Multivalent vaccines for paramyxoviruses and uses thereof - Patents.com

Abstract

A pharmaceutical composition is provided that includes two or more virus-like particles (VLPs), a first virus-like particle (VLP) that includes a first component that includes a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, and a second virus-like particle (VLP) that includes a first component that includes a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof. Additionally, a method of using the composition for vaccination is provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 342,953, filed May 17, 2022, U.S. Provisional Patent Application No. 63 / 367,109, filed June 27, 2022, U.S. Provisional Patent Application No. 63 / 378,151, filed October 3, 2022, and U.S. Provisional Patent Application No. 63 / 387,092, filed December 12, 2022, each of which is incorporated by reference in its entirety into this specification.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING This application contains a Sequence Listing that was submitted in .XML format via EFS-WEB and is hereby incorporated by reference in its entirety. The .XML copy, created on May 16, 2023, is named 061291-508001WO_SeqList_ST26.xml and is 309 kilobytes in size. [Background technology]

[0003] Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus of the Pneumoviridae family. There are two major genetic lineages, A and B, which are antigenically related (subgroups A and B, respectively), and infection with either subgroup induces cross-neutralizing antibodies. RSV is a seasonal epidemic and is the leading cause of lower respiratory tract infections (LRTIs) in all age groups worldwide. Accumulating data identify that the disease burden in adults is comparable to influenza, with most hospitalizations and deaths occurring in older adults over 65 years of age. Immunologic data suggest that RSV may cause more than 170,000 hospitalizations and approximately 14,000 deaths annually in the United States alone. RSV is an important cause of respiratory disease in Europe as well. The incidence and severity of RSV disease is particularly high in frail older adults and older adults with cardiopulmonary disease who are considered at high risk for complications and hospitalization. RSV is spread by respiratory droplets and close contact with infected individuals or fomites. In temperate climates, epidemics typically occur annually in the winter, whereas in tropical regions, seasonality is less clear, with infection occurring most frequently during the rainy season. The severity of RSV disease is determined primarily by the extent of viral replication following infection. Induction of neutralizing antibodies (NAb) is associated with protection from disease and should therefore be the goal of vaccination. However, seropositivity confers only incomplete protection from infection.

[0004] Human metapneumovirus (hMPV) is a single-stranded, negative-sense RNA virus in the genus Metapneumovirus, family Pneumoviridae. The most closely related human pathogen is RSV. Like RSV, there are two major genetic lineages, antigenically related A and B (subgroups A and B, respectively), and infection with either subgroup induces cross-neutralizing antibodies. In mouse models, hMPV infection provides short-term protection against reinfection. Macaques infected with hMPV showed seroconversion and temporary protection from subsequent infections that waned over several months. Like RSV, NAbs alone can protect against hMPV disease. Despite the presence of protective antibody titers in adults, reinfection with hMPV occurs in both healthy and immunocompromised humans. In older adults, hMPV is responsible for a significant proportion of severe respiratory infections, with a similar infection rate to RSV. In the Etiology of Pneumonia in the Community (EPIC) study, hMPV was identified in 4% of adults hospitalized with community-acquired pneumonia, whereas RSV was identified in 3% of adults. Currently, there are no licensed vaccines against RSV or hMPV.

[0005] Thus, there is a long unmet need for a multivalent vaccine for paramyxoviruses, such as a bivalent RSV-hMPV vaccine. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure relates to a multivalent vaccine for paramyxoviruses and uses thereof. In one embodiment, the present disclosure provides a composition or pharmaceutical composition comprising two or more virus-like particles (VLPs). Each VLP comprises a first component and optionally one or more additional components, which collectively assemble to form a VLP. The paramyxoviruses can be respiratory syncytial virus (RSV) and metapneumovirus (hMPV), respectively. However, other combinations of paramyxoviruses are also contemplated. The first virus-like particle (VLP) can comprise a first component (of this first VLP) comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof. The second virus-like particle (VLP) can comprise a first component (of this second VLP) comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof. The two VLPs can be mixed together to form a composition or pharmaceutical composition, which can then be used for vaccination or other purposes.

[0007] In another aspect, the disclosure provides a composition or pharmaceutical composition, a virus-like particle (VLP) comprising a plurality of first components. Some first components may comprise a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, and other first components may comprise a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof. The VLP is assembled from a mixture of the first component and optionally one or more additional components. These "mosaic" VLPs may be used for vaccination or other purposes.

[0008] The disclosure provides compositions or pharmaceutical compositions comprising two or more virus-like particles (VLPs), wherein a first virus-like particle (VLP) comprises a first component comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, and a second virus-like particle (VLP) comprises a first component comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof, and / or virus-like particles (VLPs) comprising a plurality of first components, wherein some of the first components comprise a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, and some of the first components comprise a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof.

[0009] In some embodiments, each of the VLPs independently comprises a first component that comprises a first multimerization domain and a second component that comprises a second multimerization domain.

[0010] In some embodiments the first multimerization domain is selected from SEQ ID NO:1, 4, 5, 7, 9, 18, 19, 21, 24, 25, 26, 29, 30, 31, 34, 36, 37, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 144, 145, or a functional variant thereof.

[0011] In some embodiments, the first multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 144) or I53-50A ΔCys (SEQ ID NO: 145).

[0012] In some embodiments, the first multimerization domain comprises the amino acid substitutions C74A and C98A, the amino acid substitutions C163A and C201A, or the amino acid substitutions C74A, C98A, C163A, and C201A relative to SEQ ID NO:144.

[0013] In some embodiments, the second multimerization domain is selected from SEQ ID NOs: 2, 3, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 20, 22, 23, 27, 28, 32, 33, 35, 38, 40, and 41, or functional variants and fragments thereof.

[0014] In some embodiments, the second multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50B (SEQ ID NO:8) or I53-50B.4PosT1 (SEQ ID NO:34).

[0015] In some embodiments, the RSV F protein ectodomain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with RSV F DS-Cav1 (SEQ ID NO: 173).

[0016] In some embodiments, the RSV F protein ectodomain includes the amino acid substitutions S155C and S290C, and / or the amino acid substitutions S190F and V207L.

[0017] In some embodiments, the first component of the first VLP comprises a polypeptide that shares at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with DS-Cav1-I53-50A (SEQ ID NO: 148).

[0018] In some embodiments, the hMPV F protein ectodomain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:174 or SEQ ID NO:175.

[0019] In some embodiments, the hMPV F protein ectodomain contains amino acid substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, and / or R102G, or the hMPV F protein ectodomain contains amino acid substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, N97G, P98G, R99G, Q100G, H386N, S101G, and / or R102G compared to a reference hMPV F protein sequence (SEQ ID NO:56).

[0020] In some embodiments, the first component of the second VLP comprises a polypeptide that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:176.

[0021] In some embodiments, the first component of the second VLP comprises a polypeptide that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:177.

[0022] In some embodiments, the composition comprises one or more pharma- ceutically acceptable diluents, adjuvants, or excipients.

[0023] In some embodiments, the composition comprises a stable emulsion. In some embodiments, the vaccine comprises one or more adjuvants. In some embodiments, the one or more adjuvants are squalene, alum, SLA, GLA, R848, IMQ, 3M-052, CpG, saponin (QS21), or combinations thereof. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is an oil-in-water emulsion containing squalene (4.3%) in a citrate buffer containing the stabilizing non-ionic surfactants Tween® 80 (0.5%) and Span 85 (0.5%). In some embodiments, the adjuvant is a squalene-based emulsion. In some embodiments, the adjuvant is a squalene-based emulsion and a TLR4 agonist.

[0024] The present disclosure provides a unit dose of a composition or pharmaceutical composition disclosed herein, comprising about 0.5 μg, about 1 μg, about 20 μg, about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 50 μg. In one embodiment, a unit dose is provided comprising about 0.0 μg of a first VLP and about 0.5 μg, about 1 μg, about 20 μg, about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP.

[0025] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0026] In some embodiments, the unit dose comprises about 100 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0027] In some embodiments, the unit dose comprises about 125 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0028] In some embodiments, the unit dose comprises about 150 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0029] In some embodiments, the unit dose comprises about 175 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0030] In some embodiments, the unit dose comprises about 200 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0031] In some embodiments, the unit dose comprises about 225 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0032] In some embodiments, the unit dose comprises about 250 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0033] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof and about 75 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0034] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising the RSV F protein ectodomain or an antigenic variant thereof and about 150 μg of a second VLP comprising the hMPV F protein ectodomain or an antigenic variant thereof.

[0035] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising the RSV F protein ectodomain or an antigenic variant thereof and about 225 μg of a second VLP comprising the hMPV F protein ectodomain or an antigenic variant thereof.

[0036] In some embodiments, the unit dose comprises about 150 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 150 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0037] In some embodiments, the unit dose comprises about 225 μg of a first VLP comprising the RSV F protein ectodomain or an antigenic variant thereof and about 75 μg of a second VLP comprising the hMPV F protein ectodomain or an antigenic variant thereof.

[0038] In some embodiments, the unit dose comprises about 150 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof and about 75 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0039] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, about 75 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0040] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, about 150 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0041] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, about 225 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0042] In some embodiments, the unit dose comprises about 150 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, about 150 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0043] In some embodiments, the unit dose comprises about 225 μg of a first VLP comprising the RSV F protein ectodomain or an antigenic variant thereof, about 75 μg of a second VLP comprising the hMPV F protein ectodomain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0044] In some embodiments, the unit dose comprises about 150 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, about 75 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0045] The present disclosure provides a method of vaccinating a subject, the method comprising administering to the subject an effective amount of a composition according to the present disclosure.

[0046] In some embodiments, a method for generating an immune response or enhancing an existing immune response in a subject comprises administering to the subject an effective amount of a composition according to the present disclosure.

[0047] In some embodiments, a method for preventing infection by a paramyxovirus comprises administering to a subject an effective amount of a composition according to the present disclosure.

[0048] In some embodiments, a method of immunizing a subject against a paramyxovirus comprises administering to the subject an effective amount of a composition according to the present disclosure.

[0049] In some embodiments, the paramyxovirus is respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV.

[0050] In some embodiments, the methods generate protective immunity against respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV.

[0051] In some embodiments, the methods generate neutralizing antibodies against respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV.

[0052] In some embodiments, the subject is at risk for severe RSV disease and / or at risk for severe hMPV disease.

[0053] In some embodiments, the vaccine is administered by subcutaneous injection. In some embodiments, the vaccine is administered by intramuscular injection. In some embodiments, the vaccine is administered by intradermal injection. In some embodiments, the vaccine is administered intranasally. In some embodiments, the vaccine is administered orally. In some embodiments, the vaccine is administered sublingually. In some embodiments, the vaccine is administered buccally. In some embodiments, the vaccine is administered by one or more of subcutaneous injection, intramuscular injection, intradermal injection, intranasal, oral, sublingual, or buccal.

[0054] In some embodiments, the subject is an adult over the age of 60. In some embodiments, the subject is a healthy adult between the ages of 18-45.

[0055] In some embodiments, the effective amount comprises about 0.5 μg, about 1 μg, about 2 μg, about 20 μg, about 25 μg, about 40 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 140 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 280 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, about 500 μg, or about 600 μg of the first VLP and / or second VLP.

[0056] In some embodiments, the effective amount comprises about 75 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0057] In some embodiments, the effective amount comprises about 100 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising an hMPV F protein ectodomain or an antigenic variant thereof.

[0058] In some embodiments, the effective amount comprises about 125 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0059] In some embodiments, the effective amount comprises about 150 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0060] In some embodiments, the effective amount comprises about 175 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0061] In some embodiments, the effective amount comprises about 200 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising an hMPV F protein ectodomain or an antigenic variant thereof.

[0062] In some embodiments, the effective amount comprises about 225 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0063] In some embodiments, the effective amount comprises about 250 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 50 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0064] In some embodiments, the effective amount comprises about 0.5 μg, about 1 μg, about 20 μg, about 25 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 200 μg, about 225 μg, about 250 μg, or about 500 μg of the first VLP and / or the second VLP.

[0065] In some embodiments, the effective amount comprises about 75 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 200 μg, about 225 μg, about 250 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0066] In some embodiments, the effective amount comprises about 150 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 25 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 200 μg, about 225 μg, about 250 μg, or about 500 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0067] In some embodiments, the effective amount comprises about 150 μg of a first VLP comprising a RSV F protein ectodomain or an antigenic variant thereof, and about 150 μg of a second VLP comprising a hMPV F protein ectodomain or an antigenic variant thereof.

[0068] In some embodiments, the method includes administering a second dose of the pharmaceutical composition.

[0069] In some embodiments, the second dose is administered within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, or about 12 months after the first dose.

[0070] In some embodiments, the method includes administering a third dose of the pharmaceutical composition. In some embodiments, the third dose is administered within about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years after the second dose.

[0071] In some embodiments, the method comprises administering subsequent doses at regular intervals of about 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years.

[0072] In some embodiments, the method limits the development of a RSV infection in a subject, and / or the method limits the development of a hMPV infection in a subject.

[0073] In some embodiments, the methods result in the production of RSV-A specific neutralizing antibodies in the subject.

[0074] In some embodiments, the method results in an increase in RSV-A specific neutralizing antibodies in a subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline.

[0075] In some embodiments, an increase in RSV-A specific neutralizing antibodies is detectable within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after administration of the pharmaceutical composition.

[0076] In some embodiments, the methods result in the production of RSV-B specific neutralizing antibodies in the subject.

[0077] In some embodiments, the method results in an increase in RSV-B specific neutralizing antibodies in a subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline.

[0078] In some embodiments, an increase in RSV-B specific neutralizing antibodies is detectable within about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after administration of the pharmaceutical composition.

[0079] In some embodiments, the methods result in the production of hMPV-A specific neutralizing antibodies in the subject.

[0080] In some embodiments, the method results in an increase in hMPV-A-specific neutralizing antibodies in a subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline.

[0081] In some embodiments, an increase in hMPV-A specific neutralizing antibodies is detectable within about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks of administration of the pharmaceutical composition.

[0082] In some embodiments, the methods result in the production of hMPV-B specific neutralizing antibodies in the subject.

[0083] In some embodiments, the method results in an increase in hMPV-B-specific neutralizing antibodies in a subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline.

[0084] In some embodiments, an increase in hMPV-B specific neutralizing antibodies is detectable within about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after administration of the pharmaceutical composition.

[0085] In some embodiments, the method prevents severe lower respiratory tract infections (LRTIs).

[0086] A pre-filled syringe containing the composition according to the present disclosure is provided.

[0087] Kits are provided that include a composition according to the present disclosure or that include a pre-filled syringe according to the present disclosure.

[0088] The present disclosure provides a kit comprising: A composition comprising a virus-like particle (VLP) comprising a first component comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof; A composition comprising a second virus-like particle (VLP) comprising a first component comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof; and A composition comprising a virus-like particle (VLP) comprising a plurality of first components, some of the first components comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, and some of the first components comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof. The kit includes one or more of the following:

[0089] In some embodiments, the kit comprises a composition comprising an adjuvant that is combined with one or more VLP compositions prior to administration to a subject. [Brief description of the drawings]

[0090] [Figure 1A] 1 shows an exemplary embodiment of a protein-based virus-like particle (VLP) according to the present disclosure.

[0091] [Figure 1B] Further exemplary embodiments of VLPs and VLP components are shown (F protein not shown).

[0092] [Diagram 2] Secondary structure map of the hMPV F protein in the prefusion configuration is shown (PDB ID: 5WB0, figure generated at www.rcsb.org).

[0093] [Diagram 3]1A-C are a series of plots showing antibody binding profiles for exemplary VLPs of the present disclosure. The antibodies are specific for various forms of the hMPV F protein. MF14 binds to the pre-fusion and post-fusion forms of the hMPV F protein at a site II epitope (A). MF16 binds to the pre-fusion and post-fusion forms of the hMPV F protein at a site IV epitope (B). MPE8 binds to the pre-fusion form of the hMPV F protein at a site III epitope (C).

[0094] [Figure 4] A and B show the immunogenicity of hMPV F protein mutants expressed on bicomponent virus-like particles (VLPs) or as soluble proteins (sol). The graphs show neutralizing antibody titers against hMPV-A (A) and hMPV-B (B) in serum samples collected 35 days after two immunizations.

[0095] [Diagram 5] A and B show the immunogenicity of hMPV008 VLP and the corresponding soluble protein, CompA-hMPV008, under different doses and adjuvant conditions. Also shown is hMPV033 when adjuvanted with squalene emulsion (SE). Graphs show neutralizing antibody titers against hMPV-A (A) and hMPV-B (B) in serum samples collected 35 days after two immunizations.

[0096] [Figure 6] 1 shows RSV neutralizing titers in sera from mice immunized with monovalent, bivalent, or mosaic vaccine compositions. Mice immunized with hMPV monovalent vaccine (IVX-241) alone exhibit a low immune response to RSV. The monovalent RSV, bivalent RSV / hMPV, and mosaic RSV / hMPV groups exhibited high anti-RSV immune responses.

[0097] [Figure 7]1 shows hMPV neutralizing titers in serum from mice immunized with monovalent or bivalent vaccine compositions. Mice immunized with only the RSV monovalent vaccine (IVX-121) show a low immune response to hMPV. The monovalent hMPV group, the bivalent RSV / hMPV group, and the mosaic RSV / hMPV group showed high anti-hMPV immune responses.

[0098] [Figure 8A] 1 shows the immunogenicity and induction of protective efficacy of bivalent RSV-hMPV VLP vaccines compared to monovalent RSV or hMPV VLP vaccines in a cotton rat live virus challenge model.

[0099] 1 shows titers of RSV-A neutralizing antibodies in sera from animals immunized with monovalent RSV ("IVX-121") or bivalent ("IVX-A12") VLPs.

[0100] [Figure 8B] 1 shows the immunogenicity and induction of protective efficacy of bivalent RSV-hMPV VLP vaccines compared to monovalent RSV or hMPV VLP vaccines in a cotton rat live virus challenge model. 2 shows the titers of hMPV-A neutralizing antibodies in the serum of animals immunized with monovalent hMPV ("IVX-241") or bivalent ("IVX-A12") VLPs.

[0101] [Figure 8C] 1 shows the immunogenicity and induction of protective efficacy of bivalent RSV-hMPV VLP vaccines compared to monovalent RSV or hMPV VLP vaccines in a cotton rat live virus challenge model. RSV-A virus titers in the lungs of animals immunized with monovalent RSV ("IVX-121") or bivalent ("IVX-A12") VLPs and then challenged with RSV virus are shown.

