-D185P. [Figure 6] Representative melting curves (upper panels), smoothed first derivative curves (middle panels), and light scattering [mAU] (lower panels) for A1-pre-F and A1-post-F [fluorescence emission at 330 and 350 nm] measured by nanoDSF. [Figure 7] Representative melting curves (top panel) and smoothed first derivative curves (middle panel) and light scattering [mAU] (bottom panel) for protein samples derived from A2-D185P and A2-T160F_N46V constructs [fluorescence emission at 330 and 350 nm] measured by nanoDSF. [Figure 8]FIG. 1 shows human IgG antibody titers measured on day 14 collected from supernatants of MIMIC co-cultures treated with either IPOL (polio vaccine) at 1:50 dilution against three polio strains - Polio 1 (Panel A), Polio 2 (Panel B) and Polio 3 (Panel C) or untreated control (no treatment, no human skeletal muscle cells in co-culture, "No Antigen (No HSK)"). [Figure 9] Figure 1 shows human IgG antibody titers measured on day 14 collected from supernatants of MIMIC co-cultures treated with 50 ng / ml RSV Pre-FNP (RSV Pre-F protein fused to ferritin nanoparticles) treatment against RSV Pre-F (Panel A) and RSV Post-F (Panel B). Panel C shows whether the antibodies were functional as measured by an RSV neutralization assay. [Figure 10] Figure 1 shows human IgG antibody titers measured on day 14 collected from supernatants of MIMO co-cultures treated with experimental groups - hMPV pre-F protein (100 ng / ml or 500 ng / ml) or hMPV post F antigen protein (100 ng / ml) or control groups - no HSK, RSV Pre-F NP, or IPOL against hMPV pre-F antigen (panel A) or hMPV post-F antigen (panel B). [Figure 11] FIG. 1 shows hMPV microneutralizing antibody titers measured on day 14 using harvested supernatants of MIMIC cocultures treated with hMPV pre-F protein (100 ng / ml or 500 ng / ml), post-F antigen protein (100 ng / ml), or no antigen without HSK. [Figure 12]Microscopic images of Vero (panel A) and A549 (panel B) cells in 24-well plates infected with eight different HMPV (CAN97-83) F versions. Cells were fixed and plaques were immunostained for PIV3 and HMPV F antigens detected with infrared dye-conjugated antibodies shown as green and red, respectively. For each virus, stained plaques are shown as overlay images of PIV3 and HMPV F staining. Plaques appear in one color when only HMPV F is detected, in a second color when only PIV3 antigen is detected, and in a third color when both HMPV F and PIV3 antigens are detected. The tabulated percentage of B / HPIV3 plaques appearing in the third color in panel A, indicating HMPV F expression, is shown in FIG. [Figure 13] FIG. 13 is a tabulated percentage of B / HPIV3 plaques from FIG. 12 that were positive for HMPV F expression as determined by double antigen staining plaque assay. [Figure 14] Figure 1: Expression of viral proteins during in vitro replication. Vero cells were infected with eight different HMPV (CAN97-83) F versions at an MOI of 3 PFU / cell, and after 48 hours, cell lysates were prepared for analysis by SDS-PAGE (under reducing and denaturing conditions) and Western blotting. BPIV3 N and P, HPIV3 F and HN, and HMPV F were detected using specific primary antibodies followed by infrared dye-conjugated secondary antibodies. GAPDH detection was included as a loading control. Non-infected Vero cell lysates were included as mock controls. [Figure 15]Schematic diagram of the experimental design for analyzing protective efficacy and immunogenicity of eight different HMPV (CAN97-83) F versions in hamsters. Groups of six golden Syrian hamsters received either a single intranasal (IN) dose of one of the eight B / HPIV3-HMPV F viruses or two doses of one of the HMPV purified proteins F-D185P / Q100R / S101R or F-N46V / T160F given with alum-85 adjuvant by intramuscular (IM) injection three weeks apart. Groups of six hamsters immunized by the IN route with B / HPIV3 vector (empty) and wt HMPV CAN97-83 (wt hMPV subgroup A) were included as controls. Serum was collected from all groups two weeks after the subunit vaccine boost and four weeks after virus immunization to evaluate hMPV neutralizing antibody responses. Hamsters were challenged with wt HMPV(A) (5×10 5 PFU); lungs and nasal turbinates were harvested 3 days after challenge to determine HMPV load by plaque assay. [Figure 16] FIG. 16 shows serum HMPV neutralizing antibody responses in hamsters immunized IN with B / HPIV3 expressing eight different HMPV (CAN97-83)F versions or two doses of one of the HMPV purified proteins described in FIG. 15. Serum samples from immunized hamsters were analyzed for HMPV neutralizing antibodies by a 60% plaque reduction neutralization assay on Vero cells. Data were analyzed by one-way ANOVA and Dunnett's multiple comparison test. Differences between the vaccine candidate and wt HMPV immunized groups were considered statistically significant at the 95% confidence interval (P≦0.05) and are indicated by asterisks (**, P≦0.01). [Figure 17]Figure 1 demonstrates protection of hamster airways from infection by wild-type (wt) HMPV challenge. Immunized hamsters were inoculated intranasally with wt HMPV CAN97-83 at a challenge dose of 5x105 PFU. Three days post-infection, animals were euthanized and nasal turbinates and lungs were subsequently harvested. Tissues were homogenized and HMPV titers were determined by plaque assay on Vero cells. Results are presented as HMPV PFU (log10) per gram of tissue in nasal turbinates (panel A) and lungs (panel B). Data were analyzed by one-way ANOVA and Tukey's multiple comparison test. Differences between each HMPV-immune group and the HMPV-naive [B / HPIV3 vector (empty)] group were considered significant at P ≤ 0.05 and are indicated with asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; or ns, not significant). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] The present disclosure relates, inter alia, to novel hMPV F protein vaccines and methods of vaccination therewith. In certain embodiments, the hMPV F protein is expressed from a recombinant chimeric bovine / human parainfluenza virus 3 (rB / HPIV3) vector ("rB / HPIV3-hMPV F" vector).

[0087] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, however, methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. In the event of a conflict, the present specification, including definitions, will control. In general, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Throughout the specification and embodiments, the terms "having" and "including" or variations such as "having", "having", "including" or "including" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited in this specification, this citation is not an admission that any of these documents form part of the general knowledge in the art.

[0088] It should be noted that the term "a" or "an" entity refers to one or more of that entity, for example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0089] Furthermore, as used herein, "and / or" should be understood as specifically disclosing each of the two specified features or components with or without the other. Thus, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" when used in phrases such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0090] Whenever an embodiment is described herein with the word "comprising," it should be understood that analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press can provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0092] Units, prefixes and symbols are shown in the format accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0093] The terms "approximately" or "about" are used herein to mean approximately, in the vicinity, or within the region. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value above and below the stated value by, for example, a variance of 10% above and below (higher or lower). In some embodiments, the term indicates a deviation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±10% from the stated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the stated numerical value. In some embodiments, "about" refers to a ±4% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±3% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±2% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±1% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" refers to a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±0.01% from the indicated numerical value.

[0094] As used herein, the term "antigen stability" refers to the stability of an antigen over time or in solution.

[0095] As used herein, the term "cavity-filling substitution" refers to engineered hydrophobic substitutions to fill a cavity present in the pre-fusion hMPV F trimer.

[0096] As used herein, the term "F protein" or "hMPV F protein" refers to the protein of hMPV involved in mediating the fusion of the viral envelope with the host cell membrane during viral entry. The F protein can mediate fusion between infected and uninfected cells to form multinucleated cells or syncytia.

[0097] As used herein, the term "hMPV F polypeptide" or "F polypeptide" refers to a polypeptide that contains at least one epitope of the hMPV F protein.

[0098] As used herein, the term "transmembrane domain" refers to an approximately 23 amino acid sequence near the c-terminus of hMPV F0 / F1 that spans the membrane of the hMPV virion. In certain embodiments, the transmembrane domain comprises the amino acid sequence GFIIVIILIAVLGSSMILVSIFII of SEQ ID NO:1.

[0099] As used herein, the term "cytoplasmic tail" refers to an approximately 25 amino acid sequence at the c-terminus of hMPV F0 / F1 that is located inside the virion. In certain embodiments, the transmembrane domain comprises the amino acid sequence IKKTKKPTGAPPELSGVTNNGFIPHN of SEQ ID NO:1.

[0100] As used herein, "foldon domain" refers to the trimerization domain of T4 fibritin.

[0101] As used herein, "signal peptide" or "signal sequence" refers to a peptide of about 16-30 amino acids in length present at the amino- or carboxy-terminus of a polypeptide that functions to translocate the polypeptide into the secretory pathway in the endoplasmic reticulum and Golgi apparatus. In certain embodiments, the signal sequence corresponds to amino acids 1-18 of any one of SEQ ID NOs: 1, 3, 5, and 7.

[0102] As used herein, "tag sequence" or "affinity tag" refers to a polypeptide sequence that can be used to purify a polypeptide or protein that contains the tag sequence. Examples of tags include polyhistidine tags (e.g., hexahistidine (6xHis tag), octahistidine (8xHis tag), etc.), glutathione S-transferase (GST), FLAG, streptavidin-binding peptide (SBP), Strep II, maltose-binding protein (MBP), calmodulin-binding protein (CBP), chitin-binding domain (CBD), S protein of RNase A, hemagglutinin (HA), c-Myc, etc.

[0103] As used herein, the term "intra-protomer stabilizing substitution" refers to an amino acid substitution in hMPV F that stabilizes the pre-fusion conformation by stabilizing interactions within the protomer of the hMPV F trimer.

[0104] As used herein, the term "inter-protomer stabilizing substitution" refers to an amino acid substitution in hMPV F that stabilizes the interaction of the protomers of the hMPV F trimer, thereby stabilizing the prefusion conformation.

[0105] As used herein, the term "protease cleavage" refers to the proteolysis (sometimes referred to as "clipping") of a sensitive residue (e.g., lysine or arginine) at a protease cleavage site of a polypeptide sequence. Protease cleavage sites include viral protease cleavage sites, such as hMPV F0 protease cleavage site, respiratory syncytial virus (RSV) F0 protease cleavage site, and human rhinovirus 3C (HRV-3C) protease cleavage site.

[0106] As used herein, the term "post-fusion" with respect to hMPV F refers to the stable conformation of hMPV F that occurs after merging of the viral and host cell membranes.

[0107] As used herein, the term "pre-fusion" with respect to hMPV F refers to the conformation of hMPV F adopted prior to virus-cell interaction.

[0108] As used herein, the term "protomer" refers to a structural unit of an oligomeric protein. In the case of hMPV F, each individual unit of the hMPV F trimer is a protomer.

[0109] As used herein, the term "immune response" refers to the response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage, or polymorphonuclear cell, to an antigen or a stimulus, such as a vaccine. An immune response can include any cell of the body that participates in a host defense reaction, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate and / or adaptive immune responses.

[0110] As used herein, a "protective immune response" refers to an immune response that protects a subject from infection (e.g., prevents infection or prevents the development of a disease associated with infection). Methods of measuring immune responses include, for example, measuring lymphocyte (such as B cells or T cells) proliferation and / or activity, cytokine or chemokine secretion, inflammation, antibody production, and the like.