[0102] [Figure 8D]1 shows the immunogenicity and induction of protective efficacy of bivalent RSV-hMPV VLP vaccines compared to monovalent RSV or hMPV VLP vaccines in a cotton rat live virus challenge model. 2 shows hMPV-A virus titers in the lungs of animals immunized with monovalent hMPV ("IVX-241") or bivalent ("IVX-A12") VLPs and then challenged with hMPV virus.

[0103] [Figure 8E] 1 shows the immunogenicity and induction of protective efficacy of bivalent RSV-hMPV VLP vaccines compared to monovalent RSV or hMPV VLP vaccines in a cotton rat live virus challenge model. 2 shows intranasal titers of RSV-A virus in animals immunized with monovalent RSV ("IVX-121") or bivalent ("IVX-A12") VLPs and then challenged with RSV virus.

[0104] [Figure 8F] 1 shows the immunogenicity and induction of protective efficacy of bivalent RSV-hMPV VLP vaccines compared to monovalent RSV or hMPV VLP vaccines in a cotton rat live virus challenge model. 2 shows intranasal titers of hMPV-A virus in animals immunized with monovalent hMPV ("IVX-241") or bivalent ("IVX-A12") VLPs and then challenged with hMPV virus.

[0105] [Figure 9] 1 is a series of plots showing the induction of neutralizing antibody titers over time in mice in response to dosing with hMPV F protein VLPs formulated with adjuvant.

[0106] [Figure 10] This is a Phase 1 / 1b randomized, observer-blinded, placebo-controlled study design to evaluate administration of IVX-121 in younger and older adult subjects.

[0107] [Figure 11]A summary of safety data is provided. There were no serious adverse events (SAEs), AEs of special interest (AESIs), or adverse events (AEs) leading to study withdrawal.

[0108] [Figure 12] Graphs of maximum severity of solicited local and systemic adverse events within 7 days of a single dose are shown ("Alum" = 500 μg / mL aluminum hydroxide). Unadjuvanted IVX-121 was mildly reactogenic in older adults and was tolerated similarly to placebo.

[0109] [Figure 13] A graph of RSV-A neutralizing antibodies (nAB) is shown. Geometric mean titers (GMTs) are expressed in international units per milliliter (IU / mL). The GMTs of unadjuvanted IVX-121 are comparable in younger and older adults.

[0110] [Figure 14] Figure 1 shows a graph of RSV-A neutralizing antibodies (nAB) unadjuvanted versus adjuvanted. Geometric mean titers (GMT) are expressed in international units per milliliter (IU / mL). Alum adjuvant had no beneficial effect in young and older adults.

[0111] [Figure 15] A table summarizing the neutralizing and binding antibody data is shown.

[0112] [Figure 16] Shown are graphs of unadjuvanted RSV-A neutralizing antibodies (nAB) out to day 180 in younger adult subjects (left panel) and older adult subjects (right panel). Geometric mean titers (GMT) are expressed in international units per milliliter (IU / mL). Lower limit of quantification (LloQ)=9.9, geometric mean fold increase from baseline (day 28). Data reflects all subjects available at the time of this 6-month analysis. Individual values ​​may differ slightly from earlier interim readings.

[0113] [Figure 17]

[0023] Figure 1 shows a graph of unadjuvanted RSV-A neutralizing antibodies (nAB) through day 180 in young adult subjects. For each treatment dose, μg of total VLPs and μg of RSV-A antigen are shown. For each treatment dose, the percentage of titers retained between study day 180 and study day 28 is shown. Geometric mean titers (GMTs) are expressed in international units per milliliter (IU / mL).

[0114] [Figure 18]

[0023] Figure 1 shows a graph of unadjuvanted RSV-A neutralizing antibodies (nAB) through day 180 in elderly adult subjects. For each treatment dose, μg of total VLPs and μg of RSV-A antigen are shown. For each treatment dose, the percentage of titers retained between study day 180 (M6) and study day 28 (M1) is shown. Geometric mean titers (GMTs) are expressed in international units per milliliter (IU / mL).

[0115] [Figure 19] A summary of safety data is provided. There were no serious adverse events (SAEs), AEs of special interest (AESIs), or adverse events (AEs) leading to study withdrawal.

[0116] [Figure 20] Graphs of non-adjuvanted RSV-A neutralizing antibodies (nAB) (left panel) and non-adjuvanted RSV-B neutralizing antibodies (nAB) (right panel) in elderly adult subjects are shown at days 28 and 180. Geometric mean titers (GMTs) are expressed in international units per milliliter (IU / mL).

[0117] [Figure 21]Study design for a Phase 1 randomized, observer-blinded, placebo-controlled study to evaluate a single dose of IVX-A12 in older adult subjects (age 60-75). Dose groups and total VLP content (top left panel). Each dose contained both RSV and hMPV VLPs. Low, medium, and high hMPV dose VLP compositions were combined with a fixed RSV VLP dose (top right panel). Interim readouts included safety and immunogenicity readouts through day 28 (D28) (bottom panel).

[0118] [Figure 22A] The demographic composition of study participants is shown. Demographic characteristics were similar between IVX-A12 and placebo treated subjects. A total of 141 subjects were enrolled, 140 were dosed (safety analysis set), 17 subjects were excluded due to protocol violations, and the remaining 123 subjects were in the per-protocol analysis set. [Figure 22B] A summary of safety data is provided. As of D28, there were no serious adverse events (SAEs), AEs of special interest (AESIs), or adverse events (AEs) leading to study withdrawal. In a subject with a history of hypertension, an unsolicited severe AE (unrelated to vaccine) was observed on Day 28. The AE resolved. Spontaneous AEs were collected through Day 28. Other AEs will be collected through Day 180.

[0119] [Figure 23A] Figure 1 shows a graph of maximum severity of non-spontaneously reported local adverse events within 7 days of a single dose. Reactogenicity of unadjuvanted IVX-A12 is mild in older adults. Dose group VLP: 150μg=75μg RSV+75μg hMPV, 225μg=75μg RSV+150μg hMPV, 300μg=75μg RSV+225μg hMPV.

[0120] [Figure 23B]Figure 1 shows a graph of non-spontaneous reported systemic adverse events within 7 days of a single dose. Unadjuvanted and adjuvanted (MF59®) IVX-A12 are tolerated similarly to placebo. Dose groups VLP: 150μg=75μg RSV+75μg hMPV, 225μg=75μg RSV+150μg hMPV, 300μg=75μg RSV+225μg hMPV.

[0121] [Figure 24A] Graph of RSV-A neutralizing antibodies (nAB) from the participant adherence analysis set. Geometric mean titers (GMT) are expressed in international units per milliliter (IU / mL). LLoQ for RSV-A=9.4. MF59®: A proprietary adjuvant from Seqirus Inc. GMFR: Geometric mean fold increase from baseline. VLP: 150μg=75μg RSV+75μg hMPV, 225μg=75μg RSV+150μg hMPV, 300μg=75μg RSV+225μg hMPV.

[0122] [Figure 24B] Graph of RSV-B neutralizing antibodies (nAB) from the participant adherence analysis set. Geometric mean titers (GMT) are expressed in international units per milliliter (IU / mL). LLoQ for RSV-B=8.0. MF59®: A proprietary adjuvant from Seqirus Inc. GMFR: Geometric mean fold increase from baseline. VLP: 150μg=75μg RSV+75μg hMPV, 225μg=75μg RSV+150μg hMPV, 300μg=75μg RSV+225μg hMPV.

[0123] [Figure 25A]Graph of hMPV-A neutralizing antibodies (nAB). Geometric mean titers (GMT) are expressed in assay units. Participant adherence analysis set. LLoQ=4.0log2 for hMPV-A and hMPV-B. GMFR: geometric mean fold increase from baseline. VLP: 150μg=75μg RSV+75μg hMPV, 225μg=75μg RSV+150μg hMPV, 300μg=75μg RSV+225μg hMPV.

[0124] [Figure 25B] Graph of hMPV-B neutralizing antibodies (nAB). Geometric mean titers (GMT) are expressed in assay units. Participant adherence analysis set. LLoQ=4.0log2 for hMPV-A and hMPV-B. GMFR: geometric mean fold increase from baseline. VLP: 150μg=75μg RSV+75μg hMPV, 225μg=75μg RSV+150μg hMPV, 300μg=75μg RSV+225μg hMPV.

[0125] [Figure 26A] Prespecified subanalysis examining subjects in the lowest tertile of baseline RSV-A titers is shown. Data are shown for RSV-A neutralizing antibody (nAB) in subjects in the lowest tertile of baseline nAB titers. Data for adjuvanted and non-adjuvanted subjects are pooled by dose. Geometric mean titers (GMT) are expressed as international units per milliliter (IU / mL). Participant adherence analysis set. LLoQ for RSV-A=9.4. LLoQ for RSV-B=8.0. GMFR: geometric mean fold increase from baseline. Treatment arms VLP: 150μg=75μg RSV+75μg hMPV, 225μg=75μg RSV+150μg hMPV, 300μg=75μg RSV+225μg hMPV. *Data pooled across adjuvanted and non-adjuvanted groups at one dose. [Figure 26B]Prespecified subanalysis examining subjects in the lowest tertile of baseline RSV-B titers is shown. Data are shown for RSV-B neutralizing antibody (nAB) in subjects in the lowest tertile of baseline nAB titers. Data for adjuvanted and non-adjuvanted subjects are pooled by dose. Geometric mean titers (GMT) are expressed as international units per milliliter (IU / mL). Participant adherence analysis set. LLoQ for RSV-A=9.4. LLoQ for RSV-B=8.0. GMFR: geometric mean fold increase from baseline. Treatment arms VLP: 150μg=75μg RSV+75μg hMPV, 225μg=75μg RSV+150μg hMPV, 300μg=75μg RSV+225μg hMPV. *Data pooled across adjuvanted and non-adjuvanted groups at one dose. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0126] Provided herein is a composition or pharmaceutical composition comprising two or more VLPs displaying the ectodomains of F proteins of various paramyxoviruses. For example, the present disclosure provides a composition comprising two VLPs, namely, a VLP having a component comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, and a VLP having a component comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof. These compositions can be used to vaccinate against RSV (e.g., RSV-A subtype and / or RSV-B subtype) and human metapneumovirus (hMPV; e.g., hMPV-A subtype and / or hMPV-B subtype).

[0127] In particular, provided herein are protein-based virus-like particle (VLP) vaccines against respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), in which the RSV F protein ectodomain and the hMPV F protein ectodomain are each independently linked to a protein-based VLP, such as a designed VLP, such as a symmetric VLP, and then presented thereon. For example, the vaccine antigen can be an N-terminal fusion of the RSV F protein or the hMPV F protein ectodomain with a protein having a multimerization domain for a one- or two-component de novo designed VLP, such as a two-component icosahedral VLP. Also provided are vaccine compositions, methods of manufacture, and methods of use, for example, for immunizing a subject to generate a protective immune response against RSV and / or hMPV viruses, or to enhance an existing immune response.

[0128] In a variant, the disclosure provides a "mosaic" VLP, which is generated by mixing components of RSV and hMPV vaccines prior to assembly into VLPs, such that each VLP contains some components that display the ectodomain of the RSV F protein and some components that display the ectodomain of the hMPV F protein. Thus, the disclosure further provides a virus-like particle (VLP) comprising a plurality of first components, wherein some of the first components comprise a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, and some of the first components comprise a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof.

[0129] definition All publications, patents, and patent applications, including any drawings and appendices therein, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0130] The term "virus-like particle" or "VLP" refers to a non-infectious molecular assembly that resembles a virus but displays antigenic proteins or antigenic fragments of viral proteins or glycoproteins. "Protein-based VLP" refers to a VLP formed from proteins or glycoproteins and substantially free of other components (e.g., lipids). Protein-based VLPs may include post-translational and chemical modifications, but should be distinguished from micellar VLPs and VLPs formed by extraction of viral proteins from preparations of live or inactivated live viruses. The term "designed VLP" refers to a VLP that includes one or more polypeptides generated by computational protein design. An exemplary designed VLP is a VLP that includes a nanostructure as shown in FIG. 1B. The term "symmetric VLP" refers to a protein-based VLP that has a symmetric core as shown in FIG. 1B. These include, but are not limited to, designed VLPs. For example, the protein ferritin has been used to generate symmetric protein-based VLPs using the naturally occurring ferritin sequence. Ferritin-based VLPs are distinct from designed VLPs in that no protein engineering is required other than fusing viral proteins to ferritin molecules to form symmetric VLPs from ferritin. Protein design methods can be used to generate similar one-component and two-component nanostructures based on template structures (e.g., structures deposited in protein data banks) or de novo (i.e., by computationally designing new proteins with desired structures but with little or no homology to naturally occurring proteins). Such one-component and two-component nanostructures can then be used as the core of designed VLPs.

[0131] The VLPs of the present disclosure present antigens capable of eliciting an immune response against paramyxoviruses, including respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) or other metapneumoviruses. As used herein and throughout this disclosure, immune response(s) includes generating an immune response or enhancing an existing immune response. The vaccines of the present disclosure are useful for preventing and / or reducing the severity of infection by respiratory syncytial virus and / or human metapneumovirus.

[0132] The term "icosahedral particle" refers to designed VLPs having a core with icosahedral symmetry (e.g., particles designated I53 and I52 in Table 1). I53 refers to an icosahedral particle constructed from pentamers and trimers. I52 refers to an icosahedral particle constructed from pentamers and dimers. T33 refers to a tetrahedral particle constructed from two sets of trimers. T32 refers to a tetrahedral particle constructed from trimers and dimers.

[0133] Antigens may be non-covalently or covalently bound to the core of the protein-based VLP, including as a fusion protein or by other means as disclosed herein. Multimeric antigens may optionally be presented along the axis of symmetry of the VLP. Also provided are proteins and nucleic acid molecules encoding such proteins, formulations, and methods of use.

[0134] The term "antigen" refers to a polypeptide or polypeptide complex that contains at least one component designed to elicit an immune response. As used herein, the term antigen is not limited to that portion of a polypeptide or polypeptide complex that contains an antigenic epitope.

[0135] The term "polypeptide" refers to a series of amino acid residues linked by peptide bonds, optionally with one or more post-translational modifications (e.g., glycosylation) and / or other modifications (including, but not limited to, conjugation of polypeptide moieties or adjuvants used as markers, such as fluorescent tags).

[0136] The term "infection" refers to both symptomatic and asymptomatic infections.

[0137] The term "ectodomain" refers to that part of a transmembrane protein or glycoprotein that, in the protein's native state, is on the outside of the cellular or viral membrane.

[0138] The term "variant" refers to a polypeptide that has one or more insertions, deletions, substitutions, or amino acid substitutions compared to a reference polypeptide, but retains one or more properties of the reference protein.

[0139] The term "antigenic variant" refers to a variant that has one or more epitopes in common with a reference polypeptide and / or that elicits the same or a similar immune response as the reference polypeptide when administered to a subject.

[0140] The term "functional variant" refers to a variant that exhibits the same or similar functional effect(s) as a reference polypeptide. For example, a functional variant of a multimerization domain can promote multimerization to the same extent or to a similar extent as the reference multimerization domain and / or can multimerize with the same cognate multimerization domain as the reference multimerization domain.

[0141] The term "linker" refers to either a chemical linkage (i.e., a covalent bond or series of covalent bonds with an intervening chemical moiety), or to polypeptides linked at their N- and C-termini by peptide bonds to generate a fusion protein.

[0142] The term "domain" refers to any portion of a polypeptide that has tertiary structure.

[0143] The terms "multimerization domain" and "multimerization" refer to the ability of a polypeptide, or a domain of a polypeptide, to form dimers, trimers, tetramers, pentamers, or hexamers, and / or to form heteromers with other multimerization domains.

[0144] The term "trimerization domain" refers to a multimerization domain that forms trimers.

[0145] The term "VLP-forming domain" refers to a multimerization domain that, alone or together with other multimerization domains, forms a symmetric protein complex.

[0146] The term "fragment" refers to a polypeptide having one or more N-terminal or C-terminal truncations compared to a reference polypeptide.

[0147] The term "functional fragment" refers to a functional variant of a fragment.

[0148] The term "substitution" or "replacement" refers to the replacement of a single amino acid in a sequence with another amino acid residue. Standard format abbreviations are used for amino acid substitutions. For example, V94R refers to the replacement of valine (V) in a reference sequence with arginine (R). The abbreviation Arg94 refers to any sequence in which the 94th residue compared to the reference sequence is arginine (Arg).

[0149] The term "helix" or "helical" refers to an α-helical secondary structure known to occur or predicted to occur in a polypeptide. For example, a sequence may be described as helical if computational modeling suggests that the sequence is likely to adopt a helical conformation.

[0150] The term "component" refers to a protein or protein complex (eg, an antigen or a polypeptide containing a multimerization domain) that is capable of assembling into a virus-like particle under appropriate conditions.

[0151] The term "vaccine" refers to a pharmaceutical composition that can be used to generate an immune response or enhance an existing immune response in a subject.

[0152] The term "pharmaceutical acceptable excipient" means an excipient that is biologically or pharmacologically compatible for in vivo use in animals or humans, and can mean an excipient approved by a regulatory agency of the U.S. Federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, or more specifically in humans.

[0153] The term "adjuvant" refers to a pharma- ceutically acceptable substance that enhances the immune response to an antigen when administered in combination with an antigen, or before, during, or after administration of the antigen to a subject.

[0154] The term "TLR4 immunostimulatory agent" refers to an adjuvant, e.g., monophosphoryl lipid A (MPL), glucopyranosyl lipid A (GLA), and / or soluble leishmania antigen (SLA), that stimulates Toll-like receptor 4 (TLR4) in a subject's immune cells to modulate the immune response.

[0155] The term "effective amount" refers to an amount of a formulation according to the invention that, when administered to a patient for treating a condition, disorder or condition, is sufficient to effect such treatment, or, when administered to a patient for generating an immune response, is sufficient to generate such an immune response, e.g., an antibody response. An "effective amount" will vary depending on the active ingredient, the condition, disorder, or condition being treated and its severity, as well as the age, weight, physical condition and responsiveness of the subject being treated.

[0156] This amount may vary depending on the health and physical condition of the individual to be treated, their age, the capacity of the individual's immune system to generate antibodies, the degree of protection desired, the formulation of the vaccine, and other relevant factors.

[0157] As used herein, "effective amount" refers to an amount of an immunogenic composition that is effective to treat and / or limit RSV infection (e.g., RSV-A infection and / or RSV-B infection) and / or hMPV infection (e.g., hMPV-A infection and / or hMPV-B infection).

[0158] The term "immune response" refers to the elicitation of activity of one or more immune cell types in a subject. Immune responses include, for example, T cell responses and B cell responses.