[0111] As used herein, an "antibody response" is an immune response in which antibodies are produced.

[0112] As used herein, "antigen" refers to an agent that elicits an immune response when exposed to or administered to an organism and / or binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody (e.g., produced by a B cell). In some embodiments, the antigen elicits a humoral response in the organism (e.g., including production of antigen-specific antibodies). Alternatively, or in addition, in some embodiments, the antigen elicits a cellular response in the organism (e.g., involving T cells whose receptors specifically interact with the antigen). A particular antigen may elicit an immune response in one or more members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target species. In some embodiments, the antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of members of the target species. In some embodiments, the antigen binds to an antibody and / or a T cell receptor and may or may not induce a specific physiological response in the organism. In some embodiments, for example, the antigen binds to an antibody and / or a T cell receptor in vitro, whether or not such interactions occur in vivo. In some embodiments, the antigen reacts with the products of specific humoral or cellular immunity. Antigens include hMPV polypeptides described herein.

[0113] As used herein, "adjuvant" refers to a substance or vehicle that enhances the immune response to an antigen. Adjuvants can include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphates) to which antigens are adsorbed; water-in-oil or oil-in-water emulsions in which the antigen solution is emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Killed mycobacteria are sometimes included to further enhance antigenicity (e.g., Freund's complete adjuvant). Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (see, for example, U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants can also include biological molecules such as Toll-like receptor (TLR) agonists and costimulatory molecules.

[0114] As used herein, an "antigenic hMPV polypeptide" refers to a polypeptide that includes all or a portion of an hMPV amino acid sequence of sufficient length that the molecule is antigenic to hMPV.

[0115] As used herein, "attenuated" or a virus having an "attenuated phenotype" refers to a virus that has reduced pathogenicity compared to a reference virus under similar infection conditions. Attenuation typically relates to a reduction in viral replication compared to the replication of a reference wild-type virus under similar infection conditions, and thus "attenuated" and "restricted replication" are often used interchangeably. In some hosts (typically non-native hosts, including laboratory animals), disease is not evident during infection with the reference virus in question, and restricted viral replication can be used as a surrogate marker of attenuation. In some embodiments, the attenuated disclosed rB / HPIV3-hMPV F vectors exhibit at least about a 10-fold or greater reduction in viral titer in the upper or lower respiratory tract of a mammal, such as at least about a 100-fold or greater reduction, in viral titer in the upper or lower respiratory tract of a mammal of the same species, respectively, under the same infection conditions. The attenuated rB / HPIV3-hMPV F vectors may exhibit a distinct phenotype, including, but not limited to, altered growth, temperature-sensitive growth, host range restricted growth, or plaque size changes.

[0116] As used herein, a "gene" of the rB / HPIV3 vectors described herein refers to a portion of the rB / HPIV3 genome that encodes an mRNA, typically beginning with a gene start (GS) signal at the upstream (3') end and ending with a gene end (GE) signal at the downstream (5') end. In this context, the term gene also encompasses what are called "translation open reading frames" or ORFs, particularly when a protein such as C is expressed from an additional ORF rather than from its own mRNA. To construct the disclosed rB / HPIV3 vectors, one or more genes or genome segments can be deleted, inserted or replaced in whole or in part.

[0117] As used herein, "host cell" refers to a cell in which a vector can be propagated and its nucleic acid expressed. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.

[0118] As used herein, an "infectious self-replicating virus" refers to a virus that can invade and replicate in cultured cells or cells of an animal or human host to produce progeny viruses that have the same activity.

[0119] As used herein, an "isolated" biological component is one that has been substantially separated or purified from other biological components, such as other biological components with which it naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA and proteins. "Isolated" proteins, peptides, nucleic acids, and viruses include those that have been purified by standard purification methods. Isolated does not require absolute purity and can include proteins, peptides, nucleic acids, or viral molecules that are at least 50% pure, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% pure.

[0120] As used herein, "linker" refers to a bifunctional molecule that can be used to link two molecules into one continuous molecule. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0121] As used herein, "nucleic acid molecule" refers to a polymeric form of nucleotides that can include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. Nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide. The term "nucleic acid molecule" as used herein is synonymous with "polynucleotide." Nucleic acid molecules are typically at least 10 bases in length, unless otherwise specified. The term includes single-stranded and double-stranded forms of DNA. Nucleic acid molecules can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages.

[0122] As used herein, a first nucleic acid sequence is "operably linked" to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to link two protein coding regions, in the same reading frame.

[0123] As used herein, "parainfluenza virus" or "PIV" refers to a number of enveloped, nonsegmented, negative-sense, single-stranded RNA viruses from the family Paramyxoviridae that are descriptively grouped together. This includes all of the members of the Respirovirus genus (e.g., HPIV1, HPIV3) and many of the members of the Rubulavirus genus (e.g., HPIV2, HPIV4, PIV5). PIVs are composed of two structural modules: (1) an internal ribonucleoprotein core or nucleocapsid that contains the viral genome; and (2) an outer, roughly spherical lipoprotein envelope. The PIV genome is approximately 15,000 nucleotides long and encodes at least eight polypeptides. These proteins include the nucleocapsid structural protein (NP, NC or N depending on the genus), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), hemagglutinin-neuraminidase glycoprotein (HN), large polymerase protein (L), and the C and D proteins. The gene order is 3'-NPMF-HN-L-5', with each gene encoding a distinct protein encoding mRNA, and the P gene containing one or more additional open reading frames (ORFs) encoding accessory proteins.

[0124] As used herein, "polypeptide" refers to any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptide" applies to amino acid polymers, including natural amino acid polymers and unnatural amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are unnatural amino acids, e.g., artificial chemical mimetics of the corresponding natural amino acids. "Residue" refers to an amino acid or amino acid mimetic incorporated into the polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminus (N-terminus) and a carboxy terminus (C-terminus). "Polypeptide" is used interchangeably with peptide or protein and is used herein to refer to a polymer of amino acid residues.

[0125] As used herein, a "recombinant" nucleic acid molecule or protein is one that has a sequence that does not occur in nature, e.g., one that contains one or more nucleic acid substitutions, deletions, or insertions, and / or one that has a sequence that is created by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by chemical synthesis, by targeted mutation of a naturally occurring nucleic acid molecule or protein, or by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques. A recombinant virus is a virus that contains a genome that includes a recombinant nucleic acid molecule.

[0126] As used herein, "viral vector" refers to an expression vector that can be used to produce an antigenic hMPV polypeptide, such as an antigenic hMPV F polypeptide. Suitable viral vectors include, but are not limited to, viral vectors based on the following: parainfluenza virus; vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol V is Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 3:1063 1064, 1997; Li et al., Invest Opthalmol V is Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999); 9:8186,1998,Flannery et al.,PNAS 94:6916 6921,1997;Bennett et al.,Invest Opthalmol V is Sci 38:2857 2863,1997;Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al.,Hum Gene Ther 10:641 648,1999;Ali et al.,Hum Mol Genet.5:591594,1996;Srivastava, Samulski et al., J.Vir.(1989)63:3822-3828;Mendelson et al. al., Virol. (1988) 166:154-165; and Flotte et al. al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., vectors derived from retroviruses such as murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentiviruses, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0127] Depending on the host / vector system utilized, any of a number of suitable transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcription terminators, etc., can be used in the expression vector (see, e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0128] In some embodiments, the nucleotide sequence encoding an antigenic hMPV polypeptide, e.g., an antigenic hMPV F polypeptide, is operably linked to a control element, e.g., a transcription control element, e.g., a promoter. The transcription control element can be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., a bacterial or archaeal cell). In some embodiments, the nucleotide sequence encoding an antigenic hMPV polypeptide, e.g., an antigenic hMPV F polypeptide, is operably linked to multiple control elements that allow for expression of the nucleotide sequence encoding the antigenic hMPV polypeptide, e.g., an antigenic hMPV F polypeptide, in both prokaryotic and eukaryotic cells.

[0129] Non-limiting examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include those derived from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. The expression vector may also include a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include a nucleotide sequence encoding a protein tag (e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.) that is fused to the site-directed modifying polypeptide, thus resulting in a chimeric polypeptide.

[0130] In certain embodiments, an antigenic hMPV polypeptide, e.g., an antigenic hMPV F polypeptide, is expressed in a live attenuated virus. As used herein, "live attenuated virus" refers to a virus that has reduced pathogenicity compared to the wild-type virus. Live attenuated vaccines tend to favor the production of CD8+ cytotoxic T lymphocytes and T-dependent antibody responses. Compared to inactivated vaccines, attenuated vaccines result in stronger and more durable immune responses with a rapid immune onset. Attenuated vaccines function by stimulating the production of antibodies and memory immune cells in the subject's response to the particular pathogen against which the vaccine protects. Live attenuated viruses useful for use as vaccines include, but are not limited to, measles virus, mumps virus, rubella virus, chickenpox virus, yellow fever virus, influenza virus, smallpox virus, poliovirus, rotavirus, parainfluenza virus, Japanese encephalitis virus, varicella virus, varicella zoster virus, and the like. In an exemplary embodiment, a live attenuated parainfluenza virus is provided.

[0131] As used herein, "recombinant chimeric bovine / human parainfluenza virus 3" or "rB / HPIV3" refers to a chimeric PIV3 comprising a genome that includes a combination of BPIV3 and HPIV3 genes that together constitute a full complement of PIV3 genes in the PIV3 genome (N, P, M, F, HN and L genes). The disclosed rB / HPIV3 vectors are based on the BPIV3 genome with the F and HN genes replaced with the corresponding genes from HPIV3 (one example is discussed in Schmidt AC et al., J. Virol. 74:8922-8929, 2000). The structural and functional genetic elements that control gene expression, such as gene start and gene end sequences and genomic and antigenomic promoters, are BPIV3 structural and functional genetic elements. The rB / HPIV3 vectors described herein are infectious, autonomously replicating and attenuated.

[0132] In some embodiments, a heterologous gene encoding an hMPV F protein or a variant thereof is inserted between the N and P genes of the rB / HPIV3 genome to generate an "rB / HPIV3-hMPV F" vector. The disclosed rB / HPIV3-hMPV F vectors are infectious, self-replicating, attenuated, and can be used to induce a bivalent immune response against hMPV and HPIV3 in a subject. Particularly suitable rB / HPIV3 vectors are described in U.S. Patent Application Publication No. 2021 / 0145958, which is incorporated herein by reference in its entirety for all purposes.

[0133] Non-limiting examples of methods for making recombinant parainfluenza viruses (such as rB / HPIV3) containing heterologous genes, methods for attenuating viruses (e.g., by recombinant or chemical means), and viral sequences and reagents for use in such methods are described in U.S. Patent Application Publication Nos. 2012 / 0045471, 2010 / 0119547, 2009 / 0263883, 2009 / 0017517, U.S. Patent Nos. 7,632,508, 7,622,123, 7,250,171, 7,208,161, 7,201,907, 7,192,593, PCT Publication WO 2016 / 118642, Liang et al. (J. Virol, 88(8):4237-4250, 2014), and Tang et al. (J Virol, 77(20):10819-10828, 2003), each of which is incorporated herein by reference. In some embodiments, these methods can be modified as needed using the descriptions provided herein to construct the disclosed rB / HPIV3-hMPV F vectors.

[0134] The genome of the rB / HPIV3-hMPV F vector may contain one or more mutations (e.g., mutations that cause deletion, substitution, or insertion of amino acids), as long as the resulting rB / HPIV3-hMPV F retains the desired biological function, such as the level of attenuation or immunogenicity. These mutations in the sequence may be naturally occurring mutations or may be engineered by using genetic engineering techniques. In the case of parainfluenza virus genomes, due to hexamer phasing, care may need to be taken that the nucleotide length of the genome is a multiple of six. Otherwise, the genome may not be functional. Calain P and Roux L, J.Virol., 1993, PMID: 8392616; Kolakofsky et al.; J.Virol. 1998; PMID 9444980.

[0135] Other mutations include replacement of the 3' end of the genome with its counterpart from the antigenome, associated with changes in RNA replication and transcription. Additionally, intergenic regions, such as the long variable non-coding regions at the 3' and 5' ends of each gene, can be shortened or lengthened or altered in sequence content, and naturally occurring gene duplications (Skiapodopoulos et al., Virology 272(1):225-34 (2000); Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598 (1986); Collins et al., Proc. Natl. Acad. Sci. USA 84:5134-5138 (1987)) can be removed or altered to different intergenic regions by the methods described herein.

[0136] In another embodiment, the sequence surrounding the translation start site (typically including nucleotide -3) of a selected viral gene is modified, either alone or in combination with the introduction of an upstream start codon, to modulate gene expression by directing translational up- or down-regulation.

[0137] Alternatively, or in combination with other modifications disclosed herein, gene expression can be modulated by altering the transcriptional GS signal of selected genes of the virus. In further embodiments, modifications to the transcriptional GE signal can be incorporated into the viral genome.

[0138] In addition to the above modifications to rB / HPIV3-hMPV F, different or additional modifications to the genome can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic regions (e.g., a unique AscI site between the N and P genes) or elsewhere. Non-translated gene sequences can be removed to increase the ability to insert foreign sequences.

[0139] The introduction of the aforementioned modifications into rB / HPIV3-hMPV F can be achieved by various well-known methods. Examples of such techniques can be found, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, NY, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Thus, defined mutations can be introduced into a cDNA copy of the genome or antigenome by conventional techniques (e.g., site-directed mutagenesis). The use of antigenome or genome cDNA subfragments to assemble a complete antigenome or genome cDNA has the advantage that each region can be manipulated separately (small cDNAs are easier to manipulate than large cDNAs) and then easily assembled into a complete cDNA. Thus, the complete antigenome or genome cDNA, or any subfragment thereof, can be used as a template for oligonucleotide-directed mutagenesis. The mutated subfragments can then be assembled into a complete antigenome or genome cDNA. Mutations can range from single nucleotide changes to replacements of large pieces of cDNA encompassing one or more genes or genomic regions.

[0140] Disclosed embodiments of rB / HPIV3-hMPV F are self-replicating, i.e., they are capable of replicating after infection of a suitable host cell and have an attenuated phenotype, for example, when administered to a human subject. In exemplary embodiments, rB / HPIV3-hMPV F is attenuated in the upper respiratory tract of a mammal by about 3-500 times or more and in the lower respiratory tract by about 100-5000 times or more, compared to the control HPIV3. In some embodiments, the level of in vitro viral replication is sufficient to provide for production of virus for use on a broad scale. In some embodiments, the level of in vitro viral replication of the attenuated paramyxovirus is at least 10 per n. 6, at least 10 7 , or at least 10 8 It is.

[0141] In some embodiments, rB / HPIV3-hMPV F vectors can be produced using reverse genetics recombinant DNA-based technology (Durbin PA et al., Virology 1997; PMID:9281512; Collins, et al. 1995. Proc Natl Acad. Sci USA 92:11563-11567). This system allows for the complete de novo recovery of infectious virus from cDNA in a competent cell substrate under defined conditions. Reverse genetics provides a means to introduce predetermined mutations into the rB / HPIV3-hMPV F genome via a cDNA intermediate. Specific attenuating mutations were characterized in preclinical trials and combined to achieve the desired level of attenuation. Derivation of vaccine virus from cDNA minimizes the risk of contamination with adventitious agents and helps to concisely and thoroughly record passage history. Once recovered, engineered virus strains grow in the same manner as biologically derived viruses. As a result of passaging and amplification, the virus does not contain recombinant DNA from the original recovery.

[0142] A number of cell lines that allow for virus growth can be used to grow rB / HPIV3-hMPV F vectors for immunization and other purposes. Parainfluenza viruses grow in a variety of human and animal cells. Exemplary cell lines for growing attenuated rB / HPIV3-hMPV F virus for immunization include HEp-2 cells, FRhL-DBS2 cells, LLC-MK2 cells, MRC-5 cells, and Vero cells. The highest virus yields are usually achieved in epithelial cell lines such as Vero cells. Cells are typically inoculated with virus at a multiplicity of infection ranging from about 0.001 to 1.0 or greater and cultured under conditions that permit virus replication, e.g., at about 30 to 37°C for about 3 to 10 days, or as long as necessary for the virus to reach a suitable titer. Temperature-sensitive viruses are often grown using 32°C as the "permissive temperature." The virus is typically removed from the cell culture by standard clarification procedures, such as centrifugation, and separated from cellular components, and can be further purified as necessary using known procedures.

[0143] The rB / HPIV3-hMPV F vector can be tested in a variety of well-known and commonly accepted in vitro and in vivo models to confirm sufficient attenuation, resistance to phenotypic reversion, and immunogenicity. In in vitro assays, the modified viruses are tested for temperature sensitivity of viral replication or "ts phenotype" and small plaque phenotype. The modified viruses can also be evaluated in an in vitro human airway epithelium (HAE) model, which appears to provide a means to rank viruses in order of relative attenuation in non-human primates and humans (Zhang et al., 2002 J Virol 76:5654-5666; Schaap-Nutt et al., 2010 Vaccine 28:2788-2798; Ilyushina et al., 2012 J Virol 86:11725-11734). The modified viruses are further tested in animal models of HPIV3 or hMPV infection. A variety of animal models are available (eg, mice, cotton rats, and primates).

[0144] The immunogenicity of the rB / HPIV3-hMPV F vector can be assessed in an animal model (e.g., a non-human primate, e.g., an African green monkey) by, for example, determining the number of animals that form antibodies to hMPV and HPIV3 after one immunization and after a second immunization, and measuring the magnitude of the response. In some embodiments, the rB / HPIV3-hMPV F has sufficient immunogenicity if about 60-80% of the animals develop antibodies after the first immunization and about 80-100% of the animals develop antibodies after the second immunization. In an exemplary embodiment, the immune response protects against infection by both hMPV and HPIV3.

[0145] Also provided are isolated polynucleotides comprising or consisting of the genome or antigenome of the disclosed rB / HPIV3-hMPV F vector, vectors comprising the polynucleotides, and host cells comprising the polynucleotides or vectors.

[0146] As used herein, a "subject" refers to any member of the animal kingdom. In some embodiments, a "subject" refers to a human. In some embodiments, a "subject" refers to a non-human animal. In some embodiments, a subject includes, but is not limited to, a mammal, a bird, a reptile, an amphibian, a fish, an insect, and / or a worm. In certain embodiments, a non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, a subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject."

[0147] In some embodiments, the "subject" comprises subjects aged 65 years or older, subjects aged 18-64 years (18-65 years), subjects aged 12 years or older, subjects aged 12-17 years (12-18 years), subjects aged 6-11 years (6-12 years), subjects aged 2-5 years (2-6 years), subjects aged 1-4 years (1-5 years), subjects aged 2 months to 1 year (2 months to 2 years), and subjects aged 0 months to 2 months (0 months to 3 months).

[0148] In some embodiments, the "subject" is selected from the group consisting of elderly (e.g., elderly or older adults), adults, adolescents, children, infants, and babies. In some embodiments, the "subject" is selected from the group consisting of elderly (e.g., 60 years or older), elderly (e.g., 65 years or older), adults (e.g., 18-50 years or 18-64 years), adolescents (e.g., 12-17 years) (e.g., 12-18 years), children (e.g., 6-11 years) (e.g., 6-12 years), children (e.g., 2-5 years) (e.g., 2-6 years), infants (e.g., 1-4 years) (e.g., 1-5 years), infants (e.g., 2 months to 2 years), newborns (e.g., 0-27 days), premature infants (e.g., gestational age less than 37 weeks). In some embodiments, the subject is in the pediatric age group defined by the US FDA. Neonates (e.g., from birth to less than 1 month ("NEO")); infants (e.g., 1 month to less than 2 years ("INF")); children (e.g., 2 years to less than 12 years ("CHI")); and adolescents (e.g., 12 years to less than 17 years ("ADO")). In some embodiments, the subject is an elderly person in the age groups defined by the US FDA as 65 years or older or 75 years or older. In particularly exemplary embodiments, the subject is an infant (e.g., 1 month to less than 2 years), a toddler (e.g., 1 year to less than 5 years), or an elderly person (e.g., 60 years or older, 65 years or older, or 75 years or older).

[0149] As used herein, the term "vaccination" or "vaccinate" refers to administration of a composition intended to generate an immune response, for example, against a disease-causing agent. Vaccination can be administered before, during and / or after exposure to the disease-causing agent, and / or before, during and / or immediately after the onset of one or more symptoms, in some embodiments before, during and / or immediately after exposure to the disease-causing agent. In some embodiments, vaccination includes multiple, appropriately or appropriately spaced administrations of the vaccination composition.

[0150] This disclosure describes nucleic acid sequences (eg, DNA and RNA sequences) and amino acid sequences that have a degree of identity to a given nucleic acid sequence or amino acid sequence, respectively (eg, a reference sequence).

[0151] "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0152] The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between the two sequences may, but do not necessarily, be randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually performed by comparing the sequences over a segment or "window of comparison" after optimal alignment in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs which employ such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0153] The percentage identity is obtained by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (eg, the number of positions in the reference sequence) and multiplying this result by 100.

[0154] In some embodiments, the degree of identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, in consecutive nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0155] A nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, can have at least one functional characteristic of the given sequence, e.g., in some instances, is functionally equivalent to the given sequence. In some embodiments, a nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0156] As used herein, the term "kit" refers to a packaged set of one or more compounds or compositions and one or more associated materials, such as solvents, solutions, buffers, instructions, or desiccants, or other associated components.

[0157] II. hMPV F Polypeptide Antigen Human metapneumovirus (hMPV) is a negative-sense single-stranded RNA virus belonging to the genus Metapneumovirus in the family Pneumoviridae. hMPV infects airway epithelial cells in the nose and lungs and is the second most common cause of lower respiratory tract infections in children after respiratory syncytial virus (RSV). hMPV is an enveloped virus with a glycoprotein (G protein), a small hydrophobic protein (SH protein) and a fusion protein (F protein) on the virion surface.