[0159] The term "humoral immune response" refers to an immune response that results in the generation of plasma or serum antibodies (eg, IgG).

[0160] The term "protective immune response" refers to an immune response that prevents infection and / or reduces the severity of a pathogen when the subject is subsequently challenged with the pathogen, or generates a level of immune response that correlates with protection. For example, vaccination can generate a protective immune response if it results in the production of neutralizing antibodies in the plasma or serum of a subject (e.g., a human, pet, or agricultural animal) that are present in an amount that protects the subject from subsequent infection and / or that is observed to protect a test subject (e.g., a New Zealand White (NZW) rabbit).

[0161] The term "polyclonal antibody response" refers to an antibody response comprising antibodies with multiple specificities and / or variations in antibody sequence.

[0162] The term "neutralization" (e.g., a "neutralizing antibody response") refers to an antibody that prevents infection by a pathogen and / or reduces the level of infection. Neutralizing antibody responses can be measured in either in vitro assays (e.g., infection of cells in culture by a pathogen in the presence of the antibody) or in vivo assays (e.g., by determining a protective dose of antibody by administering the antibody to a subject prior to challenge with an infectious dose of the pathogen).

[0163] The terms "binding" or "specific" or "specifically binding" (used interchangeably herein) of an antibody to a target (e.g., the F protein of RSV or hMPV) are well understood in the art, and methods for determining such specific or preferential binding are also well understood in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, with a longer duration, and / or with a higher affinity than with alternative cells or substances. For example, an immunoglobulin that specifically or preferentially binds to thymocytes is an immunoglobulin that binds to thymocytes with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other cells. An immunoglobulin that specifically binds to a first cell or substance may or may not specifically or preferentially bind to a second cell or substance. Thus, "specific binding" does not necessarily require (but may include) exclusive binding. References to binding often, but do not necessarily, imply specific binding.

[0164] The term "predefined time" refers to a time interval appropriately selected for observing a particular effect. The predefined time can be selected before or during an experiment or procedure.

[0165] The term "post-exposure prophylaxis" refers to the administration of an antigenic composition (e.g., a vaccine) to a subject previously exposed to and / or infected with a pathogen to induce an immune response that protects against infection by the pathogen and / or to reduce the severity of one or more symptoms of infection by the pathogen.

[0166] The term "administering" refers to providing a composition to a subject in a manner that allows the composition to exert its intended effect. Administration for vaccination or post-exposure prophylaxis can be by intramuscular injection, intravenous injection, intraperitoneal injection, or any other suitable route.

[0167] The terms "immunization" and "immunizing" refer to administering a composition (e.g., virus-like particles) to a subject in an amount sufficient to elicit a desired immune response (e.g., a humoral immune response to a VLP) after one or more administration steps. Immunization can include 1-10 or more administrations (e.g., injections) of the composition, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more administrations. Generally, the first administration may not elicit a detectable immune response, as each subsequent administration enhances the immune response generated by the previous administration. As used herein, the term "immunizing" includes post-exposure prophylaxis.

[0168] The term "subject" refers to a human or non-human animal to which a composition may be administered for vaccination, treatment, or other purposes. In some embodiments, the non-human animal is a non-human primate, including, but not limited to, a rabbit, hamster, gerbil, pig, cow, sheep, goat, guinea pig, rat, mouse, squirrel, wolf, fox, horse, zebra, giraffe, elephant, cat, dog, llama, or ferret.

[0169] The term "manufacturing" refers to producing a recombinant polypeptide or virus-like particle at any scale, including but not limited to at least 25 mL, 50 mL, 1 L, 1,000 L, 50,000 L, or greater.

[0170] The terms "culturing" and "culture medium" refer to standard cell culture and recombinant protein expression techniques.

[0171] The term "host cell" refers to any cell that can be used for the expression of a recombinant polypeptide.

[0172] The term "secrete" refers to the ability of a host cell to secrete a polypeptide into the medium in which the host cell is cultured.

[0173] The term "signal sequence" refers to a polypeptide sequence that is typically at the N-terminus of a polypeptide expressed in a host cell and directs the polypeptide to a specific cellular compartment. The signal sequence may be a secretion signal that causes the host cell to secrete the polypeptide into the medium in which the host cell is cultured. A variety of signal sequences are known, and it is within the skill of the art to select an appropriate signal sequence.

[0174] The term "mixing" refers to bringing two solutions into contact and allowing the solutions to mix.

[0175] The term "purify" refers to separating a molecule from other substances present in a composition. Polypeptides can be purified by affinity (e.g., using an antibody or tag, e.g., His-tag capture resin), by charge (e.g., ion exchange chromatography), by size (e.g., preparative ultracentrifugation, size exclusion chromatography), or by other methods.

[0176] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides of greater than about 100 nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. An "oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides, either single-stranded or double-stranded DNA. However, for purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as "oligomers" or "oligos" and may be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides, as applicable to the embodiments described.

[0177] The terms "identical", "identity", "percent identity", "sequence identity" or "percent sequence identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a certain percentage of identical amino acid residues or nucleotides when compared and aligned to maximize correspondence. Methods for aligning sequences for comparison are well known in the art. After alignment, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues occur in both sequences. Percent sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence and then multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554×100=75.0). As these terms are used herein, gaps in the alignment do not reduce the percent sequence identity.

[0178] Unless otherwise specified, optimal alignment of sequences for comparison is performed by the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970), as implemented by EMBOSS Needle (World Wide Web ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al. Nucleic Acids Res. 50(W1):W276-W279(2022)). In embodiments, Devereux, et al, Nucleic Acids Res. 12:387-95 (1984), Atschul et al. J. Mo. Biol. 215:403-10 (1990) (BLAST), Carrillo and Lipman Siam J. Appl. Biology (Lesk, AM, ed., 1989), Biocomputing Informatics and Genome Projects, (Smith, DW, ed., 1993), Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds., 1994), Sequence Analysis in Molecular Biology (von Heinje, 2012), Sequence Analysis Primer (Gribskov and Other alignment methods may be used, including, but not limited to, those described in (Devereux, J., eds. 1993). In embodiments, sequence identity is calculated using an implementation of the Needleman-Wunsch algorithm provided by the National Library of Medicine (World Wide Web blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln).

[0179] For example, sequence identity can be determined by standard methods commonly used to compare the similarity of two polypeptide sequences or two polynucleotide sequences.Using computer programs such as EMBOSS Needle or BLAST, two polypeptide sequences or two polynucleotide sequences are aligned for optimal matching of each residue (along the entire length of one or both sequences, or along a predefined portion of one or both sequences).These programs provide default opening penalties and default gap penalties that can be used in conjunction with the computer programs, as well as scoring matrices such as PAM 250 (standard scoring matrices; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol.5, supp.3 (1978)).

[0180] The term "treating" refers to one or more of alleviating, ameliorating, delaying, reducing, reversing, ameliorating, or managing at least one symptom of a condition in a subject. The term "treating" can also refer to one or more of arresting the condition, delaying its onset (i.e., the period before clinical manifestation of the condition), or reducing the risk of its occurrence or worsening.

[0181] The terms "a" or "an" refer to one or more of an applicable entity, i.e., may refer to multiple referents. Thus, the terms "a," "an," "one or more," and "at least one" are used interchangeably herein. Furthermore, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility of a plurality of elements being present, unless the context clearly requires that only one of the element be present.

[0182] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean, for example, within ±20%, within ±10%, or within ±5% range. When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range, or each of the values ​​recited in the series, unless otherwise specified. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0183] All weight percentages referred to herein (i.e., "weight %" and "wt.%" and w / w) are measured relative to the total weight of the pharmaceutical composition, unless otherwise specified.

[0184] As used herein, "substantially" or "substantially" refers to the complete or nearly complete extent or degree of an action, feature, characteristic, state, structure, item, or result. For example, an object that is "substantially" enclosed means that the object is completely enclosed or nearly completely enclosed. The exact degree of acceptable deviation from absolute completeness may depend on the specific context, in some cases. Generally speaking, however, the closeness of completion is such that the overall result is the same as if absolute and total completion had been obtained. The use of "substantially" is equally applicable when used in the negative sense to refer to the complete or nearly complete lack of an action, feature, characteristic, state, structure, item, or result. For example, a composition that is "substantially free" of other active agents is completely devoid of other active agents or nearly completely devoid of other active agents, so that the effect is the same as if it were completely devoid of other active agents. In other words, a composition that is "substantially free" of a component or element or another active agent may still contain such item, so long as there is no measurable effect of it.

[0185] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the present claimed invention, or that any publications specifically or implicitly referenced are prior art.

[0186] Overview Vaccination is a treatment modality used to prevent or reduce the severity of infection by a variety of infectious agents, including bacteria, viruses, and parasites. The development of new vaccines has important commercial and public health implications. In particular, Lyme disease, whooping cough, herpesvirus, orthomyxovirus, paramyxovirus, pneumovirus, filovirus, flavivirus, reovirus, retrovirus, coronavirus, and malaria are among the infectious agents for which vaccines already exist, are in development, or are desired.

[0187] Subunit vaccines are vaccines made from isolated antigens, usually proteins recombinantly expressed in bacterial, insect, or mammalian cell hosts. Typically, the antigenic components of subunit vaccines are selected from among proteins of infectious agents that have been observed to elicit natural immune responses upon infection, although in some cases, other components of infectious agents may be used. Typical antigens for use in subunit vaccines include proteins expressed on the surface of the target infectious agent, and thus surface-expressed envelope glycoproteins of viruses. Preferably, the antigen is a target for neutralizing antibodies. More preferably, the antigen is a target for broadly neutralizing antibodies, so that the immune response to the antigen covers immunity to multiple strains of infectious agents. In some cases, glycans that are N-linked or O-linked to the subunit vaccine may also be important in vaccination by contributing to the epitopes of the antigen or by directing the immune response to specific epitopes on the antigen through steric hindrance. The immune response that occurs in response to vaccination may be against the protein itself, the glycan, or both the protein and the linked glycan. Subunit vaccines have several advantages, including the absence of live pathogens, thus eliminating concerns about vaccine-induced patient infection, the ability to be designed using standard genetic engineering techniques, greater homogeneity than other forms of vaccines, and the ability to be produced in standardized recombinant protein expression and production systems using well-characterized expression systems. In some cases, antigens can be genetically engineered to favor the generation of desired antibodies, such as neutralizing or broadly neutralizing antibodies. In particular, structural information about the antigen of interest obtained by X-ray crystallography, electron microscopy, or nuclear magnetic resonance experiments can be used to guide the rational design of subunit vaccines.

[0188] A known limitation of subunit vaccines is that the immune response elicited may be weaker than that elicited by other types of vaccines, such as whole virus vaccines, live vaccines, or attenuated vaccines. The inventors have recognized and disclosed herein that designed VLP vaccines and / or protein-based VLP vaccines may be able to take advantage of the advantages of subunit vaccines while increasing the potency and breadth of vaccine-induced immune responses through multivalent presentation of antigens in a symmetrically ordered arrangement. In the present disclosure, protein-based VLPs are distinguished from nanoparticle vaccines, since the term nanoparticle vaccines is used in the art to refer to protein- or glycoprotein-based vaccines (see, e.g., U.S. Pat. No. 9,441,019), polymerized liposomes (see, e.g., U.S. Pat. No. 7,285,289), surfactant micelles (see, e.g., U.S. Pat. Publication No. US2004 / 0038406 A1), and synthetic biodegradable particles (see, e.g., U.S. Pat. No. US8,323,696).

[0189] The protein-based VLPs of the present disclosure are distinguishable from VLPs that present antigens on the surface of micellar particles containing detergents (e.g., NP-9), or alternatively, those made by extracting antigenic proteins from live viruses while preserving the lipid components of the viral envelope. In contrast, the protein-based VLPs described herein are free or substantially free of lipids and detergents. Furthermore, the symmetric presentation of the RSV and / or hMPV F proteins in some embodiments of the protein-based VLPs of the present disclosure may generate a superior immune response to the F proteins compared to other VLPs.

[0190] The present disclosure relates in part to combination vaccines for paramyxoviruses such as RSV and hMPV, e.g., bivalent (or multivalent) vaccines having two (or more) VLPs displaying paramyxovirus antigens, such as the ectodomain of the F protein of each paramyxovirus. In some variations, the VLPs are mosaic VLPs displaying multiple antigens. In further variations, the bivalent displays antigens from two strains of the same paramyxovirus (e.g., RSV-A and RSV-B, or hMPV-A and hMPV-B). In further variations, the multivalent vaccine includes VLPs displaying antigens from three or more paramyxoviruses, such as RSV-A, RSV-B, hMPV-A, and hMPV-B.

[0191] In some embodiments, the F protein(s) of each paramyxovirus(es) can be engineered to preferentially adopt a pre-fusion conformation. Thus, the present disclosure provides a pre-fusion stabilized hMPV F protein ectodomain and a pre-fusion stabilized RSV F protein ectodomain for use in VLPs.

[0192] Metapneumovirus virus-like particles Further provided is a protein-based virus-like particle (VLP) vaccine against human metapneumovirus, in which the hMPV F protein ectodomain is linked to and displayed on the protein-based VLP.

[0193] In one embodiment, the disclosure provides a virus-like particle (VLP) comprising a first component and optionally a second component, wherein the first component is a fusion protein comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof and a first multimerization domain, and the second component, if present, is a protein comprising a second multimerization domain.

[0194] In some embodiments, the first multimerization domain is a trimerization domain. In some embodiments, the multimerization domain is selected from SEQ ID NO: 1, 4, 5, 7, 9, 18, 19, 21, 24, 25, 26, 29, 30, 31, 34, 36, 37, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 144, 145, or functional variants thereof. In some embodiments, the multimerization domain is I53-50A (SEQ ID NO: 7 or SEQ ID NO: 144) or functional variants or variants thereof. In some embodiments, the first multimerization domain shares at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:7, SEQ ID NO:53, SEQ ID NO:144, or SEQ ID NO:145. In some embodiments, the first multimerization domain comprises amino acid substitutions C74A and C98A, amino acid substitutions C163A and C201A, or amino acid substitutions C74A, C98A, C163A, and C201A, relative to SEQ ID NO:144. In some embodiments, the first multimerization domain has a polypeptide sequence identical to SEQ ID NO:7 or SEQ ID NO:144. In some embodiments, the first multimerization domain has a polypeptide sequence identical to SEQ ID NO:53 or SEQ ID NO:145. In some embodiments, the first multimerization domain is a VLP-forming domain. In some embodiments, the first multimerization domain, optionally together with the second multimerization domain, is adapted to drive the assembly of icosahedral or tetrahedral particles.

[0195] In some embodiments, the second multimerization domain is selected from SEQ ID NO: 2, 3, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 20, 22, 23, 27, 28, 32, 33, 35, 38, 40, and 41, or functional variants and fragments thereof. In some embodiments, the second multimerization domain is I53-50B (SEQ ID NO: 8), I53-50B.4PosT1 (SEQ ID NO: 34), or functional variants thereof.

[0196] In some embodiments, the first and second components are linked by a linker sequence. In some embodiments, the linker sequence comprises a foldon, and the foldon sequence is EKAAKAEEAARK (SEQ ID NO: 125). In some embodiments, the linker sequence comprises GSGGSGSGSGGS (SEQ ID NO: 126).

[0197] In some embodiments, the hMPV F protein ectodomain comprises one or more substitutions or deletions selected from A185P, Q100R, S101R, T127C, N153C, V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G, R102G, A63C, K188C, K450C, S470C, G106 deletion, A113C, A120C, A339C, Q426C, T160F, Q100K, S101A, I177L, K450A, S470A, G294E, T365C, V463C, L219K, H368N, and / or V231I. In some embodiments, the hMPV F protein ectodomain comprises one or more substitutions or deletions selected from V84C, A140C, A147C, N97G, P98G, Q100G, S101G, R102G, A63C, K188C, K450C, S470C, R99G, A113C, A120C, A339C, Q426C, T160F, Q100K, S101A, Q100R, S101R, G106 deletion, S101R, A185P, I177L, and / or G294E.

[0198] In some embodiments, the hMPV F protein ectodomain comprises two or more substitutions or deletions selected from V84C, A140C, A147C, N97G, P98G, Q100G, S101G, R102G, A63C, K188C, K450C, S470C, R99G, A113C, A120C, A339C, Q426C, T160F, Q100K, S101A, Q100R, S101R, G106 deletion, S101R, A185P, I177L, and / or G294E. In some embodiments, the hMPV F protein ectodomain comprises one or more substitutions selected from A185P, Q100R, S101R, T127C, N153C, T365C, V463C, L219K, V231I, G294E, N153C, N97G, P98G, R99G, Q100G, S101G, H368N, and / or R102G.

[0199] In some embodiments, the hMPV F protein ectodomain comprises two or more of A185P, Q100R, S101R, T127C, N153C, T365C, V463C, L219K, V231I, G294E, N153C, N97G, P98G, R99G, Q100G, S101G, H368N, and / or R102G. The F protein ectodomain contains substitutions Q100R and S101R; substitutions A185P, Q100R, and S101R; substitutions A185P, T127C, N153C, Q100R, and S101R; substitutions V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G, and R102G; substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S 101G, and R102G; substitutions and deletions A63C, A140C, A147C, K188C, G106 deletion, N97G, P98G, R99G, Q100G, S101G, and R102G; substitutions A113C, A120C, A339C, Q426C, T160F, I177L, Q100K, and S101A; substitutions V84C, A140C, A147C, A249C, Q100R, and S101R; substitutions A63C, A140C, A147C, K188C, K450C , S470C, Q100R, and S101R; substitutions and deletions A63C, A140C, A147C, K188C, G106 deletion, Q100R, and S101R; substitutions A113C, A120C, A339C, Q426C, T160F, I177L, Q100R, and S101R; substitutions A185P, A113C, A339C, Q100R, and S101R; substitutions A185P, T160F, I177L, Q100R, and S101R; substitutions A185P, A113C, A339C, T160F, I177L, Q100R, and S101R; substitutions A63C, K188C, N97G, P98G, R99G, Q100G, S101G, and R102G; substitutions A63C, K188C, K450A, S470A, N97G, P98G, R99G, Q100G, S101G, and R102G; substitutions A63C, A140C, A147C, K188C, G294E, N97G, P98G, R99G, Q100G, S101G, and R102G;Substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, R102G, and G294E; Substitutions A63C, K188C, N97G, P98G, R99G, Q100G, S101G, R102G, and G294E; Substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, H368N, Q100R, and S101R; Substitutions A63C, A140C , A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, and R102G; substitutions V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G, and R102G; substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, H368N, N97G, P98G, R99G, Q100G, S101G, and R102G.

[0200] In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 58. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:59. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 60. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:61. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 62. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 63.In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:64.

[0201] In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 65. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:66. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 67. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 68. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 69. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 70.In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 71. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 72. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 73. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 74. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 75. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 76.In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 77. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 78. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 79. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:80. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 81. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:82.In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 83. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:84. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 85. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:86. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 87. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:88.In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 89. In some embodiments, the hMPV F protein ectodomain comprises a sequence that shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:90. In some embodiments, the hMPV F protein ectodomain shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 58, 60, 61, 77, 78, 86, 87, 88, or 89. In some embodiments, the hMPV F protein ectodomain shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 59, 62, 63, 64, 71, 76, 81, or 83. In some embodiments, the hMPV F protein ectodomain shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 72, 79, or 80. In some embodiments, the hMPV F protein ectodomain shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 65, 66, 82, 84, 85, 90, or 91. In some embodiments, the hMPV F protein ectodomain shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:65, 79, or 80.In some embodiments, the first component shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs: 92-124. In some embodiments, the first component shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NOs: 97-116, 119, or 122. In some embodiments, the first component shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 92-94, 96, 117, 118, 120, 121, 123, or 124. In some embodiments, the first component shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 99, 112, 117, or 118.

[0202] In some embodiments, the VLPs bind one or more hMPV F protein antibodies selected from MF1, MF2, MF3, MF9, MF12, MF14, MF15, MF11, MF16, MF20, MF17, MF18, MF19, MF10, or MPE8. In some embodiments, the VLPs bind two or more antibodies selected from MF1, MF2, MF3, MF9, MF12, MF14, MF15, MF11, MF16, MF20, MF17, MF18, MF19, MF10, MPE8. In some embodiments, the VLPs bind three or more antibodies selected from MF1, MF2, MF3, MF9, MF12, MF14, MF15, MF11, MF16, MF20, MF17, MF18, MF19, MF10, MPE8.

[0203] In some embodiments, the hMPV F protein ectodomain binds to an antibody that preferentially binds to the pre-fusion form of the hMPV F protein ectodomain. In some embodiments, the first component shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 42, 45, 58, 61, 63, or 64. In some embodiments, the hMPV F protein ectodomain has no or low binding affinity to an antibody that preferentially binds to the post-fusion form of the hMPV F protein ectodomain. In some embodiments, the VLP sequence shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 99, 115, 117, or 118.

[0204] In some embodiments, the first component shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:99.

[0205] In some embodiments, the first component is a single strand. In some embodiments, the single strand shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:123.

[0206] In some embodiments, the first component shares at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:117.

[0207] In one embodiment, the disclosure provides a polynucleotide encoding a VLP as described herein.

[0208] In one aspect, the disclosure provides a host cell comprising a polynucleotide described herein.

[0209] Respiratory syncytial virus virus-like particles In one aspect, provided herein is a pharmaceutical composition comprising a virus-like particle (VLP) comprising a first component comprising a RSV F protein and a first multimerization domain and a second component comprising a second multimerization domain, and one or more pharma- ceutically acceptable diluents or excipients. In some embodiments, the VLP is an icosahedral VLP. In some embodiments, the VLP comprises 20 copies of the first component and 12 copies of the second component.

[0210] In some embodiments, the RSV F protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 14, 34, and 35. In some embodiments, the first multimerization domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 24 and 30-31, and / or the second multimerization domain comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 22-23, 25-29, and 32. In some embodiments, the first component comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, and the second component comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:26.

[0211] Embodiment The present disclosure relates to protein-based VLPs and protein-based VLP vaccines. The VLPs of the present disclosure present antigens capable of eliciting an immune response against paramyxoviruses (e.g., RSV and hMPV). Some vaccines of the present disclosure are useful for preventing or reducing the severity of infection by paramyxoviruses (e.g., RSV and hMPV). In particular, the antigens of the present disclosure present the ectodomain of a paramyxovirus (e.g., RSV and hMPV) F protein. The ectodomain may be non-covalently or covalently linked to the core of the VLP, including as a fusion protein or by other means disclosed herein. In some embodiments, a linker connects the ectodomain to a first polypeptide that includes a multimerization domain. The linker may be any chemical linkage, including but not limited to a polypeptide used to form a fusion at the N-terminus or C-terminus of the ectodomain with the first polypeptide. The F protein of RSV and / or hMPV may optionally be presented along the axis of symmetry of the VLP. In some embodiments, the F protein is C-terminally linked to a first polypeptide comprising a trimerization domain and, optionally, an additional trimerization tag (e.g., a FoldOn tag). Also provided are proteins and nucleic acid molecules encoding such proteins, formulations, and methods of use.

[0212] Protein-based virus-like particles The present disclosure relates, in part, to vaccinating a subject with a protein-based virus-like particle (VLP) comprising a first component comprising an RSV F protein and a first multimerization domain in combination with a (VLP) comprising a first component comprising an hMPV F protein and a first multimerization domain. The protein complex may comprise two or more F proteins of RSV-A, RSV-B, hMPV-A and hMPV-B. The F protein portion and the first multimerization domain may be linked by any suitable means, including co-expression as a fusion protein. The protein complex may optionally comprise a second component comprising a second multimerization domain. Pharmaceutical compositions typically include one or more pharma- ceutical acceptable diluents or excipients.

[0213] The VLPs of the invention may comprise a multimeric protein assembly adapted for the presentation of the ectodomain of the F protein of RSV or hMPV, or an antigenic fragment thereof. The VLPs of the invention comprise at least a first plurality of polypeptides. The first plurality of polypeptides (also referred to as the "first component") may be derived from a naturally occurring protein sequence by substitution of at least one amino acid residue or by addition of one or more residues at the N-terminus or C-terminus. In some cases, the first component comprises a protein sequence determined by computational methods. This first component may form the entire core of the VLP. Alternatively, the core of the VLP may comprise one or more additional polypeptides (also referred to as the "second component", or third, fourth, fifth component, etc.), such that the VLP comprises 2, 3, 4, 5, 6, 7, or more polypeptides. In some cases, the first plurality forms trimers related by a three-fold rotational symmetry, and the second plurality forms pentamers related by a five-fold rotational symmetry. In such cases, the VLP forms an "icosahedral particle" with I53 symmetry. The one or more polypeptides can be arranged together such that the members of the various polypeptides are related to each other by symmetric operators. A general computational method for designing self-assembling protein materials, including symmetric docking of protein components in a target symmetric structure, is disclosed in US Patent Publication No. 2015 / 0356240 A1.

[0214] In some cases, the VLP is adapted to display F proteins from two or more different RSV strains. In a non-limiting example, the same VLP displays a mixed population of protein antigens or a mixed heterotrimer of protein antigens from different RSV strains. The sequences of F proteins of various RSV strains are known in the art. For example, see NCBI Accession Nos. QFX69124.1, QFX69112.1, APW78900.1, APW78889.1, APW78878.1, APW78867.1, APW78856.1, APW78845.1, APW78834.1, APW78823.1, APW78812.1, APW78801.1, APW78790.1, APW78779.1, APW787 See AAR14266.1, APW78680.1, APW78669.1, APW78658.1, APW78647.1, APW78636.1, APW78625.1, APW78614.1, and AAR14266.1.

[0215] The "core" of a VLP is used herein to describe the central portion of the VLP that links together several copies of the RSV and / or hMPV F protein ectodomain or an antigenic fragment thereof presented by the VLP. In one embodiment, the first component comprises a first polypeptide comprising an RSV and / or hMPV F protein ectodomain or an antigenic variant thereof, a linker, and a first polypeptide comprising a multimerization domain. Thus, the "antigen" is a single polypeptide that can self-assemble into a VLP, either alone or with a second component (such as a third, fourth, or fifth component). The advantage of designing the antigen itself to self-assemble is that the entire VLP thereby acts as the antigenic component of the vaccine. In other embodiments, the ectodomain or an antigenic fragment thereof is non-covalently or covalently linked to the first polypeptide comprising the multimerization domain. For example, an antibody or an antigenic fragment thereof can be fused to the first polypeptide and configured to bind to a portion of the first polypeptide or to a chemical tag on the first polypeptide. A streptavidin-biotin (or neuravidin-biotin) linker can be used. Alternatively, various chemical linkers can be used. The advantage of designing the core to be a universal platform is that one or more multiple polypeptides comprising the core can be designed and optimized in advance, and then these can be sequentially applied to the ectodomain to identify VLP(s) with superior efficacy as a vaccine. Of course, in some cases, the same polypeptide can form part of the "core" and then extend outward to include the ectodomain (i.e., a fusion protein) as either an adaptor for binding the F protein ectodomain of RSV and / or hMPV. In embodiments of the present disclosure, the antigen comprises additional polypeptide sequences in addition to the F protein ectodomain of RSV and / or hMPV. In certain embodiments, the ectodomain is glycosylated, either naturally or with alternative oligosaccharides (e.g., oligosaccharides specific to the host cell used to express the antigen).

[0216] In some cases, self-assembly can be further promoted by ectodomain multimerization, even though the core can independently self-assemble in the absence of the ectodomain. The F proteins of RSV and / or hMPV are found as trimers in the native state (Cseke et al. J. Virol. 21(2):698-707 (2007)). Presentation of the ectodomain on the VLP, at least in some embodiments, reduces the thermodynamic barrier to assembly and / or the equilibrium ratio of assembled and disassembled VLP components. In some cases, the trimeric ectodomain located along the three-fold axis of the VLP is properly trimerized in part because the ectodomain is presented on the three-fold axis of the VLP core, promoting proper folding and conformational stability of the ectodomain and making VLP self-assembly a cooperative process, in that the VLP is stabilized in the organized form, at least in part, by non-covalent or covalent interactions between the trimeric units. In some cases, especially when cysteine ​​residues are positioned to generate intramolecular disulfide bonds, the assembly can be optionally further stabilized by introducing mutations into the antigen or VLP components. In some examples, dimeric, trimeric, tetrameric, pentameric, or hexameric antigens are displayed on a core designed to have matching two-, three-, four-, five-, or six-fold symmetry axes, such that the core accommodates the arrangement of multimeric antigens with the antigen's natural symmetry.

[0217] Various Non-Limiting Examples of VLPs A non-limiting example of one embodiment is shown in Figure 1A. This figure shows an ectodomain antigen genetically fused to a component of a VLP (first polypeptide) optionally recombinantly expressed in a host cell (e.g., 293F or CHO cells), together with a pentameric protein assembly (second polypeptide) optionally recombinantly expressed in the same or a different host cell (e.g., E. coli cells), where the two polypeptides self-assemble into a VLP displaying 20 antigen trimers around an icosahedral core. In this embodiment, the core has a universal design. As described below, in other embodiments, the antigen of the present disclosure is mixed with another antigen protein, such as an ectodomain of a protein from a second virus, or a trimeric glycoprotein from another virus, in the same VLP. In some embodiments, the VLP comprises a trimeric glycoprotein of HIV-1, HIV-2, EBV, CMV, RSV, influenza, Ebola, Marburg, Dengue, SARS, MERS, Hanta, or Zika virus in addition to the F protein ectodomain of RSV and / or hMPV. In some embodiments, the VLP comprises a trimeric glycoprotein of a virus related evolutionarily or in sequence identity to any of these exemplary viruses, including but not limited to herpesvirus, orthomyxovirus, paramyxovirus, pneumovirus, filovirus, flavivirus, reovirus, or retrovirus. In one embodiment, the VLP comprises the extracellular domain(s) of a transmembrane protein or glycoprotein, or an antigenic fragment thereof. In some embodiments, the VLP is further linked to a polypeptide or other agent that can act as an adjuvant. In some embodiments, the antigen (first component) and / or the second component comprises one or more T cell epitopes, optionally including heterologous T cell epitopes.

[0218] Trimeric antigens that may be used with the VLPs of the present disclosure to form mixed VLPs are optionally, but not limited to, SARS-CoV-2, respiratory syncytial virus, HIV gp140, influenza HA, dengue E protein, or Ebola sGP. Mixed VLPs of the present disclosure include those that contain both the F protein ectodomain of RSV and / or hMPV and antigenic protein(s) from one or more of vesicular stomatitis virus, herpes simplex virus, baculovirus, thogotovirus, and Bornaviridae. If other trimeric antigens are used, they may optionally be positioned on the threefold symmetry axis of the VLP. In some cases, the selected antigens are monomeric and still positioned on the threefold axis. Thus, the VLP shown in FIG. 1A can present 20 trimeric antigens or 60 monomeric antigens. Additionally or alternatively, a pentameric complex of VLPs is used to present 12 pentameric antigens or 70 monomeric antigens. In one embodiment, the VLP comprises 20 copies of the trimeric antigen and 12 copies of the pentameric antigen.

[0219] VLP Core Other possible arrangements of the polypeptides of the present disclosure are shown in Figure IB. In some embodiments, the VLP is adapted for displaying up to 8 trimers; 8 trimers and 12 dimers; 6 tetramers and 12 dimers; 6 tetramers and 8 trimers; 20 trimers and 30 dimers; 4 trimers and 6 dimers; 4 first trimers and 4 second trimers, or 8 trimers; 12 pentamers and 20 trimers; or 12 pentamers and 30 dimers; or 4 trimers. In some cases, one of the symmetry axes is not used for antigen presentation, and thus in some embodiments, the VLP is adapted for displaying up to 8 trimers, 12 dimers, 6 tetramers, 20 trimers, 30 dimers, 4 trimers, 6 dimers, 8 trimers, or 12 pentamers. In some cases, monomeric antigens are displayed, and thus the VLP is adapted for displaying up to 12, 24, 60, or 70 monomeric antigens. In some cases, the VLP comprises multiple polypeptides mixed together, such that otherwise identical polypeptides in the core of the VLP display different antigens or no antigens. Thus, depending on the ratio of polypeptides, the VLP is adapted for displaying (e.g., on an I52 particle) of potentially 1 to 130 antigens, and each of the displayed antigens may be the same or different members of the mixed population in proportion to any ratio selected. These antigens may be co-expressed in a recombinant expression system and allowed to self-assemble prior to purification. Alternatively, the antigens may be expressed separately and then mixed together, either before or after purification from the expression host and associated contaminants. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more antigens are presented.Non-limiting examples of VLPs are provided in Bale et al. Science 353:389-94 (2016), Heinze et al. J. Phys. Chem B. 120:5945-5952 (2016), King et al. Nature 510:103-108 (2014), and King et al. Science 336:1171-71 (2012).

[0220] Mixed VLP In some cases, the VLP is adapted to present the same antigen from two or more different hMPV strains. In a non-limiting example, the same VLP presents a mixed population of homotrimeric protein antigens or mixed heterotrimers of protein antigens from different hMPV strains. In one embodiment, the VLP presents the F protein or antigenic fragments thereof disclosed in any of the sequences in GenBank found by searching the protein database with the keyword "human metapneumovirus F", either individually or in a mixed VLP.

[0221] When mixed VLPs are made, it may be advantageous to ensure the formation of homomultimers in a strain-specific manner rather than allowing heteromultimerization, for example, all strain 1 F proteins are displayed on one three-fold axis of the T33 particle, while all strain 2 F proteins are displayed on the other three-fold axis of the T33 particle. This can be achieved by using a VLP containing two or more types of multiple polypeptides as the core of the VLP to bind each type of multiple polypeptide to different antigens. Alternatively, the VLP may be engineered with one or more symmetry-breaking mutations, such as knob-in-hole mutations or intermolecular disulfide mutations, which have the effect of preventing trimerization between different antigens. In that case, the VLP will display multimeric antigens from different strains at symmetrically equal positions on the VLP, but each position on the VLP will be occupied by a homomultimer from the same strain, with only a small proportion of inter-strain heteromeric antigens. In some cases, the antigens themselves can be genetically engineered to prevent inter-strain heteromultimerization. In one embodiment, the VLP is engineered to prevent heteromultimerization of two structurally conserved but antigenically distinct antigenic proteins, such as the F protein of strain 1 and the F protein of strain 2, or the F protein of hMPV and a non-hMPV antigenic protein. Furthermore, when mixed VLPs are made and the antigens are presented as fusion proteins, the VLP will contain three or more different proteins, since the fusion proteins will share the same (or equivalent) domains used to form the core of the VLP, with one different antigenic domain for each antigen presented on the VLP.

[0222] Linker The VLPs of the present disclosure display antigenic proteins in a variety of ways, including genetic fusion or other means as disclosed herein. As used herein, "linked to" or "bound to" refers to any means known in the art for associating two polypeptides. The association can be direct or indirect, reversible or irreversible, weak or strong, covalent or non-covalent, and selective or non-selective.

[0223] In some embodiments, the binding is achieved by genetic engineering to create an N- or C-terminal fusion of the antigen with one of the polypeptides that constitute the VLP. Thus, the VLP can consist of or essentially consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides that present 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigens, where at least one antigen of the plurality is genetically fused to at least one polypeptide of the plurality. In some cases, the VLP consists essentially of one polypeptide, which can self-assemble and contains antigenic proteins genetically fused thereto. In some cases, the VLP consists essentially of a first polypeptide that contains antigens and a second polypeptide that can co-assemble into a two-component VLP, where one polypeptide links antigenic proteins to the VLP and the other polypeptide promotes self-assembly of the VLP.

[0224] In some embodiments, the binding is achieved by post-translational covalent bonds between one or more of the multiple polypeptides and one or more of the multiple antigenic proteins. In some cases, chemical cross-linking is used to non-specifically bind the antigen to the VLP polypeptide. In some cases, chemical cross-linking is used to specifically bind the antigenic protein to the VLP polypeptide (e.g., to the first polypeptide or the second polypeptide). A variety of specific and non-specific cross-linking chemistries are known in the art, such as click chemistry and other methods. Generally, any cross-linking chemistry used to link two proteins can be adapted for use in the VLPs disclosed herein. In particular, chemistries used to create immunoconjugates or antibody drug conjugates can be used. In some cases, the VLPs are created using cleavable or non-cleavable linkers. Processes and methods for conjugating antigens to carriers are provided, for example, by US Patent Publication No. 2008 / 0145373 A1.

[0225] In one embodiment, the binding is achieved by non-covalent binding between one or more of the multiple polypeptides and one or more of the multiple antigens. In some cases, the antigenic protein is engineered to be negatively charged on at least one surface, and the core polypeptide is engineered to be positively charged on at least one surface, or positively and negatively charged, respectively. This promotes intermolecular association between the antigenic protein and the core polypeptide by electrostatic forces. In some cases, shape complementarity is used to cause the linkage of the antigen protein to the core. The shape complementarity may be pre-existing or rationally designed. In some cases, computational design of the protein-protein interface is used to achieve the binding. In one embodiment, the antigen is biotinylated and the polypeptide comprises streptavidin, or vice versa. In one embodiment, the streptavidin is presented as a tetramer on the four-fold axis of the core, by genetic fusion or otherwise, and the biotinylated antigen is a monomer, dimer, or tetramer, allowing association to the core in a configuration appropriate for natural multimerization of the antigen. In some cases, a protein-based adaptor is used to capture the antigenic protein. In some cases, the polypeptide is fused to a protein that can bind to a complementary protein that is fused to the antigenic protein.