[0158] Because hMPV is an enveloped virus, entry of hMPV into host cells requires fusion of the viral and cellular membranes. Pneumovirus attachment and entry typically require two viral glycoproteins, the fusion (F) and attachment (G) proteins, and membrane fusion, promoted by all pneumovirus fusion glycoproteins tested, occurs at neutral pH. In addition to virus-cell membrane fusion, pneumovirus glycoproteins also promote cell-cell fusion. Multinucleated giant cells, called syncytia, can be found in cell cultures following infection with various pneumoviruses. Cultured cells infected with hMPV form syncytia, but examination of primary human airway epithelial cells infected with hMPV suggests that syncytium formation by this virus may not be a common in vivo occurrence.

[0159] hMPV F is a class I fusion glycoprotein that is synthesized as an inactive precursor (F0) that must be cleaved to become fusion-competent. Proteolytic cleavage generates two disulfide-linked subunits (N-terminus of F2 to F1) that assemble into a homotrimer. Cleavage occurs at a monobasic cleavage site immediately upstream of the hydrophobic fusion peptide. Cleavage can be achieved in tissue culture by adding exogenous trypsin to the medium or by adding a furin-expressing plasmid. However, in vivo, other serine proteases such as TMPRSS2 are more likely to be involved in the cleavage. The F trimer is incorporated into the virus particle in a metastable "pre-fusion" or "pre-F" conformation. To initiate membrane fusion, hMPV F is activated and undergoes a series of stepwise conformational changes in the F protein that drive membrane fusion and result in hMPV F adopting a highly stable "post-fusion" or "post-F" conformation.

[0160] In certain exemplary embodiments, proteolytic cleavage of F0 is achieved by co-transfecting a plasmid encoding an hMPV F polypeptide with a plasmid encoding furin in a 4:1 ratio of hMPV plasmid:furin plasmid.

[0161] Provided herein are antigenic hMPV polypeptides, including hMPV F polypeptides. The hMPV F polypeptides can include the entire sequence of hMPV F or a portion of hMPV F. In certain embodiments, the portion is the ectodomain.

[0162] In some embodiments, the hMPV F polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to any one of SEQ ID NOs:1, 3, 5 and 7.

[0163] In some embodiments, the hMPV F polypeptide comprises a modified hMPV F polypeptide having at least 80% identity to the polypeptide of any one of SEQ ID NOs: 1, 3, 5, and 7, wherein the hMPV F polypeptide is antigenic.

[0164] In some embodiments, the hMPV F polypeptide comprises only a portion of the ectodomain of the F protein.

[0165] The amino acid sequence of F0 of A2-CAN97-83 is as follows: [ka] (Accession no. AAN52910; version AAN52910.1; DB source accession no. AY145296.1) The transmembrane is bold and underlined, the cytoplasmic tail is in bold.

[0166] The nucleotide sequence of F0 of A2-CAN97-83 is as follows: [ka]

[0167] In some embodiments, an epitope of the hMPV F protein shared between pre-F and post-F is blocked. Blocking the epitope reduces or eliminates the production of antibodies against the epitope when an RNA (e.g., mRNA) encoding an antigenic hMPV F polypeptide is administered to a subject, or when an antigenic hMPV F polypeptide is administered to a subject. This can increase the proportion of antibodies targeting epitopes specific to a particular conformation of F, such as the pre-fusion conformation (e.g., antibodies targeting site Φ and / or site V). Since F has a pre-fusion conformation in viruses that have not yet entered a cell, increasing the proportion of antibodies targeting pre-F can provide a greater degree of neutralization (e.g., expressed as a neutralization-to-binding ratio as described herein).

[0168] The hMPV F polypeptides described herein can have deletions or substitutions compared to the wild-type hMPV F protein (eg, SEQ ID NO:1).

[0169] For example, in certain embodiments, the hMPV polypeptide (a) lacks a transmembrane domain, lacks a cytoplasmic tail, and comprises a human rhinovirus 3C (HRV-3C) protease cleavage site; (b) comprises an F0 cleavage site mutation comprising amino acid substitutions Q100R and S101R relative to SEQ ID NO:1, which substitute arginine for glutamine at amino acid position 100 and arginine for serine at amino acid position 101; (c) comprises a heterologous signal peptide; (d) optionally comprises at least one tag sequence, which is a polyhistidine tag (e.g., a 6xHis tag, an 8xHis tag, etc.) and / or a Strep II tag; and / or (e) comprises a foldon domain.

[0170] In certain embodiments, the hMPV polypeptide lacks a transmembrane domain, lacks a cytoplasmic tail, and comprises an F0 cleavage site mutation that includes amino acid substitutions Q100R and S101R relative to SEQ ID NO:1, which replace glutamine at amino acid position 100 with arginine and replace serine at amino acid position 101 with arginine; a human rhinovirus 3C (HRV-3C) protease cleavage site; a heterologous signal peptide; a polyhistidine tag (e.g., 6xHis tag, 8xHis tag, etc.) and / or a Strep II tag; and a foldon domain.

[0171] In certain embodiments, the hMPV polypeptide includes a valine, alanine, glycine, isoleucine, leucine, or proline substitution at position 185 of SEQ ID NO:1.

[0172] In certain embodiments, the pre-fusion F2 polypeptide comprises a phenylalanine, tryptophan, tyrosine, valine, alanine, isoleucine, or leucine substitution at position 160 of SEQ ID NO:1, and / or a valine, alanine, isoleucine, leucine, phenylalanine, tyrosine, or proline substitution at position 46 of SEQ ID NO:1.

[0173] In certain embodiments, the hMPV polypeptide is derived from an A strain hMPV (eg, an A1 subtype or an A2 subtype).

[0174] In certain embodiments, an amino acid sequence comprising a "backbone" F0 polypeptide sequence is provided and is described as follows: [ka]

[0175] In certain embodiments, a nucleotide sequence encoding a "backbone" F0 polypeptide sequence is provided and is described as follows: [ka]

[0176] In certain embodiments, an hMPV polypeptide comprises the "backbone" hMPV sequence set forth as SEQ ID NO:3, and may include one or more amino acid substitutions. For example, in certain embodiments, an hMPV polypeptide comprises a valine, alanine, glycine, isoleucine, leucine, or proline substitution at position 185 of SEQ ID NO:3.

[0177] In certain embodiments, the hMPV polypeptide includes a phenylalanine, tryptophan, tyrosine, valine, alanine, isoleucine, or leucine substitution at position 160 of SEQ ID NO:3, and / or a valine, alanine, isoleucine, leucine, phenylalanine, tyrosine, or proline substitution at position 46 of SEQ ID NO:3.

[0178] In certain embodiments, the hMPV polypeptide includes an arginine substitution at one or both of positions 100 and 101 of SEQ ID NO:3.

[0179] In certain embodiments, the hMPV polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3.

[0180] In certain embodiments, an amino acid sequence is provided that includes an hMPV polypeptide sequence, described as follows: [ka] (D185P). (The amino acids in lower case indicate the linker, foldon motif, linker, HRV-3C cleavage site, linker, 8X-His-tag and strep-tag II regions.)

[0181] In certain embodiments, a nucleotide sequence encoding an hMPV polypeptide sequence is provided and is described as follows: [ka] (D185P).

[0182] In certain embodiments, the hMPV polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5. In certain embodiments, the hMPV polypeptide comprises SEQ ID NO:5.

[0183] In certain embodiments, an amino acid sequence is provided that includes an hMPV polypeptide sequence, described as follows: [ka] (T160F_N46V). (The amino acids in lower case indicate the linker, foldon motif, linker, HRV-3C cleavage site, linker, 8X-His-tag and strep-tag II regions.)

[0184] In certain embodiments, a nucleotide sequence encoding an hMPV polypeptide sequence is provided and is described as follows: [ka] (T160F_N46V).

[0185] In certain embodiments, the hMPV polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7. In certain embodiments, the hMPV polypeptide comprises SEQ ID NO:7.

[0186] In general, positions in the constructs described herein can be mapped to a reference sequence, e.g., the wild-type sequence of SEQ ID NO:1 or the backbone sequence of SEQ ID NO:3, by pairwise alignment, e.g., using the Needleman-Wunsch algorithm with standard parameters (EBLOSUM62 matrix, gap penalty of 10, gap extension penalty of 0.5).

[0187] III. Immunogenic pharmaceutical compositions Immunogenic compositions are also provided that include the disclosed rB / HPIV3-hMPV F vector and a pharma- ceutically acceptable carrier. Such compositions can be administered to a subject in a variety of ways, for example, by the intranasal route. Standard methods for preparing administrable immunogenic compositions are described in publications such as, for example, Remington's Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995.

[0188] Carriers include, but are not limited to, physiologically balanced media, phosphate buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryoprotectant additives or stabilizers, such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., monosodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for immediate use or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple doses.

[0189] The immunogenic compositions may contain bacteriostatic agents to prevent or minimize degradation during storage, including, but not limited to, benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben at effective concentrations (typically 1% w / v). For some patients, bacteriostasis may be contraindicated, and therefore the lyophilized formulations may be reconstituted in solutions with or without such ingredients.

[0190] The immunogenic composition may contain as a pharma- ceutically acceptable vehicle such substances as are required to approximate physiological conditions, e.g., pH adjusting agents and buffers, isotonicity adjusting agents, wetting agents, etc., e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0191] The immunogenic composition may optionally include an adjuvant to enhance the immune response of the host. Suitable adjuvants are, for example, Toll-like receptor agonists, alum, AlPO4, Alhydrogel, Lipid-A and its derivatives or variants, oil emulsions, saponins, neutral liposomes, liposomes containing recombinant viruses, and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.) and IL-12, among many other suitable adjuvants known in the art, may be used as adjuvants (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage that they stimulate the immune system in a non-specific manner and thus help to enhance the immune response to pharmaceutical agents.

[0192] In some instances, it may be desirable to combine immunogenic compositions comprising rB / HPIV3-hMPV F with other pharmaceutical agents (e.g., vaccines) that induce protective responses against other viral agents, particularly those that cause other pediatric diseases. For example, compositions comprising rB / HPIV3-hMPV F described herein may also include other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov / vaccines / acip / index.html) for the target age group (e.g., infants about 1-6 months of age). These additional vaccines include, but are not limited to, IN-administered vaccines. Thus, rB / HPIV3-hMPV F described herein may be administered simultaneously with vaccines against, for example, Hepatitis B (HepB), Diphtheria, Tetanus and Pertussis (DTaP), Pneumococcal Vaccine (PCV), Haemophilus influenzae type b (Hib), Polio, Influenza, and Rotavirus.

[0193] In some embodiments, the immunogenic compositions can be provided in a unit dosage form for use in inducing an immune response in a subject, e.g., for preventing HPIV3 and / or hMPV infection in a subject. The unit dosage form includes a suitable single preselected dose for administration to a subject, or a suitable marked or measured multiple of two or more preselected unit doses, and / or a metering mechanism for administering the unit dose or multiples thereof.

[0194] IV. Packaging and Use of Immunogenic Pharmaceutical Compositions The hMPV vaccines described herein can be formulated or packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) or nasopharyngeal (e.g., intranasal) administration. In various embodiments, the hMPV vaccines can be formulated or packaged for pulmonary administration. In various embodiments, the hMPV vaccines can be formulated or packaged for intravenous administration. The vaccine compositions can be in the form of an extemporaneous preparation, where the compositions are lyophilized and reconstituted with a physiological buffer (e.g., PBS) immediately prior to use. The vaccine compositions can also be shipped and provided in the form of an aqueous or frozen aqueous solution, and can be administered directly to a subject without reconstitution (after thawing, if previously frozen).

[0195] Thus, the disclosure provides articles of manufacture such as kits that provide the hMPV vaccine in a single container, or that provide the hMPV vaccine in one container (e.g., a first container) and a physiological buffer for reconstitution in another container (e.g., a second container). The containers may contain single-use dosages or multi-use dosages. The containers may be pre-processed glass vials or ampoules. The articles of manufacture may also include instructions for use.

[0196] Methods of administration of the vaccine include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intratracheal, epidural and oral routes. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents.

[0197] The pharmaceutical compositions described herein can be delivered, for example, intramuscularly, subcutaneously, or intravenously, using a standard needle and syringe, which is optionally pre-filled. Additionally, pen delivery devices (e.g., injectors (e.g., single or multi-chamber) or autoinjector pens) have utility for delivering the pharmaceutical compositions described herein. Such pen delivery devices can be reusable or disposable. In some embodiments, the vaccine is provided for use in inhalation and is provided in a pre-filled pump, aerosolization device, or inhaler. In certain embodiments, a pre-filled syringe can be utilized for dropwise administration for intranasal delivery.

[0198] The hMPV vaccines disclosed herein can be administered to a subject to induce an immune response against the hMPV F protein, where anti-antigen antibody titers in the subject are increased following vaccination compared to anti-antigen antibody titers in a subject not vaccinated with the hMPV vaccine disclosed herein, or compared to an alternative vaccine against hMPV. An "anti-antigen antibody" is a serum antibody that specifically binds to an antigen.

[0199] Provided herein are methods of eliciting an immune response in a subject by administering to the subject an immunogenic composition containing the disclosed rB / HPIV3-hMPV F. Upon immunization, the subject responds by producing antibodies specific for the hMPV F protein and one or more of the HPIV3 HN and F proteins. Additionally, innate and cell-mediated immune responses are induced, which can provide antiviral effectors as well as modulate the immune response. As a result of immunization, the host becomes at least partially or completely immune to HPIV3 and / or hMPV infection, or resistant to development of moderate or severe HPIV3 and / or hMPV disease, particularly of the lower respiratory tract.

[0200] An immunogenic composition containing rB / HPIV3-hMPV F is administered to a subject susceptible to or otherwise at risk of hMPV and / or HPIV3 infection in an "effective amount" sufficient to induce or enhance the individual's immune response capability to hMPV and / or HPIV3. The immunogenic composition may be administered by any suitable method, including but not limited to injection, aerosol delivery, nasal spray, nasal drops, oral inoculation, or topical application. In an exemplary embodiment, the attenuated virus is administered according to established human intranasal administration protocols (e.g., discussed in Karron et al. JID191:1093-104, 2005). Briefly, an adult or child is inoculated intranasally via droplets with an effective amount of rB / HPIV3-hMPV F in a physiologically acceptable diluent or carrier, typically in a volume of 0.5 ml. This has the advantages of simplicity and safety compared to parenteral immunization with non-replicating viruses. It also provides a direct stimulation of local airway immunity, which plays a major role in resistance to hMPV and HPIV 3. Moreover, this mode of vaccination effectively circumvents the immunosuppressive effects of HPIV 3- and hMPV-specific maternal serum antibodies typically found in the very young.

[0201] The embodiments of rB / HPIV3-hMPV F and immunogenic compositions thereof described herein are administered to a subject in an amount effective to induce or enhance the subject's immune response to the HPIV3 and hMPV antigens contained in rB / HPIV3-hMPV F. An effective amount allows some growth and proliferation of the virus to produce the desired immune response, but does not cause virus-associated symptoms or disease.

[0202] The desired immune response is to inhibit subsequent infection by hMPV and / or HPIV3. hMPV and / or HPIV3 infection need not be completely inhibited for the method to be effective. For example, administration of an effective amount of the disclosed rB / HPIV3-hMPV F can reduce subsequent hMPV and / or HPIV3 infection (e.g., as measured by infection of cells or by the number or percentage of subjects infected with hMPV and / or HPIV3) by a desired amount, e.g., at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to a suitable control (prevention of detectable hMPV and / or HPIV3 infection).

[0203] Administration of rB / HPIV3-hMPV F to a subject can induce the production of an immune response that is protective against severe lower respiratory tract disease, such as pneumonia and bronchiolitis, or croup, if the subject is subsequently infected or reinfected with wild-type hMPV or HPIV3. Naturally circulating viruses can cause infections, particularly in the upper respiratory tract, but immunization may result in a decreased likelihood of rhinitis and subsequent infection with wild-type virus may increase resistance. After immunization, there are detectable levels of host-infected serum and secretory antibodies that can neutralize the homologous (same subgroup) wild-type virus in vitro and in vivo. In many cases, host antibodies also neutralize wild-type viruses of different non-vaccine subgroups. For example, to achieve a higher level of cross-protection against heterologous strains of another subgroup, a subject can be immunized with multiple immunogenic compositions that include rB / HPIV3-hMPV F with genomes encoding hMPV F proteins from at least one predominant strain of both hMPV subgroups A and B.

[0204] Immunogenic compositions comprising one or more of the disclosed rB / HPIV3-hMPV F viruses can be used in concerted (or prime-boost) immunization protocols or combination formulations. It is contemplated that there can be several boosts and that each boost can be a different disclosed immunogen. In some instances, it is also contemplated that a boost can be the same immunogen or prime as another boost. In certain embodiments, the novel combinatorial immunogenic compositions and concerted immunization protocols use separate immunogens or formulations aimed at eliciting anti-viral immune responses, such as immune responses against hMPV and HPIV3 proteins, respectively. The separate immunogenic compositions that elicit anti-viral immune responses can be combined in a multivalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in a monovalent immunogenic composition) in a concerted (or prime-boost) immunization protocol.

[0205] The resulting immune response can be characterized by a variety of methods. These include taking samples of nasal washes or serum for analysis of hMPV-specific antibodies, which can be detected by tests including, but not limited to, complement fixation, plaque neutralization, enzyme-linked immunosorbent assay, luciferase immunoprecipitation assay, and flow cytometry. Additionally, immune responses can be detected by assays of cytokines in nasal washes or serum, ELISPOT of immune cells from either source, quantitative RT-PCR or microarray analysis of nasal wash or serum samples, and restimulation of immune cells from nasal washes or serum by re-exposure to viral antigens in vitro, as well as analysis of production or display of cytokines, surface markers, or other immune correlates by flow cytometry, or cytotoxic activity against indicator target cells displaying hMPV antigens. In this regard, individuals are also monitored for signs and symptoms of upper respiratory tract disease.

[0206] In order that the present invention may be better understood, the following examples are set forth below, which are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. EXAMPLES

[0207] The foregoing description of specific embodiments sufficiently reveals the general nature of the present disclosure so that others may easily modify and / or adapt such specific embodiments to various applications by applying knowledge within the skill of those of ordinary skill in the art without departing from the general concept of the present disclosure and without undue experimentation. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It should be understood that the expressions or terms used herein are intended to be descriptive rather than limiting, as the terms or terms used herein would be interpreted by those of ordinary skill in the art in light of the teaching and guidance.

[0208] Example 1: Design of a stable B / HPIV3-hMPV F construct Recombinant bovine human parainfluenza virus type 3 (B / HPIV3) was used to express the human metapneumovirus (HMPV) fusion (F) protein. B / HPIV3 is a chimeric virus consisting of bovine PIV3 (Kansas strain; GenBank accession number: AF178654) in which the fusion (F) gene and the hemagglutinin neuraminidase (HN) gene have been replaced by those from human PIV3 (JS strain; GenBank accession number: Z11575). Schmidt et al. (2001), J Virol 75:4594-603.

[0209] In this study, B / HPIV3 was used to express the HMPV F protein (strain CAN97-83, subgroup A, GenBank accession number: AY297749) from a gene added at the second gene location between the N and P genes of BPIV3. This resulted in a live attenuated intranasal (IN) bivalent vaccine against HPIV3 and HMPV. The high degree of conservation of the F gene nucleotide sequence and F protein amino acid sequence (95% amino acid identity) among all HMPV subgroups and strains indicates that the results with this strain are generally applicable to HMPV strains. It is also generally true that if a foreign protein can be successfully expressed from a paramyxovirus (e.g., PIV3), it can be expressed from various gene junctions, as previously shown with this same B / HPIV3 vector. Liang et al. (2014), J Virol 88: 4237-50.

[0210] As shown in Figure 1, eight different versions of the HMPV F protein were constructed to identify forms that were stable and well accommodated by the vector. Construct 1 expressed the unmodified wild type of HMPV F protein of the CAN97-83 strain. Constructs 2 and 3 expressed forms of this protein that were codon-optimized (without changes in the amino acid code) by Biobasic, Markham, ON (BBopt; construct 2) or Genscript, Piscataway, NJ (GSopt; construct 3) to increase F expression. Construct 4 (GSopt-TMCT) was a variant of GSopt (construct 3) that replaced the transmembrane and cytoplasmic tail (TMCT) domain (nt 1474-1620) of the HMPV F ORF with that of BPIV3F, potentially enhancing packaging into vector particles. This was based on previous findings that TMCT modifications in the human respiratory syncytial virus (RSV) F protein significantly enhanced its packaging into B / HPIV3 vector virions and increased its immunogenicity (Liang et al. (2016), J Virol 90:10022-10038). Constructs 5-8 contained amino acid substitutions (either D185P / Q100R / S101R or N46V / T160F) introduced into HMPV F to stabilize it in the pre-fusion (pre-F) form. Construct 5 had D185P / Q100R / S101R added to BBopt, construct 6 had D185P / Q100R / S101R added to GSopt, construct 7 had N46V / T160F added to BBopt, and construct 8 had N46V / T160F added to GSopt.

[0211] Each version of HMPV F was inserted into the B / HPIV3 antigenome at an AscI site located in the untranslated region downstream of the N gene and was designed to allow transcription as a separate mRNA by initiating and terminating at the provided P gene start and N gene end transcription signals, respectively. The B / HPIV3 vectors were used to express eight versions of the F protein of HMPV strain CAN97-83 (subgroup A) from a second gene location between the BPIV3 N and P genes.