[0226] The immune response to RSV and / or hMPV F can be controlled by altering the positioning of the ectodomain relative to the core. Depending on how the antigenic protein is attached to the core of the VLP, the antigenic protein can be presented in various positions. In some embodiments, the antigenic protein is presented such that one or more known epitopes are positioned at or toward the distal end of the antigenic protein, making these epitope(s) preferentially accessible to the immune system. In some cases, the positioning recapitulates the positioning of the viral protein with respect to the virus. The choice of positioning can direct the immune system to one epitope or the other.

[0227] In some embodiments, epitope preference is controlled by other means, such as positioning of glycans on the VLP by addition or removal of the N-linked glycan sequence motif NX-[T / S] at defined positions within the amino acid sequence of any of the polypeptides of the VLP, including within the amino acid sequence of the antigenic protein.

[0228] In some cases, epitopes found at intermediate distances from the proximal to the distal end are preferred over more distally located epitopes, depending on a variety of considerations, including, but not limited to, the overall shape of the VLP, surface hydrophobicity, surface charge, and competitive binding of proteins endogenously present in the subject or exogenously provided in the vaccine composition. The present disclosure encompasses all known methods for the rational design of protein structures, and the foregoing is not intended to be limiting.

[0229] VLP Polypeptide Sequences The polypeptides of the present disclosure may have any of a variety of amino acid sequences. US Patent Publication No. 2015 / 0356240 A1 describes various methods for designing protein assemblers. As described in US Patent Publication No. US2016 / 0122392 A1 and International Patent Publication No. WO2014 / 124301 A1, isolated polypeptides of SEQ ID NO: 1-51 have been designed to be pairwise self-assembled to form VLPs, such as icosahedral particles. This design required the design of suitable interface residues for each member of the polypeptide pair that can be assembled to form a VLP. The VLP thus formed contains symmetrically repeated, non-natural, non-covalent inter-polypeptide interfaces that position the first and second assemblers into a VLP, such as one with icosahedral symmetry. Thus, in one embodiment, the first and second polypeptides (i.e., the two polypeptides of the core of the VLP) are selected from the group consisting of SEQ ID NO: 1-51. In either case, the sequences do not include the N-terminal methionine residue that is present in the full-length protein but that may be removed to create a fusion. The identified residues in Table 1 are numbered starting from the N-terminal methionine (not shown). In various embodiments, one or more additional residues are deleted from the N-terminus and / or additional residues are added to the N-terminus (e.g., to form a helical extension). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0230] Table 1 shows the amino acid sequences of the first and second polypeptides of embodiments of the present disclosure. In each case, the pair of sequences together form an I53 multimer with icosahedral symmetry. The right-most column of Table 1 identifies the residue numbers in each of the exemplary polypeptides identified as being present at the interface of the resulting assembled virus-like particle (i.e., "Identified Interface Residues"). As can be seen from the table, the number of interface residues in the exemplary polypeptides of SEQ ID NOs: 1-34 ranges from 4-13. In various embodiments, the first and second polypeptides comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 identified interface positions (depending on the number of interface residues in a given polypeptide) to an amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 1-34. SEQ ID NOs: 35-51 represent other amino acid sequences of the first and second polypeptides according to embodiments of the present disclosure. In other embodiments, the first polypeptide and / or the second polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length and at least 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NOs: 1-51.

[0231] As is true for proteins in general, polypeptides are expected to tolerate some variation in designed sequences that do not interfere with subsequent assembly into virus-like particles, particularly when such variations involve conservative amino acid substitutions. As used herein, "conservative amino acid substitutions" means that hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) may only be substituted with other hydrophobic amino acids, hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) may only be substituted with other hydrophobic amino acids with bulky side chains, amino acids with positively charged side chains (Arg, His, Lys) may only be substituted with other amino acids with positively charged side chains, amino acids with negatively charged side chains (Asp, Glu) may only be substituted with other amino acids with negatively charged side chains, and amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) may only be substituted with other amino acids with polar, uncharged side chains.

[0232] In some cases, for example, when it is desired to eliminate flexible portions of the secondary structure of the native RSV F protein, for example by the addition of cysteine ​​residues, non-conservative amino acid substitutions may be preferred (or vice versa). "Non-conservative substitution" refers to the substitution of one class of amino acid with another class of amino acid, for example, Ala with Asp, Asn, Glu, or Gln. Further non-limiting examples of non-conservative substitutions include the substitution of non-polar (hydrophobic) amino acid residues, such as isoleucine, valine, leucine, alanine, methionine, etc., with polar (hydrophilic) residues, such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of polar residues with non-polar residues. Substitution of D-Cys with D-Ala, D-Ser, or D-Tyr (or another residue) can be used to eliminate intramolecular disulfide bonds, which in some cases can improve protein stability or expression. Substitution with D-Cys can be used to create disulfide bonds to stabilize proteins or to lock proteins into desired conformations.

[0233] In various embodiments of the VLP of the invention, the first polypeptide and the second polypeptide (or vice versa) comprise polypeptides having an amino acid sequence selected from the following pairs or modified versions thereof (i.e., permissible modifications disclosed for the polypeptides of the invention: an isolated polypeptide comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length to the amino acid sequence set forth by a SEQ ID NO: and / or identical at at least one of the identified interface positions): SEQ ID NO: 1 and SEQ ID NO: 2 (I53-34A and I53-34B), SEQ ID NO: 3 and SEQ ID NO: 4 (I53-40A and I53-40B), SEQ ID NO: 3 and SEQ ID NO: 24 (I53-40A and I53-40B.1), SEQ ID NO: 23 and SEQ ID NO: 4 (I53-40A.1 and I53-40B), SEQ ID NO: 35 and SEQ ID NO: 36 (I53-40A and I53-40B genera); SEQ ID NO:5 and SEQ ID NO:6 (I53-47A and I53-47B), SEQ ID NO:5 and SEQ ID NO:27 (I53-47A and I53-47B.1), SEQ ID NO:5 and SEQ ID NO:28 (I53-47A and I53-47B.1NegT2), SEQ ID NO: 25 and SEQ ID NO: 6 (I53-47A.1 and I53-47B), SEQ ID NO: 25 and SEQ ID NO: 27 (I53-47A.1 and I53-47B.1), SEQ ID NO: 25 and SEQ ID NO: 28 (I53-47A.1 and I53-47B.1NegT2), SEQ ID NO: 26 and SEQ ID NO: 6 (I53-47A.1NegT2 and I53-47B), SEQ ID NO: 26 and SEQ ID NO: 27 (I53-47A.1NegT2 and I53-47B.1), SEQ ID NO: 26 and SEQ ID NO: 28 (I53-47A.1NegT2 and I53-47B.1NegT2), SEQ ID NO: 37 and SEQ ID NO: 38 (I53-47A and I53-47B genera); SEQ ID NO: 7 and SEQ ID NO: 8 (I53-50A and I53-50B), SEQ ID NO: 7 and SEQ ID NO: 32 (I53-50A and I53-50B.1), SEQ ID NO: 7 and SEQ ID NO: 33 (I53-50A and I53-50B.1NegT2), SEQ ID NO: 7 and SEQ ID NO: 34 (I53-50A and I53-50B.4PosT1), SEQ ID NO: 29 and SEQ ID NO: 8 (I53-50A.1 and I53-50B), SEQ ID NO: 29 and SEQ ID NO: 32 (I53-50A.1 and I53-50B.1), SEQ ID NO: 29 and SEQ ID NO: 33 (I53-50A.1 and I53-50B.1NegT2), SEQ ID NO: 29 and SEQ ID NO: 34 (I53-50A.1 and I53-50B.4PosT1), SEQ ID NO: 30 and SEQ ID NO: 8 (I53-50A.1NegT2 and I53-50B), SEQ ID NO: 30 and SEQ ID NO: 32 (I53-50A.1NegT2 and I53-50B.1), SEQ ID NO: 30 and SEQ ID NO: 33 (I53-50A.1NegT2 and I53-50B.1NegT2), SEQ ID NO: 30 and SEQ ID NO: 34 (I53-50A.1NegT2 and I53-50B.4PosT1), SEQ ID NO: 31 and SEQ ID NO: 8 (I53-50A.1PosT1 and I53-50B), SEQ ID NO: 31 and SEQ ID NO: 32 (I53-50A.1PosT1 and I53-50B.1), SEQ ID NO: 31 and SEQ ID NO: 33 (I53-50A.1PosT1 and I53-50B.1NegT2), SEQ ID NO: 31 and SEQ ID NO: 34 (I53-50A.1PosT1 and I53-50B.4PosT1), SEQ ID NO: 39 and SEQ ID NO: 40 (I53-50A and I53-50B genera); SEQ ID NO: 9 and SEQ ID NO: 10 (I53-51A and I53-51B), SEQ ID NO:11 and SEQ ID NO:12 (I52-03A and I52-03B), SEQ ID NO: 13 and SEQ ID NO: 14 (I52-32A and I52-32B), SEQ ID NO:15 and SEQ ID NO:16 (I52-33A and I52-33B) SEQ ID NO: 17 and SEQ ID NO: 18 (I32-06A and I32-06B), SEQ ID NO: 19 and SEQ ID NO: 20 (I32-19A and I32-19B), SEQ ID NO: 21 and SEQ ID NO: 22 (I32-28A and I32-28B), SEQ ID NO: 23 and SEQ ID NO: 24 (I53-40A.1 and I53-40B.1), SEQ ID NO: 41 and SEQ ID NO: 42 (T32-28A and T32-28B), SEQ ID NO: 43 and SEQ ID NO: 44 (T33-09A and T33-09B), SEQ ID NO: 45 and SEQ ID NO: 46 (T33-15A and T33-15B), SEQ ID NO: 47 and SEQ ID NO: 48 (T33-21A and T33-21B), SEQ ID NO: 49 and SEQ ID NO: 50 (T33-28A and T32-28B), and SEQ ID NO:51 and SEQ ID NO:44 (T33-31A and T33-09B (also referred to as T33-31B)).

[0234] In some embodiments, one or more RSV and / or hMPV F proteins, or antigenic fragments thereof, are expressed as a fusion protein with the first multimerization domain. In some embodiments, the first multimerization domain and the RSV and / or hMPV F protein ectodomain are linked by a linker sequence. In some embodiments, the linker sequence comprises a foldon, and the foldon sequence is EKAAKAEEAARK (SEQ ID NO: 125).

[0235] Non-limiting examples of designed protein complexes that are useful in the protein-based VLPs of the present disclosure include those disclosed in U.S. Pat. No. 9,630,994, International Patent Publication No. WO2018187325 A1, U.S. Patent Publication No. 2018 / 0137234 A1, and U.S. Patent Publication No. 2019 / 0155988 A2, each of which is incorporated herein by reference in its entirety.

[0236] In various embodiments of the VLPs of the present disclosure, the multimerization domain is a polypeptide having an amino acid sequence selected from the following pairs or modified versions thereof (i.e., permissible modifications as disclosed for the polypeptides of the present invention: an isolated polypeptide comprising an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over its entire length and / or identical at at least one of the identified interface positions to the amino acid sequence set forth by a SEQ ID NO): SEQ ID NO: 135 and SEQ ID NO: 136 (T33_dn2A and T33_dn2B), SEQ ID NO: 137 and SEQ ID NO: 138 (T33_dn5A and T33_dn5B), SEQ ID NO: 139 and SEQ ID NO: 140 (T33_dn10A and T33_dn10B), or SEQ ID NO: 141 and SEQ ID NO: 142 (I53_dn5A and I53_dn5B).

[0237] Antigenic Proteins The present disclosure provides protein-based VLP vaccines against RSV and / or hMPV in humans or animals (particularly pets, farm animals, and any animals associated with the spread of disease). The present disclosure relates to incorporating any antigenic fragment of the hMPV F protein (e.g., the ectodomain or an antigenic fragment thereof) into the VLP vaccine. Guidance is available, in particular, from studies of immune responses to infection or vaccination, such as isolation of binding or neutralizing antibodies, genetic analysis of the F protein sequence, structural studies of antigenic proteins and antibodies, and most particularly from clinical and veterinary experience with subunit vaccines. By using the above-mentioned presentation formats, subunit vaccines against RSV or hMPV can be adapted for use with the VLPs of the present disclosure. Although the present disclosure refers to the ectodomain of RSV or hMPV F, it is understood that a portion of the transmembrane domain may also be included.

[0238] The term "antigenic fragment" refers to any fragment of a protein that generates an immune response (humoral or T-cell response) against the protein in vivo. An antigenic fragment can be a linear epitope, a discontinuous epitope, or a conformational epitope (e.g., a folded domain). An antigenic fragment can preserve the secondary, tertiary, and / or quaternary structure of the full-length protein. In some embodiments, an antigenic fragment contains a neutralizing epitope. In such cases, the VLP can generate a neutralizing antibody response. An antigenic fragment can be computationally designed, such as by predicting the secondary structure and rationally removing N- or C-terminal unstructured regions or internal loops, or entire structural elements (alpha helices and / or beta sheets). Figure 2 provides an exemplary secondary structure map. In some embodiments, the ectodomain comprises a truncation or deletion of part or all of the N-terminal signal peptide (e.g., MSWKVVIIFSLLITPQHGL (SEQ ID NO: 133) or MSWKVMIIISLLITPQHGL (SEQ ID NO: 134).

[0239] In some embodiments, the hMPV F protein ectodomain is C-terminally truncated. In some embodiments, the hMPV F protein ectodomain sequence ends at residue 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, or 490 of the native sequence.

[0240] The native sequence of the hMPV F protein of the A strain (GenBank AY145297) is shown below, with the signal sequence underlined and italicized, and the transmembrane and intracellular portions underlined (SEQ ID NO:56). [ka]

[0241] The native signal sequence is post-translationally cleaved when the protein is expressed. The native signal sequence may be replaced with another signal sequence for expression of the ectodomain, or in some embodiments, no signal sequence is used. Thus, in some embodiments, the hMPV F protein ectodomain is SEQ ID NO: 57, or a variant thereof. [ka]

[0242] In some embodiments, the hMPV F protein ectodomain shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:57, or an antigenic fragment thereof.

[0243] Additional hMPV F protein sequences are shown in Table 2. In some embodiments, the hMPV F protein ectodomain shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an ectodomain in Table 2, or an antigenic fragment thereof.

[0244] Various signal sequences can be used, paired with native ectodomain sequences, or swapped between different hMPV sequences. Thus, in some embodiments, the antigen comprises a sequence that shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a signal sequence in Table 2.

[0245] The hMPV F protein can contain one or more substitutions.

[0246] Exemplary substitutions include A185P, Q100R, S101R, T127C, N153C, V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G, R102G, A63C, K188C, K450C, S470C, G106 deletion, A113C, A120C, A339C, Q426C, T160F, Q100K, S101A, I177L, K450A, S470A, G294E, T365C, V463C, L219K, H368N, and / or V231I relative to SEQ ID NO:57. Thus, in various embodiments, the hMPV F protein ectodomain comprises one or more substitutions selected from this list.

[0247] Exemplary substitutions include V84C, A140C, A147C, N97G, P98G, Q100G, S101G, R102G, A63C, K188C, K450C, S470C, R99G, A113C, A120C, A339C, Q426C, T160F, Q100K, S101A, Q100R, S101R, G106 deletion, S101R, A185P, I177L, and G294E relative to SEQ ID NO: 57. Thus, in various embodiments, the hMPV F protein ectodomain comprises one or more substitutions selected from this list.

[0248] Exemplary substitutions include V84C, A140C, A147C, N97G, P98G, Q100G, S101G, R102G, A63C, K188C, K450C, S470C, R99G, A113C, A120C, A339C, Q426C, T160F, Q100K, S101A, Q100R, S101R, G106 deletion, S101R, A185P, I177L, and G294E. Thus, in various embodiments, the hMPV F protein ectodomain comprises two or more substitutions selected from this list.

[0249] Exemplary substitutions include A185P, Q100R, S101R, T127C, N153C, T365C, V463C, L219K, V231I, G294E, N153C, N97G, P98G, R99G, Q100G, S101G, and R102G. Thus, in various embodiments, the hMPV F protein ectodomain comprises one or more substitutions selected from this list.

[0250] Exemplary substitutions include A185P, Q100R, S101R, T127C, N153C, T365C, V463C, L219K, V231I, G294E, N153C, N97G, P98G, R99G, Q100G, S101G, and R102G. Thus, in various embodiments, the hMPV F protein ectodomain comprises two or more substitutions selected from this list.

[0251] In some embodiments, the hMPV F protein ectodomain comprises substitutions Q100R and S101R. In some embodiments, the hMPV F protein ectodomain comprises substitutions A185P, Q100R, and S101R. In some embodiments, the hMPV F protein ectodomain comprises substitutions A185P, T127C, N153C, Q100R, and S101R. In some embodiments, the hMPV F protein ectodomain comprises substitutions V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein ectodomain comprises the substitutions and deletions A63C, A140C, A147C, K188C, G106 deletion, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A113C, A120C, A339C, Q426C, T160F, I177L, Q100K, and S101A. In some embodiments, the hMPV F protein ectodomain comprises the substitutions V84C, A140C, A147C, A249C, Q100R, and S101R. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A63C, A140C, A147C, K188C, K450C, S470C, Q100R, and S101R. In some embodiments, the hMPV F protein ectodomain comprises the substitutions and deletions A63C, A140C, A147C, K188C, G106 deletion, Q100R, and S101R. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A113C, A120C, A339C, Q426C, T160F, I177L, Q100R, and S101R. In some embodiments, the hMPV F protein ectodomain contains the substitutions A185P, A113C, A339C, Q100R, and S101R.In some embodiments, the hMPV F protein ectodomain comprises the substitutions A185P, T160F, I177L, Q100R, and S101R. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A185P, A113C, A339C, T160F, I177L, Q100R, and S101R. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A63C, K188C, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A63C, K188C, K450A, S470A, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A63C, A140C, A147C, K188C, G294E, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, R102G, and G294E. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A63C, K188C, N97G, P98G, R99G, Q100G, S101G, R102G, and G294E. In some embodiments, the hMPV F protein ectodomain comprises the substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, H368N, Q100R, and S101R. In some embodiments, the hMPV F protein ectodomain comprises the substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein ectodomain comprises the substitutions V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G, and R102G.In some embodiments, the hMPV F protein ectodomain contains the substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, N97G, P98G, R99G, Q100G, S101G, H368N, and R102G.

[0252] In some embodiments, the substitution has an intended effect on the VLP, although of course the intended effect is not achieved in all embodiments of the VLP having the substitution. In some embodiments, the intended effect is increased expression in a production cell line (Mas et al. PLoS Pathog. 12: e1005859 (2016)). In some embodiments, the intended effect is to stabilize the hMPV F protein ectodomain in post-fusion or pre-fusion form (Battles et al. Nat Commun. 8: 1528 (2017)). In some embodiments, the intended effect is to bind neutralizing antibodies against the hMPV F protein. The antigen may further comprise a leucine at the N-terminus, as set forth in SEQ ID NOs: 146-178. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12]

[0253] Thus, in some embodiments, the hMPV F protein ectodomain shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NOs:58-90, or an antigenic fragment thereof.

[0254] In some embodiments, the hMPV F protein contains one or more furin cleavage sites, and optionally one or more copies of an Arg-XX-Arg motif (SEQ ID NO:52), such as the native sequence RQSR (SEQ ID NO:54). When a furin cleavage site is present in the hMPV ectodomain, expression yields can be increased, in some cases, by co-expression of furin or a functional variant thereof from a transfected plasmid or from a stably integrated polynucleotide sequence in the host cell. In some embodiments, the furin cleavage site of the hMPV F protein is modified by mutating the RQSR (SEQ ID NO:54) motif to RRRR (SEQ ID NO:55) or is removed by substituting a Gly linker or making other amino acid substitutions.

[0255] hMPV neutralizing activity is mediated by antibodies that recognize both pre- and post-fusion F protein conformations, and antibodies generated in an in vivo immunological response may selectively recognize pre- or post-fusion specific sites, or may recognize both pre- and post-fusion forms of the F protein. Battles et al. Nat Commun. 8:1528 (2017). DS7, MPV196, MPV201, and MPV314 neutralizing antibodies bind to antigenic sites accessible in both pre- and post-fusion forms of the F protein, which are located on the DI and DII head domains of the F protein. Wen et al. Nat Struct Mol Biol. 19(4):461-463 (2012), Bar-Peled et al. J Virol. 93:e00342-19 (2019)). MPE8 neutralizing antibodies bind epitopes on the F protein spanning the DI, DII, and DIII subunits, making contacts that are dependent on the prefusion F conformation (Wen et al. Nat Microbiol. 2:16272 (2017)). Several antigenic epitopes are shared with the respiratory syncytial virus (RSV) F protein, such as site III and site IV epitopes (Huang et al. Front Immunol. 10:2778 (2019)). These are examples and are not a complete list of epitopes reported for neutralizing antibodies against the hMPV F protein.

[0256] Neutralizing epitopes are generally conformational epitopes, so peptides corresponding to the epitopes do not bind to antibodies and therefore do not induce neutralizing titers. Neutralizing antibodies can be isolated from healthy donors who are seropositive for hMPV (Battles et al.) or generated by immunization in animal models (Gabriella et al. J Virol. 81(2):698-707(2007)).

[0257] In some embodiments, the VLPs of the present disclosure comprising an hMPV F protein ectodomain can bind to neutralizing antibodies against hMPV F protein, also referred to as anti-hMPV F protein antibodies. In some embodiments, the VLPs described herein can bind to anti-hMPV F protein antibodies known to selectively bind to the pre-fusion form of the hMPV F protein. Examples of antibodies that selectively bind to the pre-fusion form of the hMPV F protein include, but are not limited to, the MF10 and MPE8 antibodies. In some embodiments, the VLPs described herein can bind to anti-hMPV F protein antibodies known to selectively bind to the post-fusion form of the hMPV F protein. Examples of antibodies that selectively bind to the post-fusion form of the hMPV F protein include, but are not limited to, the MF1, MF2, MF3, MF11, MF17, MF18, and MF19 antibodies. In some embodiments, the VLPs described herein can bind to antibodies that bind to both the pre-fusion and post-fusion forms of the hMPV F protein. Examples of antibodies that bind to pre-fusion and post-fusion forms of the hMPV F protein include, but are not limited to, MF9, MF12, MF14, MF15, MF16, and MF20 antibodies. In some embodiments, the VLPs described herein bind to one or more anti-hMPV F protein antibodies. In some embodiments, the VLPs described herein bind to two or more anti-hMPV F protein antibodies.

[0258] Multimerization Domains and Linkers In some embodiments, the VLP comprises a trimeric assembly of antigens, comprising a first polypeptide comprising a first multimerization domain and a first polypeptide comprising a second multimerization domain. The first multimerization domain comprises a protein-protein interface that induces three copies of the first polypeptide to self-associate to form a trimeric component. In a VLP with two or more components, each copy of the first multimerization domain further comprises a surface-exposed interface that interacts with a complementary surface-exposed interface on the second multimerization domain. Similarly described, the second multimerization domain is adapted to multimerize with the first multimerization domain (of the first polypeptide of the first component). As described by King et al. (Nature 510,103-108,2014), Bale et al. (Science 353,389-394,2016), and patent publications WO2014124301A1 and US20160122392A1, the complementary protein-protein interface between the first and second multimerization domains drives the assembly of multiple copies of the trimer assembly domain and the second assembly domain into a target VLP. In some embodiments, each copy of the trimer assembly domain of a VLP has an antigenic protein or antigenic fragment thereof linked thereto (e.g., as a gene fusion), and these VLPs display the full valency of the protein. In other embodiments, the VLPs of the present disclosure contain one or more copies of the first multimerization domain with an antigenic protein or antigenic fragment thereof (e.g., as a genetic fusion), as well as one or more first multimerization domains without an antigenic protein, and these VLPs display partial valency G proteins. The first multimerization domain can be any polypeptide sequence that forms a trimer and interacts with the second multimerization domain to drive assembly into a target VLP. In some embodiments, the VLP comprises a first polypeptide and a second polypeptide selected from those disclosed in US20130274441A1, US2015 / 0356240A1, US2016 / 0122392A1, WO2018 / 187325A1, each of which is incorporated herein by reference in its entirety.

[0259] In some embodiments of the VLP of the present disclosure, the antigenic protein and the core of the VLP may be genetically fused such that they are both present in a single polypeptide (also referred to as a single chain polypeptide). The binding between the protein and the core allows the antigenic protein or antigenic fragment thereof to be presented on the outside of the VLP. Thus, the connection point to the core must be on the outside of the core of the formed virus-like particle. A wide variety of polypeptide sequences may be used to link the protein or antigenic fragment thereof to the core of the virus-like particle. In some cases, the linker comprises the polypeptide sequence. Any suitable linker polypeptide may be used. In some embodiments, the linker imposes a strict relative positioning of the antigenic protein (e.g., ectodomain) or antigenic fragment thereof with respect to the core. In some embodiments, the linker allows for flexible linking of the antigenic protein (e.g., ectodomain) or antigenic fragment thereof with respect to the core. In some embodiments, the linker comprises an additional trimerization domain (e.g., the foldon domain of T4 fibritin) to help stabilize the trimeric form of the F protein, e.g., GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 91) or a functional variant thereof.

[0260] In some embodiments, the linker may comprise a Gly-Ser linker of any suitable length (i.e., a linker comprised of glycine and serine residues). In some embodiments, the Gly-Ser linker may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In some embodiments, the Gly-Ser linker may comprise or consist of the amino acid sequence GSGGSGSGSGGSGSG (SEQ ID NO: 127), GGSGGSGS (SEQ ID NO: 128), or GSGGSGSG (SEQ ID NO: 129). In some embodiments, the linker comprises the sequence GSGSGSGSG (SEQ ID NO: 130). In some embodiments, the linker comprises the sequence GSGSGSGSGSGSGSSG (SEQ ID NO: 131). In some embodiments, the linker comprises the sequence GSGGSGSGSGGS (SEQ ID NO: 126). In some embodiments of the VLPs of the present disclosure, the first component may optionally include a poly-His tag, HHHHHH (SEQ ID NO: 143). Tables 3A and 3B show illustrative examples of VLP sequences described herein. These sequences include a multimerization domain, an RSV or hMPV F protein ectodomain, a linker, a polyHis tag, and a signal sequence. The signal and polyHis tag sequences are underlined and are optionally included in the sequence. [Table 3A-1] [Table 3A-2] [Table 3A-3] [Table 3A-4] [Table 3A-5] [Table 3A-6] [Table 3A-7] [Table 3A-8] [Table 3A-9] [Table 3A-10] [Table 3A-11] [Table 3A-12] [Table 3A-13] [Table 3A-14] [Table 3A-15]

[0261] In some embodiments, the RSV protein ectodomain comprises S46G, K66E, I76V, K77E, K80E, N88C, E92C, E92D, Q98C, A149C, E161G, E161P, E161Q, S173P, N175P, S182P, G184N, V185N, K201Q, L203I, K209Q, S215P, S238C, 254C, Q279C, Q361C, K421N, N426S, N428C, K465E, K465Q, Y458C, K508E substitutions, or any combination thereof. In some embodiments, the RSV protein ectodomain comprises substitutions of Q98C and Q361C, A149C and Y458C, N183GC and N428C, N88C and N254C, E92C and N254C, and / or S238C and Q279C, or any combination thereof. [Table 3B-1]

Table 3B-2

Table 3B-3

Table 3B-4

Table 3B-5

Table 3B-6

Table 3B-7

Table 3B-8

Table 3B-9

[0262] Organization of VLP In some embodiments, a single component self-assembles into a VLP. In some embodiments, one or more purified samples of the first and second components for use in forming a VLP are mixed in an approximately equimolar molar ratio under aqueous conditions (e.g., I53-50A / B icosahedral VLP). The first and second components interact with each other (through the multimerization domain, and optionally through the ectodomain) to drive the assembly of the target VLP. The successful assembly of the target VLP can be confirmed by analyzing the in vitro assembly reaction by common biochemical or biophysical methods used to examine the physical size of proteins or protein assemblies, including, but not limited to, size exclusion chromatography, native (non-denaturing) gel electrophoresis, dynamic light scattering, multi-angle light scattering, analytical ultracentrifugation, negative staining electron microscopy, cryo-electron microscopy, or X-ray crystallography. If necessary, the assembled VLPs can be purified from other species or molecules present during the in vitro assembly reaction using preparative techniques commonly used to isolate proteins by their physical size, including but not limited to size exclusion chromatography, preparative ultracentrifugation, tangential flow filtration, or preparative gel electrophoresis. The presence of antigenic proteins in the VLPs can be examined by techniques commonly used to determine the identity of protein molecules in aqueous solutions, including but not limited to SDS-PAGE, mass spectrometry, protein sequencing, ELISA, surface plasmon resonance, biolayer interferometry, or amino acid analysis. The accessibility of the protein outside the particle, and its conformation or antigenicity, can be examined by techniques commonly used to detect the presence and conformation of antigens, including but not limited to binding by monoclonal antibodies, conformation-specific monoclonal antibodies, surface plasmon resonance, biolayer interferometry, or antisera specific for the antigen.

[0263] In various embodiments, the VLPs of the present disclosure comprise two or more separate first polypeptides with different antigenic proteins as gene fusions. These VLPs co-present multiple different proteins on the same VLP. These multi-antigen VLPs are produced by in vitro assembly with a mixture of two or more antigens, each of which contains a multimerization domain. The proportion of each antigen in the mixture determines the average valency of each antigenic protein in the resulting VLP. The presence and average valency of each antigen in a given sample can be examined by quantitative analysis using the techniques described above to assess the presence of antigenic proteins in full valency VLPs.

[0264] In various embodiments, the VLPs are about 20 nanometers (nm) to about 40 nm in diameter, the lumen is about 15 nm to about 32 nm in diameter, and the pore size within the protein shell is about 1 nm to about 14 nm in its longest dimension.

[0265] In some embodiments, the VLP has icosahedral symmetry. In such an embodiment, the VLP may comprise 60 copies of the first component and 60 copies of the second component. In one such embodiment, the number of identical first polypeptides in each first assembly is different from the number of identical first polypeptides in each second assembly. For example, in some embodiments, the VLP comprises 12 first assembly and 20 second assembly, in such an embodiment, each first assembly may comprise, for example, 5 copies of the same first component, and each second assembly may comprise, for example, 3 copies of the same second component. In some embodiments, the VLP comprises 12 first assembly and 30 second assembly, in such an embodiment, each first assembly may comprise, for example, 5 copies of the same first component, and each second assembly may comprise, for example, 2 copies of the same second component. In a further embodiment, the VLP comprises 20 first assemblers and 30 second assemblers, where each first assembler may, for example, comprise 3 copies of the same first component, and each second assembler may, for example, comprise 2 copies of the same second component, all of which are capable of forming protein-based VLPs with icosahedral symmetry.

[0266] In various further embodiments, the oligomeric state of the first and second multimerization domains is as follows: I53-34A: trimer + I53-34B: pentamer, I53-40A: pentamer + I53-40B: trimer, I53-47A: trimer + I53-47B: pentamer, I53-50A: trimer + I53-50B: pentamer, I53-51A: trimer + I53-51B: pentamer, I32-06A: dimer + I32-06B: trimer, I32-19A: trimer + I32-19B: dimer, I32-28A: trimer + I32-28B: dimer, I52-03A: pentamer + I52-03B: dimer, I52-32A: dimer + I52-32B: pentamer, and I52-33A: Pentamer + I52-33B: Dimer.

[0267] In some embodiments, the second multimerization domain of the second polypeptide comprises a sequence that shares at least 95% identity with I53-50A or a variant thereof. [ka]

[0268] In some embodiments, the second multimerization domain shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:7 or SEQ ID NO:144, or an antigenic fragment thereof.

[0269] The I53-50A protein sequence has two intramonomer disulfide bonds. In some embodiments, the cysteine ​​residues are mutated to residues that do not contain thiol groups (e.g., alanine or serine). Removal of the thiol groups can promote correct protein folding without impairing multimerization. In some embodiments, the multimerization domain of the first polypeptide comprises amino acid substitutions at one or more of positions 74, 98, 163, and 201 relative to SEQ ID NO: 144, as shown herein. [ka]

[0270] In some embodiments, the multimerization domain of the first polypeptide comprises one or more amino acid substitutions of C74A, C98A, C163A, and C201A compared to SEQ ID NO: 132. In some embodiments, the multimerization domain of the first polypeptide comprises SEQ ID NO: 132 or a variant thereof. [ka]

[0271] In some embodiments, the multimerization domain shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 144 or SEQ ID NO: 132, or an antigenic fragment thereof, and comprises one, two, three or four amino acid substitutions selected from C74A, C98A, C163A, and C201A. Alternatively, the substitutions can be from C to any amino acid other than A, T, S, L, I, or C.

[0272] nucleic acid In another aspect, the present disclosure provides isolated nucleic acids encoding the antigen, first component, and / or second component of the present disclosure. The isolated nucleic acid sequence may comprise RNA or DNA. As used herein, an "isolated nucleic acid" is a nucleic acid that is removed from its normal surrounding nucleic acid sequence in a genome or cDNA sequence. Such isolated nucleic acid sequences may include additional sequences useful for facilitating expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, transport signals, and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it will be clear to one of skill in the art which nucleic acid sequences encode the proteins of the present disclosure.

[0273] In a further aspect, the present disclosure provides a recombinant expression vector comprising an isolated nucleic acid of any embodiment or combination of embodiments of the present disclosure operably linked to a suitable control sequence. A "recombinant expression vector" includes a vector that operably links a nucleic acid coding region or gene to any control sequence that can effect expression of the gene product. A "control sequence" operably linked to a nucleic acid sequence of the present disclosure is a nucleic acid sequence that can effect expression of a nucleic acid molecule. Control sequences need not be contiguous with a nucleic acid sequence, so long as they function to induce expression of the nucleic acid sequence. Thus, for example, intervening sequences that are not translated but are transcribed may be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence may still be considered to be "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including, but not limited to, plasmids and viral-based expression vectors. The control sequences used to drive expression of the nucleic acid sequences of the present disclosure in mammalian systems can be constitutive (driven by any of a variety of promoters, including but not limited to CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters, including but not limited to tetracycline, ecdysone, steroid responsive). The construction of expression vectors for use in transfection of prokaryotic cells is also well known in the art and can thus be accomplished via standard techniques.(See, e.g., Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, NJ; and Ambion 1998 Catalog (Ambion, Austin, TX).) Expression vectors must be replicable in the host organism either as episomes or by integration into the host chromosomal DNA. In preferred embodiments, the expression vector comprises a plasmid. However, the present disclosure is intended to include other expression vectors that serve equivalent functions, such as viral vectors.

[0274] In another aspect, the disclosure provides a host cell transfected or transduced with a recombinant expression vector disclosed herein, which may be either a prokaryotic or eukaryotic cell. The cell may be transiently or stably transfected or transduced. Such transfection or transduction of the expression vector into prokaryotic and eukaryotic cells may be accomplished by any technique known in the art, including but not limited to standard bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome-mediated, DEAE-dextran-mediated, polycation-mediated, or viral-mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (RI Freshney. 1987. Liss, Inc. New York, NY)).

[0275] In another aspect, the disclosure provides a method of producing an antigen, component, or VLP according to the disclosure, hi some embodiments, the method comprises (a) culturing a host according to this aspect of the disclosure under conditions conducive to expression of the polypeptide, and (b) optionally recovering the expressed polypeptide.

[0276] In some embodiments, the disclosure provides a method of producing a vaccine comprising culturing a host cell comprising a polynucleotide comprising a sequence encoding an antigen of the disclosure in a culture medium such that the host cell secretes the antigen into the culture medium; optionally purifying the antigen from the culture medium; mixing the antigen with a second component, where the second component multimerizes with the antigen to form a VLP; and optionally purifying the VLP.

[0277] In some embodiments, the disclosure provides a method of producing a vaccine comprising culturing a host cell comprising one or more polynucleotides comprising sequences encoding both components of any one of the VLPs of the disclosure, such that the host cell secretes the first component and the second component into the culture medium; and optionally purifying the VLP from the culture medium.

[0278] Exemplary host cells include E. coli cells, 293 and 293F cells, HEK293 cells, Sf9 cells, Chinese Hamster Ovary (CHO) cells, as well as any other cell line used for the production of recombinant proteins.