[0212] Materials and Methods: Cells and viruses. Vero (African green monkey epithelial) and LLC-MK2 (rhesus monkey kidney epithelial) cell lines were cultured in Opti-MEMI medium (Life Technologies, Gaithersburg, MD) containing 5% fetal bovine serum (FBS; Hyclone, Marlborough, MA). The BHK (baby hamster kidney) BSR-T7 / 5 cell line, stably expressing T7 RNA polymerase, was maintained as previously described (Buchholz et al. (1999), J Virol 73:251-259). Culture medium was supplemented with 2% geneticin every other passage to maintain T7 transgene expression. The human lung epithelial A549 cell line (CCL-185; ATCC, Manassas, VA) was grown in F12 medium (ATCC) containing 5% FBS. B / HPIV3 P1 and P2 stocks were propagated in LLC-MK2 cells in Opti-MEMI medium containing 2.5% FBS and incubated at 32°C for 7 days. Wild-type HMPV (Canadian HMPV isolate CAN97-83; subgroup A; GenBank accession number: AY297749) was propagated in Vero cells by infection at an MOI of 0.01 PFU / cell using Opti-MEMI medium without FBS and containing 2% TrypLE Select (Life Technologies), followed by incubation at 32°C. Peret et al. (2002), J Infect Dis 185:1660-1663. An additional 2% TrypLE Select was added on day 4, and virus was harvested on day 7. Infected cell supernatants were clarified by centrifugation, flash frozen on dry ice, and stored at -80°C.

[0213] Virus rescue. Virus was recovered by co-transfecting BHK BSR-T7 / 5 cells with the full-length viral antigenome plasmid together with a support plasmid expressing the N, P and L proteins of BPIV3. 48 hours after transfection, the transfected cells were co-cultured with 50% confluent LLC-MK2 cell monolayers and incubated at 32°C for 7 days, after which the virus-containing culture supernatant was clarified and stored at -80°C. This P1 strain was passaged once more in LLC-MK2 cells by infection at an MOI of 0.01 PFU / cell to obtain the P2 strain. The viral genome sequence was determined by sequencing uncloned overlapping RT-PCR products derived from viral RNA extracted from the P2 virus stock.

[0214] Stability of HMPV F expression by B / HPIV3 in vitro. Viral stocks were assessed for stability of HMPV F expression by a double antigen immunostaining plaque assay detecting HMPV F and B / HPIV3 antigens. Vero and A549 cells were infected with serially diluted viruses, overlaid with medium containing 0.9% methylcellulose, and incubated at 32°C for 6 days. For staining, cells were fixed with cold 80% methanol, blocked by incubation with Odyssey blocking buffer (Licor Biosciences, Lincoln NE) for 1 h at room temperature, and then incubated with a mixture of three HMPV F-specific human monoclonal antibodies (Adi15614, MPE33, and MPF5h) at 1:2500 dilution each in Odyssey blocking buffer (Licor Biosciences) and rabbit hyperimmune serum prepared against sucrose gradient-purified HPIV3 (MS456) at 1:5000 dilution for 1 h at room temperature. Human- and rabbit-specific infrared dye-conjugated secondary antibodies (Licor Biosciences) were then used for detection. Plaques were visualized using an Odyssey infrared scanner (Licor Biosciences) and pseudocolored to appear red and green for HMPV F antigen and HPIV3 antigen, respectively. PFUs that appeared yellow when the red and green images were overlaid indicated expression of the HMPV F protein by the B / HPIV3 vector.

[0215] result: All vectors were readily recovered in transfected BHK BSR-T7 / 5 cells and then passaged in LLC-MK2 cells. To visualize the plaque phenotype and evaluate the stability of HMPV F expression, P1 and P2 virus stocks were subjected to plaque assays in Vero and A549 cells with dual staining for HPIV3 protein and HMPV F protein. Results for the P2 stock are shown in FIG. 12.

[0216] Plaques were generally uniform in size for each virus, but appeared slightly larger in Vero (Figure 12, panel A) compared to A549 (Figure 12, panel B) cells. Without intending to be bound by scientific theory, this may be due to the lack of interferon beta induction in Vero cells, in contrast to A549, which are interferon competent. Interestingly, in both A549 and Vero cells, plaques of all B / HPIV3 vectors expressing HMPV F were much smaller than the empty vector, suggesting a growth restriction contributed by the HMPV F insert. Furthermore, viruses expressing GSopt-TMCT developed substantially smaller plaques compared to other B / HPIV3 / HMPV vectors in both Vero and A549 cells (Figure 12), indicating a further restrictive effect of the TMCT mutation on viral growth.

[0217] In double antigen staining plaque assays (Figure 12), immunostaining for HMPV F protein was the first color, immunostaining with HPIV3-specific antiserum was the second color, and plaques expressing both HMPV F protein and PIV3 protein were the third color in both Vero and A549 cells (Figure 12, panels A and B). For most BHPIV3 / HMPV vectors, 95-99% of plaques from P2 virus stocks (grown in LLC-MK2 cells) were the third color, indicating expression of HMPV F against a background of PIV3 antigens (Figure 13; derived from data in panel A of Figure 12). Thus, expression of the insert was generally very stable. This was despite the apparent growth restriction conferred by the added HMPV genes, allowing selective pressure to be exerted to delete or mutate the added HMPV genes. The instability of expression of the foreign RSV F protein gene by B / HPIV3 vectors contributed to poor immunogenicity in previous studies. Bernstein et al. (2012), Pediatric Infect Dis J 31:109-114. One exception was the construct expressing GSopt-TMCT, where stocks from the first recovery had a significant proportion of green plaques, indicating loss of expression of the HMPV F protein. However, upon further recovery, a P2 stock was identified in which 96% of the plaques were positive for the HMPV F protein (Figure 13). The smaller plaque size and instability of expression of the GSopt-TMCT insert strongly indicated that it was not well tolerated by the B / HPIV3 vector, resulting in growth inhibition and negative selection pressure that favored silencing of expression of the insert.

[0218] Viral RNA was extracted from P2 stocks for all vectors and subjected to complete genome sequencing, which was performed by automated sequencing of uncloned overlapping RT-PCR products covering the entire genome except for 28 and 35 nucleotides at the primed 3' and 5' genomic ends, respectively. No adventitious mutations were identified in any of the viruses, confirming the accuracy of the dual-stain plaque assay to detect instability.

[0219] Example 2: Prefusion stabilized hMPV F glycoprotein antigen constructs To improve the stability of the prefusion conformation, enhance purification, and induce higher neutralizing antibody titers, a panel of candidate hMPV prefusion F antigen constructs was designed with mutations in the wild-type hMPV-F antigen based on the A2 subtype from Canada, designated A2-CAN97-83 (SEQ ID NO: 1).

[0220] A graphical representation of the design considerations for a panel of candidate hMPV pre-fusion F antigen constructs is shown for two exemplary constructs, D185P (SEQ ID NO:5) and T160F / N46V (SEQ ID NO:7), in Figure 2. Each construct contained the following features: (1) a signal peptide; (2) a pre-F cleavage site mutation (QS to RR) at amino acids 100-101; (3) removal of the transmembrane domain and cytoplasmic tail; (4) addition of a fibritin motif (i.e., a foldon domain); (5) an HRV-3C cleavage site; (6) an 8xHis tag and a Strep II tag; and (7) an appropriate linker for items (4)-(6) (SEQ ID NO:3).

[0221] From this scaffold, in silico analysis was performed to determine single or double point mutations that would increase pre-F conformational stability by adding either packed cavity mutations or interface stabilizing mutations. In total, the panel of candidate hMPV pre-fusion F antigens consisted of 21 different constructs, as shown in column 1 of Table 1.

[0222] Example 3: Evaluation of protein expression of prefusion stabilized hMPV F antigen constructs Nucleic acid molecules for each of the candidate hMPV pre-fusion F antigen constructs were isolated and cloned into an expression vector. Production of protein expression of each construct was assessed upon mammalian transient transfection using Expi293F human cells. 24 hours after transfection of the constructs, cell lysates or supernatants were harvested for analysis by Western blot.

[0223] Of the 21 candidate designs, nine protein antigens could be produced. However, only four protein antigens had a purity of 90% or higher as determined by SDS-PAGE from 1 L cultures. Protein expression characteristics of all 21 constructs are shown in Table 1. Constructs with high protein production and purity had the following mutations: D185P, T160F_N46V, K138F, and G366F_K362F.

[0224] [Table 1]

[0225] Example 4: Immunogenicity of prefusion stabilized hMPV F antigen protein constructs in mice Four candidate hMPV F antigen constructs with purities of 90% or higher, as described in Table 1, were then evaluated for immunogenicity in mice in comparison to the reference hMPV-F protein from the A1 strain.

[0226] Groups of eight BALB / c mice (N=8) shown in Table 2 were administered a 0.5 μg dose of protein antigen adjuvanted with aluminum hydroxide (Al(OH)3) by intramuscular (IM) injection on days 0 (D) and 21 (D). All mice were bled and serum was extracted before each vaccine administration and 2 weeks after the last vaccination (D35). Serum was then used to determine circulating anti-hMPV-F IgG titers as measured by enzyme-linked immunosorbent assay (ELISA) (Figure 3) and hMPV microneutralization assay (Figure 4) to determine the neutralizing activity of the antibody response. To ensure that all proteins in the post-F group were indeed in the post-F conformation, the proteins were heated to 70°C for 10 min prior to preparation for administration.

[0227] [Table 2]

[0228] The data show that the construct carrying the A2-K138F mutation induced the highest binding antibody titers by hMPV-F ELISA, followed by A2-T160F_N46V, A2-G366F_K362F, and finally A2-D185P (Figure 3). As assessed by microneutralization with hMPV A2-GFP virus, A2-T160F_N46V had the highest neutralization titers, followed by A2-K138F, A2-D185P, and A2-G366F_K362F (Figure 4).

[0229] Although A2-K138F had the highest binding and second highest neutralizing antibody titers, this construct was found to form aggregates in solution, indicating possible improper protein folding, and was therefore excluded from further evaluation. A2-G366F_K362F also had the second lowest binding and lowest neutralizing antibody titers, and was therefore excluded from further evaluation. Thus, A2-D185P and A2-T160F_N46V were found to induce the highest quality antibodies and were selected for advanced analytical analysis to assess purity, size, and thermal stability as described in Example 5.

[0230] Example 5: Physicochemical characterization of prefusion stabilized hMPV F antigen constructs To further characterize the purity, size and thermostability of proteins produced from the A2-D185P and A2-T160F_N46V constructs, HP-SEC, SEC-HPLC, SEC-MALS and nanoDSF analyses were performed.

[0231] Purity and Size The results of the HP-SEC, SEC-HPLC, and SEC-MALS analyses are summarized in Table 3 below.

[0232] [Table 3]

[0233] Molecular weights (MW) from MALS were determined for the trimer peak. SEC-HPLC conditions were as follows: TSK 3000SWxl SEC column, phosphate buffer (0.2 M NaH2PO4, 0.1 M arginine, 1% IPA, pH 6.5), flow rate 0.5 ml / min. SEC-MALS conditions were as follows: 1.7 mM, 200 Å BEH protein column, 50 mM Tris buffer pH 7.5, flow rate 0.3 ml / min.

[0234] Figure 5 shows the SEC-MALS results for the reference A1 proteins, A1-A185P and A1-post-F, and the following A2 protein antigen candidates, A2-T160F_N46V and A2-D185P. Data for all four proteins are also summarized in Table 3. Both A1 reference proteins show more than 98.8% trimer formation and MWs of 224 and 283 kDa for A1-A185P and A1-post-F, respectively. Proteins from the A2-T160F_N46V and A2-D185P constructs were composed of 97.4% and 97.1% trimers with MWs of 267 and 224 kDa, respectively.

[0235] thermal stability The onset temperature of protein denaturation (Tonset) and melting temperature (Tm) were determined for both large and small batch lots of A1-Pre-F and A1-Post-F proteins, as well as the A2 candidate protein antigens, A2-T160F_N46V and A2-D185P, using nano-differential scanning fluorimetry (nanoDSF). Samples were diluted in formulation buffer to a final concentration of 0.5 pg / ml and loaded in duplicate into nanoDSF capillaries. All measurements were performed using the nanoDSF instrument. The heating rate was 1.5 °C per minute from 20 °C to 95 °C. Data were recorded and analyzed using PR.Stability Analysis v1.01.

[0236] Figure 6 shows the melting curves of A1-preF(A185P) and A2-postF (n=3), resulting in Tm values ​​of 60.12°C and 86.7°C, respectively. This data indicates that nanoDSF can distinguish between pre- and post-fusion antigens of A1, with melting temperatures differing by approximately 27°C.

[0237] Interestingly, when comparing the thermostability profiles of the A2 hMPV-F candidate protein antigens, as seen in Figure 7, the protein derived from the A2-T160F_N46V construct was found to be more thermostable than the more minimally engineered protein produced from the A2-D185P construct, with an increased melting point of approximately 9°C (Tm's of 70.4°C and 79.3°C, respectively).

[0238] Example 6: Immunogenicity of hMPV F antigen protein constructs before and after stabilization in the MIMIC system Introduction The MIMIC© (Modular Immune In vitro Construct) system can stimulate in vitro innate and adaptive immune responses that occur in vivo at the vaccination / inflection site. Williams et al. (2015) Sanofi Pasteur poster, “In vitro differentiation of class-switched YF specific antibody secreting cells from naive B cells”. Using the MIMIC system, it is possible to recapitulate several aspects unique to human physiology, such as HLA haplotype, age, autoimmune profile, and sex, thereby complementing immunogenicity studies performed in animal models. Higbee et al. (2009) ATLA 37:19-27.

[0239] To this end, pre- and post-hMPV F antigen protein constructs were tested in the MIMIC system to assess the quality of the immunogenic response compared to controls, which included: untreated control (no antigen without human skeletal muscle cells (HSK)), reference antigen-RSV pre-F protein fused to ferritin nanoparticles (pre-F NP) and polio vaccine (IPOL).

[0240] Materials and Methods Briefly, PBMCs were collected from 22 different human blood donors via magnetic bead separation kit. Human dendritic cells (DCs) and selected B cells were added to human skeletal muscle cells (HSKMCs) and co-cultured and stimulated with either hMPV pre-F antigen protein (100ng / ml or 500ng / ml) or hMPV post-F antigen protein (100ng / ml). For B cell responses, supernatants were collected after 14 days of co-culture and analyzed for antibody specificity and function.

[0241] result: To confirm the activation of MIMIC co-cultures, previously analyzed polio vaccine (IPOL) and antigen (RSV pre-F-NP) were used as positive controls. As shown in FIG. 8, IPOL treatment at 1:50 dilution induced antibody responses against three polio strains (polio 1, 2, and 3) compared to untreated controls. Similarly, 50 ng / ml RSV Pre-F NP treatment of the co-cultures induced IgG-specific antibody responses against both RSV Pre-F (FIG. 9, panel A) and RSV Post-F (FIG. 9, panel B). Furthermore, these antibodies were also functional as measured by RSV neutralization assay (FIG. 9, panel C). Supernatants from co-cultures treated with the experimental groups, hMPV pre-F antigen protein (100 ng / ml or 500 ng / ml) or hMPV post F antigen protein (100 ng / ml), elicited robust IgG antibody responses to both hMPV pre-F antigen (Figure 10, panel A) and hMPV post F antigen (Figure 10, panel B) compared to the no antigen control. These antibodies were also functional as measured by hMPV neutralization assays (Figure 11). Antibodies from all three treatment groups bound to hMPV pre- and post-fusion F antigens and neutralized viral infectivity, supporting the idea that pre- and post-fusion hMPV share neutralizing epitopes.

[0242] Example 7: Evaluation of protein expression from an optimized panel of rB / HPIV3-hMPV F constructs Introduction Expression of vector proteins and HMPV F from B / HPIV3 vectors expressing eight different HMPV (CAN97-83) F versions designed in Example 1 was evaluated. Vero cells were infected with the eight B / HPIV3 / HMPV constructs, empty B / HPIV3 vector, wild-type HMPV CAN97-83, or mock infected at a multiplicity of infection (MOI) of 3 PFU per cell. Cells were incubated at 32° C. for 48 hours and cell lysates were prepared and analyzed by Western blotting.

[0243] Materials and Methods Cells and viruses. Cells were cultured and viruses were propagated as described in Example 1.

[0244] Analysis of viral protein expression by Western blotting. Expression of PIV3 vector proteins and HMPV F protein was assessed. Vero cells were infected with the indicated viruses at an MOI of 3 PFU / cell and incubated at 32 °C for 48 h, followed by cell lysis, SDS-PAGE (4–12% Bis-Tris gels under reducing and denaturing conditions), and Western blotting as previously described (Liu et al. (2020), PLoS One 15:e0228572). HMPV F protein was detected using a hamster monoclonal antibody (mAb1017). Corresponding species-specific infrared dye-conjugated secondary antibodies were used to visualize proteins with an infrared scanner as described above.

[0245] result: As shown in Figure 14, all eight HMPV F versions were efficiently expressed by the B / HPIV3 vector, and both the F0 precursor and F1 protein subunits were detected using a monoclonal antibody specific for HMPV F. No increase in expression of HMPV F was observed with BBopt or GSopt HMPV F compared to wild-type HMPV F. Specifically, expression of HMPV F with the GSopt construct was similar to that of wild-type HMPV F, while expression of HMPV F with the BBopt construct was somewhat reduced compared to wild-type and GSopt. Thus, contrary to expectations, codon optimization did not increase expression, and in fact, BBopt optimization unpredictably decreased expression. Other constructs based on GSopt (GSopt-D185P / Q100R / S101R, GSopt-N46V / T160F) also had F expression levels similar to those of wild-type HMPV, whereas other constructs based on BBopt (BBopt-D185P / Q100R / S101R, BBopt-N46V / T160F) had reduced F expression levels.

[0246] The amount of vector-expressed BPIV3 N and P proteins and HPIV3 F and HN proteins was reduced for all viruses expressing HMPV F compared to the empty B / HPIV3 control. The reduction was modest for the N protein but was greater for the P, F and HN proteins. This likely reflects the placement of the HMPV F gene in the second gene position immediately following the N gene. In this position, due to the 3'-5' gradient of viral transcription, the insert may have minimal effect on the expression of the upstream N gene but may cause a greater reduction in the expression of other viral genes located downstream. This may be the basis for the reduction in plaque size associated with the insertion of the HMPV F gene. The GSopt-TMCT construct had a greater reduction in overall expression of PIV3 proteins compared to the other constructs, which is consistent with and may be the basis for the greater reduction in plaque size compared to the other viruses. Despite this, the expression level of the HMPV F protein was comparable to the other GSopt versions.

[0247] Example 8: Immunogenicity of an optimized panel of rB / HPIV3-hMPV F constructs in hamsters Introduction The immunogenicity and efficacy of B / HPIV3 vectors expressing eight different HMPV (CAN97-83) F versions designed in Example 1 were tested.

[0248] The experimental design is shown in Figure 15. Briefly, groups of six hamsters were each immunized with a single intranasal dose of one of the eight virus B / HPIV3-hMPV F constructs, wild-type HMPV, or an empty vector control, or with two intramuscular doses, three weeks apart, of either two purified HMPV F subunit recombinant proteins (F-D185P / Q100R / S101R and F-N46V / T160F). Serum samples were collected from all animals four weeks after virus immunization or two weeks after the second intramuscular protein dose, and serum HMPV and HPIV3 neutralizing antibody titers were determined by 60% plaque reduction neutralization assays.

[0249] Materials and Methods The animal study protocol was approved by the NIH Animal Care and Use Committee. Groups of six 6-week-old golden Syrian hamsters were administered 10 mg of 10 ... 5 PFU dose of rB / HPIV3-HMPV F vector was inoculated intranasally. B / HPIV3 vector without HMPV F insert and wt HMPV (subgroup A, strain CAN97-83, GenBank accession number: AY297749) were included as controls. Two groups of six hamsters were immunized intramuscularly with 20 μg per dose of either of two purified HMPV F proteins, F-D185P / Q100R / S101R or F-N46V / T160F, mixed 1:1 (volume:volume) with alum-85 adjuvant as two doses administered 3 weeks apart. Serum samples were collected from immunized hamsters 4 weeks after virus immunization or 2 weeks after the second IM protein administration. Two days after serum collection, all hamsters were immunized with 5 × 10 6 of wt HMPV (CAN97-83) intramuscularly. 5 Three days after challenge, animals were euthanized, and nasal turbinates and lungs were harvested to quantify HMPV replication in those tissues. Tissue homogenates were prepared and titrated by HMPV plaque assay on Vero cells, and data were reported as PFU / g of each tissue.

[0250] HMPV- and HPIV3-specific 60% plaque reduction neutralization titers (PRNT60) of hamster sera were determined as previously described using wild-type HMPV strain CAN97-83 and HPIV3 expressing green fluorescent protein, respectively, and reported as Log2 PRNT60. Liu et al. (2020), PLoS One 15:e0228572; Skiadopoulos et al. (2004), J Virol 78:6927-37.; Bernstein et al. (2012), Infect Dis J 31:109-14.

[0251] result: As shown in Figure 16, all B / HPIV3 vector constructs expressing HMPV F induced serum HMPV neutralizing antibody titers comparable to those induced by wild-type HMPV A, with no statistically significant difference. Subunit protein vaccines also induced serum HMPV neutralizing antibodies, with F-N46V / T160F conferring significantly higher titers (p<0.01), whereas F-D185P / Q100R / S101R titers were similar to the wild-type HMPV A control.

[0252] Thirty-seven days after vaccination, all hamsters were vaccinated with 5 × 10 5 PFU of wild-type HMPV A (CAN97-83) was challenged intranasally. Three days after challenge, nasal turbinates and lungs were harvested and challenge HMPV load was quantified by HMPV plaque assay.

[0253] In the nasal turbinates, wild-type HMPV replicated to high titers in B / HPIV3 vector (empty)-immunized animals (mean: 10 6 PFU / g). (Figure 17, Panel A) On the other hand, all rB / HPIV3 vector constructs expressing different forms of HMPV F were highly protective with either no or significantly reduced HMPV replication. The wt HMPV A immunized control group was fully protected with no detectable infectious virus. B / HPIV3 vector constructs expressing the native forms of HMPV F protein, i.e., wild-type HMPVA, BBopt, and GSopt, were the most protective, with no detectable infectious HMPV in the nasal turbinates (p<0.0001). Similarly, GSopt-TMCT also provided complete protection with no detectable infectious virus in the nose (p<0.0001). Vectors expressing BBopt or GSopt versions carrying the pre-F stabilizing D185P / Q100R / S101R or N46V / T160F mutations also provided complete protection after 10 min. 1.8 ~10 2.1The HMPV F-expressing B / HPIV3 vector constructs demonstrated significantly reduced HMPV titers with averages ranging from PFU / g (P<0.0001 to p<0.001). Each of the B / HPIV3 vector constructs expressing HMPV F demonstrated that the majority of animals had no detectable virus, with only one or two animals showing very low HMPV replication.