[0279] In various embodiments, the first component is expressed at about 0.5 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, about 2.5 mg / mL, about 5 mg / mL, about 10 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, or more in a manufacturing method according to the present disclosure (e.g., 293F cells grown in suspension). In various embodiments, the first component expresses at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% expression levels of the RSV and / or hMPV F protein (optionally the same ectodomain as that of the VLP) in the same or a similar expression system. In various embodiments, the first component expresses an expression level of at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 150%, at least 175%, or at least 200% of the RSV and / or hMPV F protein (optionally the same ectodomain as that of the VLP) in the same or a similar expression system.

[0280] In some embodiments, the RSV and / or hMPV F protein ectodomain of the first component is in a prefusion conformation, or a substantial proportion of the RSV and / or hMPV F protein ectodomain is in a prefusion conformation. In various embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the RSV and / or hMPV F protein ectodomain of the first component is in a prefusion conformation. In some embodiments, the RSV and / or hMPV F protein ectodomain of the VLP is in a prefusion conformation, or a substantial proportion of the RSV and / or hMPV F protein ectodomain is in a prefusion conformation. In various embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the RSV and / or hMPV F protein ectodomain of the VLP is in a prefusion conformation.

[0281] In some embodiments, the percentage of RSV and / or hMPV F protein ectodomain in the prefusion state is determined by binding to conformation-specific antibodies (e.g., D1-25 and 1112-1). In some embodiments, the percentage of hMPV F protein ectodomain in the first component in the prefusion conformation is at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 150%, at least 175%, or at least 200% higher than the percentage in the reference component or the percentage in the reference protein not linked to a multimerization domain. In some embodiments, the percentage of hMPV F protein ectodomain in the first component in the prefusion conformation is at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 150%, at least 175%, or at least 200% higher than the percentage in a VLP (e.g., a micellar VLP).

[0282] Vaccines and Administration The present disclosure also provides a vaccine comprising the VLPs described herein. Such compositions can be used to generate antibodies in mammals (e.g., humans). Vaccine compositions of the present disclosure typically include a pharma- ceutically acceptable carrier, a detailed description of which is available in Remington: The Science and Practice of Pharmacy.

[0283] The pH of the composition is usually about 4.5 to about 11, for example, about 5 to about 11, about 5.5 to about 11, about 6 to about 11, about 5 to about 10.5, about 5.5 to about 10.5, about 6 to about 10.5, about 5 to about 10, about 5.5 to about 10, about 6 to about 10, about 5 to about 9.5, about 5.5 to about 9.5, about 6 to about 9.5, about 5 to about 9, about 5.5 to about 9, about 6 to about 9, about 5 to about 8.5, about 5.5 to about 8.5, about 6 to about 8.5, about 5 to about 8, about 5.5 to about 8, about 6 to about 8, about 4.5, about 5, about 6.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, etc. A stable pH may be maintained through the use of a buffer such as a Tris buffer, a citrate buffer, a phosphate buffer, or a histidine buffer. Thus, the composition generally includes a buffer.

[0284] In some embodiments, the pH of the formulation is about pH 6.2 to about pH 8.0. In some embodiments, the pH is about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, or about 8.0. Of course, the pH may be within a range of values. Thus, in some embodiments, the pH is about 6.2 to about 8.0, about 6.2 to 7.8, about 6.2 to 7.6, about 6.2 to 7.4, about 6.2 to 7.2, about 6.2 to 7.0, about 6.2 to 6.8, about 6.2 to about 6.6, or about 6.2 to 6.4. In other embodiments, the pH is 6.4 to about 8.0, about 6.4 to 7.8, about 6.4 to 7.6, about 6.4 to 7.4, about 6.4 to 7.2, about 6.4 to 7.0, about 6.4 to 6.8, or about 6.4 to about 6.6. In still other embodiments, the pH is about 6.6 to about 8.0, about 6.6 to 7.8, about 6.6 to 7.6, about 6.6 to 7.4, about 6.6 to 7.2, about 6.6 to 7.0, or about 6.6 to 6.8. In still other embodiments, the pH is about 6.8 to about 8.0, about 6.8 to 7.8, about 6.8 to 7.6, about 6.8 to 7.4, about 6.8 to 7.2, or about 6.8 to 7.0. In still other embodiments, it is about 7.0 to about 8.0, about 7.0 to 7.8, about 7.0 to 7.6, about 7.0 to 7.4, about 7.0 to 7.2, about 7.2 to 8.0, about 7.2 to 7.8, about 7.2 to about 7.6, about 7.2 to 7.4, about 7.4 to about 8.0, about 7.4 to about 7.6, or about 7.6 to about 8.0.

[0285] In some embodiments, the formulation may include one or more salts, such as sodium chloride, sodium phosphate, or a combination thereof. Generally, each salt is present in the formulation at about 10 mM to about 200 mM. Thus, in some embodiments, any salt present is present at about 10 mM to about 200 mM, about 20 mM to about 200 mM, about 25 mM to about 200 mM, about 30 mM to about 200 mM, about 40 mM to about 200 mM, about 50 mM to about 200 mM, about 75 mM to about 200 mM, about 100 mM to about 200 mM, about 125 mM to about 200 mM, about 150 mM to about 200 mM, or about 175 mM to about 200 mM. In other embodiments, any salt present is present at about 10 mM to about 175 mM, about 20 mM to about 175 mM, about 25 mM to about 175 mM, about 30 mM to about 175 mM, about 40 mM to about 175 mM, about 50 mM to about 175 mM, about 75 mM to about 175 mM, about 100 mM to about 175 mM, about 125 mM to about 175 mM, or about 150 mM to about 175 mM. In still other embodiments, any salt present is present at about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 25 mM to about 150 mM, about 30 mM to about 150 mM, about 40 mM to about 150 mM, about 50 mM to about 150 mM, about 75 mM to about 150 mM, about 100 mM to about 150 mM, or about 125 mM to about 150 mM. In still other embodiments, any salts present are present at about 10 mM to about 125 mM, about 20 mM to about 125 mM, about 25 mM to about 125 mM, about 30 mM to about 125 mM, about 40 mM to about 125 mM, about 50 mM to about 125 mM, about 75 mM to about 125 mM, or about 100 mM to about 125 mM. In some embodiments, any salts present are present at about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 25 mM to about 100 mM, about 30 mM to about 100 mM, about 40 mM to about 100 mM, about 50 mM to about 100 mM, or about 75 mM to about 100 mM. In still other embodiments, any salt present is present at about 10 mM to about 75 mM, about 20 mM to about 75 mM, about 25 mM to about 75 mM, about 30 mM to about 75 mM, about 40 mM to about 75 mM, or about 50 mM to about 75 mM.In still other embodiments, any salts present are present at about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 30 mM to about 50 mM, or about 40 mM to about 50 mM. In other embodiments, any salts present are present at about 10 mM to about 40 mM, about 20 mM to about 40 mM, about 25 mM to about 40 mM, about 30 mM to about 40 mM, about 10 mM to about 30 mM, about 20 mM to about 30, about 25 mM to about 30 mM, about 10 mM to about 25 mM, about 20 mM to about 25 mM, or about 10 mM to about 20 mM. In some embodiments, sodium chloride is present in the formulation at about 100 mM. In some embodiments, sodium phosphate is present in the formulation at about 25 mM.

[0286] The composition may be sterile and / or pyrogen-free. The composition may be isotonic with respect to human standards.

[0287] The vaccine composition may include an immunological adjuvant. Exemplary adjuvants include: mineral-containing compositions, oil emulsions, squalene emulsions, saponin formulations, virosomes and virus-like particles, bacterial or microbial derivatives, bioadhesives and mucoadhesives, liposomes, polyoxyethylene ether and polyoxyethylene ester formulations, polyphosphazenes (PCPPs), muramyl peptides, imidazoquinolone compounds, thiosemicarbazone compounds, tryptanthrin compounds, human immunomodulators, lipopeptides, benzonaphthyridines, microparticles, immunostimulatory polynucleotides (such as RNA or DNA; e.g., CPG-containing oligonucleotides).

[0288] For example, the composition may include an aluminum salt adjuvant, an oil-in-water emulsion (e.g., an oil-in-water emulsion with squalene, such as MF59® or AS03), a TLR7 agonist (such as imidazoquinoline or imiquimod), or a combination thereof. Suitable aluminum salts include hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates) (see, e.g., Vaccine Design. (1995) eds. Powell & Newman, Chapters 8 and 9, ISBN: 030644867X. Plenum), or mixtures thereof. The salt may be in any suitable form (e.g., gel, crystalline, amorphous, etc.), one example being adsorption of an antigen to the salt. The Al salt in the composition for administration to the patient may be used in combination with an adsorbent such as a phosphate salt. +++ The concentration may be less than 5 mg / ml, e.g., <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. A preferred range is 0.3-1 mg / ml. A maximum of 0.85 mg / dose is preferred. Aluminum hydroxide and aluminum phosphate adjuvants are suitable for use in the present disclosure.

[0289] Exemplary adjuvants that may be used in the pharmaceutical compositions provided herein include 3M-052, Adju-Phos™, Alhydrogel™, Adjumer™, Albumin-Heparin Microparticles, Algal Glucan, Algammulin, Alum, Antigen Preparations, AS-2 Adjuvant, ASO1, ASO3, Autologous Dendritic Cells, Autologous PBMCs, Avridine™, B7-2, BAK, BAY R1005, BECC TLR-4 agonists, bupivacaine, bupivacaine-HCl, BWZL, calcitriol, calcium phosphate gel, CCR5 peptide, CFA, cholera holotoxin (CT) and cholera toxin B subunit (CTB), cholera toxin A1-subunit-protein AD fragment fusion protein, CpG, CPG-1018, CPG-1018 with aluminum salt, CRL1005, cytokine-containing liposomes, D-murapalmitin, DDA, DHEA, diphtheria toxoid, DL-PGL, DMPC, DMPG, DOC / alum complex, fowlpox, Freund's complete adjuvant, gamma inulin, Gerbu adjuvant, GM-CSF, GMDP, hGM-CSF, hIL-12 (N222L), hTNF-alpha, IFA, IFN-gamma in pcDNA3, IL-12 DNA, IL-12 plasmid, IL-12 / GMCSF plasmid (Sykes), IL-2 in pcDNA3, IL-2 / Ig plasmid, IL-2 / Ig protein, IL-4, IL-4 in pcDNA3, Imiquimod™, ImmTher™, immunoliposomes containing antibodies to costimulatory molecules, interferon-gamma, interleukin-1 beta, interleukin-12, interleukin-2, interleukin-7, ISCOM(s)™, Iscoprep 7.0.3™, keyhole limpet hemocyanin, lipid-based adjuvants, liposomes, loxoribine, LT(R192G), LT-OA or LT oral adjuvant, LT-R192G, LTK63, LTK72, Matrix-M™ adjuvant, MF59, MONTANIDE ISA 51, MONTANIDE ISA 720, M.P.L.(trademark), MPL-SE, MTP-PE, MTP-PE liposomes, Murametide, Murapalmitin, NAGO, nCT native cholera toxin, non-ionic surfactant vesicles, non-toxic mutant E112K of cholera toxin mCT-E112K, p-hydroxybenzoic acid methyl ester, pCIL-10, pCIL12, pCMVmCAT1, pCMVN, Peptomer-NP, Pleuran, PLG, PLGA, PGA, and PLA, Pluronic® L121, PMMA, PODDS™, Poly rA:Poly rU, Polysorbate 80, Protein Cochleate, QS-21, Quadri A saponin, Quil-A, Rehydragel HPA, Rehydragel These include, but are not limited to, LV, RIBI, Ribi-like adjuvant systems (MPL, TMD, CWS), S-28463, SAF-1, Sclavo peptides, Sendai proteoliposomes, Sendai-containing lipid matrix, Span 85, Specol, squalane 1, squalene 2, stearyl tyrosine, SWE, tetanus toxoid (TT), Theramide™, threonyl muramyl dipeptide (TMDP), Ty particles, and Walter Reed liposomes.

[0290] In a preferred embodiment, the adjuvant is aluminum hydroxide gel (e.g., Alhydrogel™). In a preferred embodiment, the adjuvant is SWE. In a preferred embodiment, the adjuvant is MF59.

[0291] MF59 is an oil-in-water emulsion containing squalene (4.3%) in a citrate buffer with the stabilizing non-ionic surfactants Tween 80 (0.5%) and Span 85 (0.5%). MF59 has been shown to be well tolerated in humans and has been used in vaccines against seasonal influenza (see Ko and Kang, Hum Vaccin Immunother. 2018;14(12):3041-3045; U.S. Patent No. 6,299,884).

[0292] For example, the composition may include an aluminum salt adjuvant, an oil-in-water emulsion (e.g., an oil-in-water emulsion with squalene, such as MF59, SWE, or AS03), a TLR4 agonist, a TLR9 agonist (such as a CpG oligodeoxynucleotide), a TLR7 agonist (such as an imidazoquinoline or imiquimod), or a combination thereof. In some embodiments, the adjuvant is a combination of an aluminum salt and CPG1018. Suitable aluminum salts include hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates) (see, e.g., Chapters 8 and 9 of Vaccine Design. (1995) eds. Powell & Newman, ISBN: 030644867X. Plenum), or mixtures thereof. The salt may be in any suitable form (e.g., gel, crystalline, amorphous, etc.), one example being adsorption of an antigen to the salt. The concentration of Al+++ in the composition for administration to a patient can be less than 5 mg / ml, e.g., <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. A preferred range is 0.3-1 mg / ml. A maximum of 0.85 mg / dose is preferred. Aluminum hydroxide and aluminum phosphate adjuvants are suitable for use in the present disclosure. In preferred embodiments, the pharmaceutical compositions provided herein include aluminum hydroxide as an adjuvant. In some embodiments, the pharmaceutical compositions provided herein include 500 μg of aluminum hydroxide.

[0293] In one aspect, the present disclosure provides a vaccine comprising the VLP described herein, which optionally comprises one or more pharma- ceutically acceptable diluents, adjuvants, or excipients. In some embodiments, the vaccine is a stable emulsion. In some embodiments, the vaccine comprises one or more adjuvants. In some embodiments, the one or more adjuvants are squalene, SLA, GLA, R848, IMQ, 3M-052, CpG, saponin (QS21), or combinations thereof. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is a squalene-based emulsion. In some embodiments, the squalene-based emulsion is MF59. The choice of adjuvant depends on the subject to be treated. Preferably, a pharma-ceutically acceptable adjuvant is used.

[0294] In some embodiments, the adjuvant is a squalene emulsion.

[0295] In some embodiments, the adjuvant is a TLR4 immunostimulant (e.g., SLA, GLA), e.g., as described in Van Hoeven at al. PLoS One. 11(2):e0149610 (2016).

[0296] In some embodiments, the adjuvant is a TLR7 / 8 immunostimulant (e.g., R848, IMQ, 3M-052), e.g., as described in Dowling D. ImmunoHorizons(6):185-197(2018).

[0297] In some embodiments, the adjuvant is a TLR9 immunostimulant (CpG), e.g., as described in Bode et al. Expert Rev Vaccines. 10(4):499-511 (2011).

[0298] In some embodiments, the adjuvant is a saponin (QS21), e.g., as described in Zhu et al. Nat Prod Chem Res. 3(4):e113 (2016).

[0299] In some embodiments, the vaccine comprises a combination of two or more adjuvants (eg, a squalene emulsion and alum or a TLR4 immunostimulant).

[0300] One suitable immunological adjuvant comprises a compound of formula (I) as defined in WO2011 / 027222, or a pharma- ceutically acceptable salt thereof, adsorbed to an aluminium salt. Many further adjuvants can be used, including those disclosed in Powell & Newman (1995).

[0301] The compositions may contain antibacterial agents, particularly when packaged in a multi-dose format. Antibacterial agents such as thiomersal and 2-phenoxyethanol are commonly found in vaccines, although sometimes it may be desirable to use mercury-free preservatives, or no preservatives at all.

[0302] The composition may include a surfactant, such as a polysorbate, for example polysorbate 80. The surfactant is generally present at low levels, for example less than 0.01%.

[0303] The composition may include a sodium salt (e.g., sodium chloride) to impart tonicity. A concentration of 10±2 mg / ml, e.g., about 9 mg / ml NaCl is typical.

[0304] In some embodiments, the buffer in the vaccine composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer, or an acetate buffer. The composition may include a lyoprotectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the composition includes a preservative, such as, for example, benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition includes a bulking agent, such as glycine. In yet other embodiments, the composition comprises a surfactant, such as, for example, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, poloxamer 188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or combinations thereof. The composition may also comprise a tonicity adjuster, e.g., a compound that renders the formulation substantially isotonic or isoosmolar with human blood. Exemplary tonicity adjusters include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the composition further comprises a stabilizer, e.g., a molecule that substantially prevents or reduces chemical and / or physical instability of the VLP in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0305] In some embodiments, the present disclosure provides vaccines (immunogenic compositions) comprising one or more pharma- ceutically acceptable excipients.

[0306] In some embodiments, the vaccine (immunogenic composition) is a stable emulsion.

[0307] In some embodiments, the disclosure provides vaccines (immunogenic compositions) that include one or more adjuvants. In some embodiments, the one or more adjuvants include a TLR4 immunostimulant, such as monophosphoryl lipid A (MPL), glucopyranosyl lipid A (GLA), and / or a soluble Leishmania antigen (SLA).

[0308] In another aspect, the disclosure provides a method for inducing an immune response or enhancing an existing immune response against RSV and / or hMPV, comprising administering to a subject in need thereof an immunologically effective amount of an immunogenic composition described herein comprising a VLP described herein.

[0309] In certain embodiments, the immune response comprises the production of neutralizing antibodies to the infectious agent, hi certain embodiments, the neutralizing antibodies are complement-independent.

[0310] The immune response may include a humoral immune response, a cellular immune response, or both. In some embodiments, an immune response is induced against each antigenic protein delivered. The cellular immune response may include a helper T cell (Th) response, a CD8+ cytotoxic T cell (CTL) response, or both. In some embodiments, the immune response includes a humoral immune response and the antibodies are neutralizing antibodies. Neutralizing antibodies block viral infection of cells. The virus also infects epithelial cells and fibroblasts. In some embodiments, the immune response reduces or prevents infection of both cell types. The neutralizing antibody response may be complement-dependent or complement-independent. In some embodiments, the neutralizing antibody response is complement-independent. In some embodiments, the neutralizing antibody response is cross-neutralizing, i.e., antibodies generated against the administered composition neutralize viruses of strains other than the strain used in the composition.