[0254] In lung homogenates, as shown in FIG. 17B, the results were less clear in the lungs due to the overall low replication of HMPV challenge virus in all groups, including the HMPV naïve (B / HPIV3 vector) group. GSopt, GSopt-D185P / Q100R / S101R, and BBopt-N46V / T160F viruses did not confer significant protection in the lungs. The remaining five rB / HPIV3 vectors expressing HMPV F were significantly more effective in the lungs than the 10 1.8 ~10 1.9 Both purified protein vaccines F-D185P / Q100R / S101R and F-N46V / T160F also provided significant protection in the lungs, with mean HMPV challenge virus titers ranging from 10 to 10 PFU / g (p<0.01 to p<0.05). 1.94 (P<0.05) and 10 1.77 (p<0.01) PFU / g, indicating that the high serum HMPV neutralizing antibody titers induced by them were protective in the lung.

[0255] In summary, the hamster studies demonstrated that B / HPIV3 vectors expressing various forms of HMPV F were at least as immunogenic as infection with wild-type HMPV. Immunization with these B / HPIV3 vectors was as protective in the upper respiratory tract as wild-type HMPV. Furthermore, a single intranasal dose of any vector was more protective in the upper respiratory tract than two large adjuvanted intramuscular doses of the corresponding purified pre-F HMPV F protein (compare panels A and B in FIG. 17). The similarity or superiority of vector versus purified protein with respect to serum HMPV neutralization titers and protective efficacy was remarkable because (i) the protein vaccine was administered with an adjuvant and (ii) the protein vaccine was administered in two doses, whereas the vector was administered in a single dose, and the secondary immune response was typically much higher and more protective than the primary response. The superior protection in the upper respiratory tract by the vector, likely due to direct stimulation of respiratory tract immunity, indicates the importance of the intranasal route of administration. In the lower respiratory tract, the level of challenge HMPV replication was generally similar in all immunized animals. This assessment was confounded by the generally low replication of challenge HMPV in the lower respiratory tract. Nevertheless, all immunogens appeared to confer approximately equal levels of protection in the lower respiratory tract.

[0256] Thus, B / HPIV3 vectors, previously shown to be fully attenuated and safe in children, can express the HMPV F protein to provide an immunogenic, protective and stable vaccine. Given the previous demonstration of safety of B / HPIV3 and B / HPIV3 / RSV vectors in children, these HMPV-F expressing derivatives are suitable for direct evaluation in young children as intranasal vaccines against HPIV3 and HMPV that are superior to two intramuscular doses of purified protein.

[0257] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the present disclosure being indicated by the appended claims.

[0258] All patents and publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A viral vector encoding a human metapneumovirus (hMPV) F polypeptide antigen that lacks a transmembrane domain, lacks a cytoplasmic tail, and contains a human rhinovirus 3C (HRV-3C) protease cleavage site.

2. 2. The viral vector of claim 1, comprising a viral vector backbone derived from a parainfluenza virus (PIV), optionally wherein the PIV is a chimeric bovine / human parainfluenza type 3 virus (rB / HPIV3) or a human parainfluenza type 3 virus (HPIV3).

3. The F polypeptide comprises the amino acid substitutions Q100R and S101R, which substitute arginine for glutamine at amino acid position 100 of SEQ ID NO:1 and arginine for serine at amino acid position 101 of SEQ ID NO:

1. 0 further comprising a cleavage site mutation; the F polypeptide further comprises a signal peptide; the F polypeptide optionally further comprises at least one tag sequence which is an 8xHis tag and / or a Strep II tag; and / or the F polypeptide further comprises a foldon domain, and / or the hMPV F polypeptide is a pre-fusion F polypeptide; The viral vector of claim 1.

4. The F polypeptide is an amino acid substitution substituting the amino acid at position 160 of SEQ ID NO:1 and an amino acid substitution substituting the amino acid at position 46 of SEQ ID NO:1; an amino acid substitution replacing the amino acid at position 160 with phenylalanine, tryptophan, tyrosine, valine, alanine, isoleucine, or leucine; an amino acid substitution at amino acid position 160 with phenylalanine; an amino acid substitution at amino acid position 46 with valine, alanine, isoleucine, leucine, phenylalanine, tyrosine, or proline; an amino acid substitution at amino acid position 46 with valine; an amino acid substitution at amino acid position 160 of SEQ ID NO:1 with phenylalanine, tryptophan, tyrosine, valine, alanine, isoleucine, or leucine and / or an amino acid substitution at amino acid position 46 of SEQ ID NO:1 with valine, alanine, isoleucine, leucine, phenylalanine, tyrosine, or proline; and / or An amino acid substitution replacing threonine at amino acid position 160 of SEQ ID NO:1 and an amino acid substitution replacing asparagine at amino acid position 46 of SEQ ID NO:1 The viral vector of claim 1 further comprising:

5. the viral vector encodes an F polypeptide antigen; the F polypeptide lacks a transmembrane domain and lacks a cytoplasmic tail; F containing the amino acid substitutions Q100R and S101R, which substitute arginine for glutamine at amino acid position 100 of SEQ ID NO:1 and arginine for serine at amino acid position 101 of SEQ ID NO:

1. 0 cleavage site mutations; protease cleavage site; heterologous signal peptide; 8xHis tag and / or Strep II tag; and foldon domain The viral vector of claim 1 further comprising:

6. A viral vector encoding an F polypeptide antigen, wherein the F polypeptide lacks a transmembrane domain, lacks a cytoplasmic tail, and comprises an amino acid substitution substituting a threonine at amino acid position 160 of SEQ ID NO:1 and an amino acid substitution substituting an asparagine at amino acid position 46 of SEQ ID NO:

1.

7. The F polypeptide is an amino acid substitution at amino acid position 160 replacing the threonine with phenylalanine, tryptophan, tyrosine, valine, alanine, isoleucine, or leucine; the amino acid substitution T160F, substituting phenylalanine for threonine at amino acid position 160; an amino acid substitution at amino acid position 46 replacing asparagine with valine, alanine, isoleucine, leucine, phenylalanine, tyrosine, or proline; the amino acid substitution N46V substituting valine for asparagine at amino acid position 46; At least 95% sequence identity with SEQ ID NO:7; F containing the amino acid substitutions Q100R and S101R, which substitute arginine for glutamine at amino acid position 100 of SEQ ID NO:1 and arginine for serine at amino acid position 101 of SEQ ID NO:

1. 0 cleavage site mutations; signal peptide; Optionally, at least one tag sequence that is an 8xHis tag and / or a Strep II tag; foldon domain; Including, Optionally, the hMPV F polypeptide is a pre-fusion F polypeptide, and / or the viral vector further comprises an hMPV F nucleic acid molecule having at least 95% sequence identity to SEQ ID NO:8, optionally wherein the nucleic acid molecule comprises SEQ ID NO:8; The viral vector of claim 6.

8. 7. The viral vector of claim 6, encoding an antigenic pre-fusion F polypeptide, wherein the pre-fusion F polypeptide lacks a transmembrane domain and lacks a cytoplasmic tail; the amino acid substitution T160F, which replaces the threonine at amino acid position 160 of SEQ ID NO:1 with phenylalanine, and the amino acid substitution N46V, which replaces the asparagine at amino acid position 46 of SEQ ID NO:1 with valine; F containing the amino acid substitutions Q100R and S101R, which substitute arginine for glutamine at amino acid position 100 of SEQ ID NO:1 and arginine for serine at amino acid position 101 of SEQ ID NO:

1. 0 cleavage site mutations; protease cleavage site; signal peptide; 8xHis tag and / or Strep II tag; and foldon domain The viral vector of claim 6, comprising:

9. the hMPV F is derived from an A strain of hMPV, and / or The viral vector of claim 1 , wherein the hMPV F is an A1 or A2 subtype hMPV.

10. 2. The viral vector of claim 1, wherein the F polypeptide has at least 95% sequence identity with or comprises SEQ ID NO:

3.

11. A viral vector encoding an F polypeptide, wherein the F polypeptide comprises at least 95% sequence identity with SEQ ID NO:

7.

12. the F polypeptide is a pre-fusion F polypeptide; the viral vector backbone is derived from (PIV), optionally the PIV is chimeric rB / HPIV3 or HPIV3; the F polypeptide comprises an amino acid substitution T160F, which substitutes the threonine at amino acid position 160 with phenylalanine, and an amino acid substitution N46V, which substitutes the asparagine at amino acid position 46 with valine; the F polypeptide comprises SEQ ID NO:7, and / or the viral vector further comprises an hMPV F nucleic acid molecule having at least 95% sequence identity to SEQ ID NO:8, optionally wherein the nucleic acid molecule comprises SEQ ID NO:8; The viral vector of claim 11.

13. 10. A live-attenuated virus or pharmaceutical composition comprising the viral vector of claim 1, optionally wherein the live-attenuated virus or pharmaceutical composition is comprised in a vaccine.

14. A pharmaceutical composition comprising the vaccine described in claim 13 for use in a method for inducing an immune response against hMPV and / or HPIV3 or protecting a subject from hMPV infection and / or HPIV3 infection, the method comprising administering the vaccine described in claim 13 to a subject.

15. the vaccine is co-administered with an adjuvant; the vaccine is administered in combination with an additional vaccine, optionally wherein the additional vaccine is a respiratory syncytial virus (RSV) vaccine or an influenza vaccine; the subject is a human, optionally the human subject is an infant, a young child or an older adult; and / or 15. The pharmaceutical composition of claim 14, wherein the vaccine increases serum concentrations of neutralizing antibodies and the subject has pre-existing hMPV immunity.

16. The viral vector of claim 1 in the manufacture of a medicament for inducing an immune response against hMPV and HPIV3 or for protecting a subject from hMPV and HPIV3 infection.

17. A pharmaceutical composition comprising the viral vector of claim 1 for use in the following method (A) or (B): (A) A method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of the viral vector of claim 1; or (B) A method for preventing hMPV infection and HPIV3 infection or alleviating one or more symptoms of hMPV infection and HPIV3 infection, the method comprising administering to a subject, optionally intramuscularly, intranasally, intravenously, subcutaneously, or intradermally, a prophylactically effective amount of the viral vector described in claim 1, comprising a pharmaceutical composition.

18. 10. The viral vector of claim 1 for use in treating a subject in need thereof.

19. 10. A kit comprising a container containing a single-use or multi-use dose of the viral vector of claim 1, optionally wherein the container is a vial or a pre-filled syringe or injector.

20. 2. The viral vector of claim 1, wherein the viral vector comprises an hMPV F nucleic acid molecule having at least 95% sequence identity to SEQ ID NO:8, and optionally, the nucleic acid molecule comprises SEQ ID NO:8.

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