[0311] A useful measure of antibody potency in the art is the "50% neutralization titer". To determine the 50% neutralization titer, serum from immunized animals is diluted to determine how much the diluted serum can retain the ability to block 50% of the virus from entering cells. For example, a titer of 700 means that the serum retains the ability to neutralize 50% of the virus after being diluted 700 times. Thus, a higher titer indicates a stronger neutralizing antibody response. In some embodiments, the titer is within a range with a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000. The range of 50% neutralization titers is about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 8000, about 9000, about 10000, about 11000, about 12000. , about 13000, about 14000, about 15000, about 16000, about 17000, about 18000, about 19000, about 20000, about 21000, about 22000, about 23000, about 24000, about 25000, about 26000, about 27000, about 28000, about 29000, or about 30000. For example, the 50% neutralization titer may be about 3000 to about 25000. "About" means the stated value ±10%.

[0312] In some embodiments, the virus-like particles of the present disclosure are 0.5×10 3 IU / mL, 1.0x10 3 IU / mL, 1.5x10 3 IU / mL, 2.0x10 3 IU / mL, 3.0x10 3 IU / mL, 4.0x10 3 IU / mL, 5.0x10 3 IU / mL, 6.0x10 3 IU / mL, 7.0x10 3 IU / mL, 8.0x10 3 IU / mL, 9.0x10 3IU / mL, 10x10 3 IU / mL, 25x10 3 IU / mL, 50x10 3 IU / mL, 100x10 3 In some embodiments, the virus-like particles of the present disclosure generate an immune response of 0.5×10 IU / mL or more. 3 Generate an immune response of IU / mL or more.

[0313] The composition of the present disclosure is generally administered directly to a subject.Direct delivery can be achieved by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, or into the interstitial space of tissue), orally, intranasally, or by any other suitable route.For example, intramuscular administration can be used, for example, in the thigh or upper arm.Injection can be performed by needle (e.g., hypodermic needle), but needle-free injection can be used instead.A typical intramuscular dose is 0.5ml.

[0314] Dosage may be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or a booster immunization schedule. In a multiple dose schedule, various doses may be given by the same or different routes, e.g., parenteral prime and transmucosal boost, transmucosal prime and parenteral boost, etc. Multiple doses are typically administered at least one week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). Multiple doses may be administered at least one month apart (e.g., about 2 months, about 3 months, about 4 months, about 6 months, about 8 months, about 10 months, about 12 months, about 16 months, etc.). A second or subsequent dose may be administered at longer intervals, e.g., about 3 to 5 years after the preceding dose, about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years after the preceding dose.

[0315] The subject may be a child (e.g., a toddler or infant), a teenager, or an adult. A pediatric vaccine may be administered to an adult, e.g., to assess safety, dosage, immunogenicity, etc.

[0316] Vaccines of the present disclosure may be prophylactic (i.e., prevent disease) or therapeutic (i.e., reduce or eliminate symptoms of disease). The term prophylactic may be considered as reducing the severity of a particular condition or preventing its onset. For the avoidance of doubt, the term prophylactic vaccine may also refer to a vaccine that ameliorates the effects of such infection, for example, by reducing the severity or duration of future infection.

[0317] The isolated and / or purified VLPs described herein may be administered alone or as either a prime or a boost in a mixed modality regimen of RNA prime or DNA primer followed by a protein boost. For example, an adenoviral vector may be used as a prime in combination with the vaccine composition of the present disclosure. The advantages of the RNA prime / protein boost strategy compared to the protein prime / protein boost strategy include, for example, increased antibody titers, a more balanced IgG1:IgG2a subtype profile, induction of a TH1-type CD4+ T cell-mediated immune response similar to that of viral particles, and reduced production of non-neutralizing antibodies. RNA prime may increase the immunogenicity of a composition, regardless of whether the composition contains an adjuvant or not.

[0318] In the RNA prime / protein boost strategy, RNA and protein are directed to the same target antigen. Examples of suitable modes of RNA delivery include viral-like replicon particles (VRP), alphavirus RNA, replicon encapsulated in lipid nanoparticles (LNP), or formulated RNA, such as replicon formulated with cationic nanoemulsion (CNE). Suitable cationic oil-in-water nanoemulsions are disclosed in WO2012 / 006380, which include, for example, an oil core (e.g., squalene) and cationic lipids (e.g., DOTAP, DMTAP, DSTAP, DC-cholesterol, etc.).

[0319] Alternatively, two doses of VLPs may be administered at a predefined interval to achieve a prime-boost effect. The predefined interval may be 1, 2, 3, 4, 6, 7, 10, or 14 days, or 3-5 days, 7-10 days, or 10-14 days, 14-21 days, etc. The predefined interval may be 1, 2, 3, 4, or 6 weeks, or 2-3 weeks, 3-4 weeks, or 5-6 weeks, etc. The predefined interval may be 1, 2, 3, or 4 months, or 1, 2, 3, or 4 years.

[0320] In some embodiments, the RNA molecule is encapsulated within, bound to, or adsorbed onto cationic lipids, liposomes, cochleates, virosomes, immune stimulating complexes, microparticles, microspheres, nanospheres, unilamellar vesicles, multilamellar vesicles, oil-in-water emulsions, water-in-oil emulsions, emulsomes, polycationic peptides, cationic nanoemulsions, or combinations thereof.

[0321] The present disclosure further provides combination vaccines, including vaccines that contain both RSV and / or hMPV VLPs and vaccines against one or more of coronaviruses (e.g., betacoronaviruses, e.g., SARS-CoV-2), respiratory syncytial virus, rabies, pneumococcal virus, typhoid, hepatitis A, polio, influenza, hepatitis B, yellow fever, Japanese encephalitis, parvovirus, distemper, adenovirus, parainfluenza, influenza, measles, Lyme disease, coronaviruses, vesicular stomatitis virus, herpes simplex virus, baculovirus, thogotovirus, and bornaviridae.

[0322] Also provided herein are kits and instructions for administering the nucleic acids (e.g., RNA), purified proteins, and purified VLPs described herein. The disclosure also provides delivery devices pre-filled with the compositions or vaccines disclosed herein.

[0323] The pharmaceutical compositions described herein can be administered in combination with one or more additional therapeutic agents, such as antiviral agents, such as palivizumab.Additional therapeutic agents can include, but are not limited to, antibiotics or antibacterial agents, antiemetics, antifungals, anti-inflammatory agents, antiviral agents, immunomodulators, cytokines, antidepressants, hormones, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, topoisomerase inhibitors, cytostatic agents, anti-invasive agents, antiangiogenic agents, growth factor function inhibitors, viral replication inhibitors, viral enzyme inhibitors, anticancer agents, α-interferon, β-interferon, ribavirin, hormones, and other toll-like receptor modulators, immunoglobulins (Ig), and antibodies that modulate Ig function, such as anti-IgE (omalizumab).

[0324] In certain embodiments, the compositions disclosed herein may be used as pharmaceuticals, e.g., for use in inducing or enhancing an immune response in a subject, e.g., a mammal, in need thereof.

[0325] In certain embodiments, the compositions disclosed herein may be used in the manufacture of a medicament for inducing or enhancing an immune response in a subject, e.g., a mammal, in need thereof.

[0326] One way to confirm the effectiveness of therapeutic treatment includes monitoring infection by an infectious agent after administration of a composition or vaccine disclosed herein. One way to confirm the effectiveness of prophylactic treatment includes monitoring the immune response to an antigen systemically (e.g., monitoring the level of IgG1 and IgG2a production) and / or mucosally (e.g., monitoring the level of IgA production). Typically, antigen-specific serum antibody responses are determined after immunization but before challenge, whereas antigen-specific mucosal antibody responses are determined after immunization and after challenge.

[0327] Treatment Method In one aspect, the present disclosure provides a method of immunizing a subject against infection by human metapneumovirus (hMPV), comprising administering a vaccine as described herein. In some embodiments, the subject is simultaneously immunized against infection by respiratory syncytial virus (RSV). In some embodiments, the vaccine is administered by subcutaneous injection. In some embodiments, the vaccine is administered by intramuscular injection. In some embodiments, the vaccine is administered by intradermal injection. In some embodiments, the vaccine is administered intranasally. In one aspect, the present disclosure provides a pre-filled syringe comprising the vaccine as described herein. In one aspect, the present disclosure provides a kit comprising the vaccine as described herein or the pre-filled syringe as described herein.

[0328] In another aspect, provided herein is a unit dose of a pharmaceutical composition comprising about 0.5 μg to about 1 μg, about 20 μg to about 25 μg, about 70 μg to about 75 μg, about 100 μg to about 125 μg, about 100 μg to about 150 μg, about 125 μg to about 175 μg, about 200 μg to about 250 μg, about 225 μg to about 300 μg, or about 250 μg to about 350 μg of VLPs.

[0329] In another aspect, provided herein is a unit dose of a pharmaceutical composition comprising about 0.5 μg to about 1 μg, about 20 μg to about 25 μg, about 25 μg to about 50 μg, about 50 μg to about 70 μg, about 70 μg to about 75 μg, about 75 μg to about 100 μg, about 100 μg to about 125 μg, about 125 μg to about 150 μg, about 150 μg to about 175 μg, about 175 μg to about 200 μg, about 200 μg to about 250 μg, or about 250 μg to about 300 μg of VLPs.

[0330] In another aspect, provided herein is a method of vaccinating a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition provided herein. In another aspect, provided herein is a method of generating an immune response in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition provided herein. In another aspect, provided herein is a method of preventing RSV disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition provided herein. In some embodiments, the subject is at risk for severe RSV disease. In another aspect, provided herein is a method of preventing hMPV disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition provided herein. In some embodiments, the subject is at risk for severe hMPV disease. In some embodiments, the subject is an adult over 60 years of age. In some embodiments, the subject is a healthy adult between 18 and 45 years of age.

[0331] In some embodiments, a composition comprising 25 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 125 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof is administered intramuscularly to a subject. In some embodiments, a composition comprising 25 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 125 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof and 9.75 mg of an oil-in-water emulsion comprising squalene, such as MF59®, is administered intramuscularly to a subject.

[0332] In some embodiments, a composition comprising 75 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 75 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof is administered intramuscularly to a subject. In some embodiments, a composition comprising 75 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 75 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof and 9.75 mg of an oil-in-water emulsion comprising squalene, such as MF59®, is administered intramuscularly to a subject.

[0333] In some embodiments, a composition comprising 75 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 150 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof is administered intramuscularly to a subject. In some embodiments, a composition comprising 75 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 150 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof and 9.75 mg of an oil-in-water emulsion comprising squalene, such as MF59®, is administered intramuscularly to a subject.

[0334] In some embodiments, a composition comprising 75 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 225 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof is administered intramuscularly to a subject. In some embodiments, a composition comprising 75 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, 225 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof, and 9.75 mg of an oil-in-water emulsion comprising squalene, such as MF59®, is administered intramuscularly to a subject.

[0335] In some embodiments, a composition comprising 112.5 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 112.5 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof is administered intramuscularly to a subject. In some embodiments, a composition comprising 112.5 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 112.5 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof and 9.75 mg of an oil-in-water emulsion comprising squalene, such as MF59®, is administered intramuscularly to a subject.

[0336] In some embodiments, a composition comprising 150 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 150 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof is administered intramuscularly to a subject. In some embodiments, a composition comprising 150 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 150 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof and 9.75 mg of an oil-in-water emulsion comprising squalene, such as MF59®, is administered intramuscularly to a subject.

[0337] In some embodiments, a composition comprising 225 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 150 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof is administered intramuscularly to a subject. In some embodiments, a composition comprising 225 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 150 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof and 9.75 mg of an oil-in-water emulsion comprising squalene, such as MF59®, is administered intramuscularly to a subject.

[0338] In some embodiments, a composition comprising 150 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 75 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof is administered intramuscularly to a subject. In some embodiments, a composition comprising 150 μg of a first VLP comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof and 75 μg of a second VLP comprising a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof and 9.75 mg of an oil-in-water emulsion comprising squalene, such as MF59®, is administered intramuscularly to a subject.

[0339] In another aspect, provided herein are methods of generating an immune response in a fetus, the methods comprising administering to the mother of the fetus an effective amount of a pharmaceutical composition provided herein, in some embodiments, the pharmaceutical composition is administered to the mother during the last trimester of pregnancy.

[0340] In some embodiments, a first dose of the pharmaceutical composition is administered to a pregnant woman, and a second dose of the composition is administered to an infant born from the pregnancy.Non-limiting examples of generating an immune response in an infant include those disclosed in International Patent Publication No. WO2012103361 A1, which is incorporated herein by reference in its entirety.

[0341] In some embodiments, an effective amount of the pharmaceutical composition comprises about 0.5 μg to about 1 μg, about 20 μg to about 25 μg, about 70 μg to about 75 μg, about 100 μg to about 125 μg, about 100 μg to about 150 μg, about 125 μg to about 175 μg, about 200 μg to about 250 μg, about 225 μg to about 300 μg, or about 250 μg to about 350 μg of VLPs.

[0342] In some embodiments, an effective amount of the pharmaceutical composition comprises about 0.5 μg to about 1 μg, about 20 μg to about 25 μg, about 25 μg to about 50 μg, about 50 μg to about 70 μg, about 70 μg to about 75 μg, about 75 μg to about 100 μg, about 100 μg to about 125 μg, about 125 μg to about 150 μg, about 150 μg to about 175 μg, about 175 μg to about 200 μg, or about 200 μg to about 250 μg, or about 250 μg to about 300 μg of VLPs.

[0343] In some embodiments, the method provided herein further comprises administering a second dose of the pharmaceutical composition provided herein. In some embodiments, the second dose is administered within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, or about 12 months after the first dose. In some embodiments, the method provided herein further comprises administering a third dose of the pharmaceutical composition provided herein. In some embodiments, the third dose is administered about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years after the second dose. In some embodiments, the method provided herein further comprises administering a subsequent dose at regular intervals of about 1, 2, 3, 4, or 5 years.

[0344] In some embodiments, the methods provided herein limit the onset of RSV infection in a subject. In some embodiments, the methods result in the production of RSV-A specific neutralizing antibodies in a subject. In some embodiments, the methods result in an increase in RSV-A specific neutralizing antibodies in a subject of at least about 2-fold, about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in RSV-A specific neutralizing antibodies is detectable within about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after administration of the pharmaceutical composition. In some embodiments, the methods result in the production of RSV-B specific neutralizing antibodies in a subject. In some embodiments, the method results in an increase in RSV-B specific neutralizing antibodies in a subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in RSV-B specific neutralizing antibodies is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition.

[0345] In some embodiments, the method results in the production of RSV F protein-specific IgG antibodies in the subject. In some embodiments, the method results in an increase in RSV F protein-specific neutralizing antibodies in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in RSV F protein-specific neutralizing antibodies is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition.

[0346] In some embodiments, the method results in the production of core VLP-specific IgG antibodies in the subject. In some embodiments, the method results in an increase in core VLP-specific IgG antibodies in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in core VLP-specific IgG antibodies is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition.

[0347] In some embodiments, the method results in the production of RSV F protein-specific memory B cells in a subject. In some embodiments, the method results in an increase in RSV F protein-specific memory B cells in a subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in RSV F protein-specific memory B cells is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition.

[0348] In some embodiments, the method results in the production of RSV F protein-specific T cells in the subject. In some embodiments, the method results in an increase in RSV F protein-specific T cells in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in RSV F protein-specific T cells is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition.

[0349] In some embodiments, the method results in the production of antibodies against human metapneumovirus in the subject. In some embodiments, the method results in an increase in antibodies against human metapneumovirus (hMPV) in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in antibodies against human metapneumovirus is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition.

[0350] In some embodiments, the methods provided herein limit the onset of hMPV infection in a subject. In some embodiments, the methods result in the production of hMPV-A specific neutralizing antibodies in a subject. In some embodiments, the methods result in an increase in hMPV-A specific neutralizing antibodies in a subject of at least about 2-fold, about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in hMPV-A specific neutralizing antibodies is detectable within about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks of administration of the pharmaceutical composition. In some embodiments, the methods result in the production of hMPV-B specific neutralizing antibodies in a subject. In some embodiments, the method results in an increase in hMPV-B specific neutralizing antibodies in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in hMPV-B specific neutralizing antibodies is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition.

[0351] In some embodiments, the method results in the production of hMPV F protein-specific IgG antibodies in the subject. In some embodiments, the method results in an increase in hMPV F protein-specific neutralizing antibodies in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in hMPV F protein-specific neutralizing antibodies is detectable within about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after administration of the pharmaceutical composition.

[0352] In some embodiments, the method results in the production of core VLP-specific IgG antibodies in the subject. In some embodiments, the method results in an increase in core VLP-specific IgG antibodies in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in core VLP-specific IgG antibodies is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition.

[0353] In some embodiments, the method results in the production of hMPV F protein-specific memory B cells in the subject. In some embodiments, the method results in an increase in hMPV F protein-specific memory B cells in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in hMPV F protein-specific memory B cells is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition.

[0354] In some embodiments, the method results in the production of hMPV F protein-specific T cells in the subject. In some embodiments, the method results in an increase in hMPV F protein-specific T cells in the subject of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. In some embodiments, the increase in hMPV F protein-specific T cells is detectable within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, within about 6 weeks, within about 7 weeks, within about 8 weeks, within about 9 weeks, within about 10 weeks, within about 11 weeks, or within about 12 weeks of administration of the pharmaceutical composition. In another aspect, the disclosure provides a method of vaccinating a subject against infection with RSV (e.g., infection with RSV-A and / or RSV-B) and / or infection with hMPV (e.g., infection with hMPV-A and / or hMPV-B), comprising administering to a subject in need thereof a polypeptide, virus-like particle, composition, nucleic acid, pharmaceutical composition, or vaccine (referred to as an "immunogenic composition") of any of the embodiments herein in an amount effective to treat or limit the onset of the infection. In some embodiments, such methods prevent disease following infection with RSV subtypes A and / or B. In some embodiments, such methods protect against the onset of RSV-associated disease (e.g., severe disease), e.g., pneumonia and / or acute respiratory disease. The subject can be any suitable mammalian subject, including, but not limited to, a human subject. In some embodiments, the subject is a human child, e.g., a child under 12 months of age. In some embodiments, the subject is a human infant, e.g., about 1 to about 3 years of age, or about 1 to about 5 years of age. In some embodiments, the subject is an adult over 50 or 60 years of age. In some embodiments, the subject is an adult over 65 years of age. In certain embodiments, the subject is dependent on the assistance of others or has serious health concerns or risks (e.g., frail elderly). In some embodiments, the subject is a healthy adult between 18 and 60 years of age. In some embodiments, the subject is a healthy adult between 18 and 45 years of age.In another embodiment, the subject is a pregnant woman. In some embodiments, the subject is an immunocompromised adult. In some embodiments, the subject is an adult with underlying chronic cardiac and / or pulmonary disease or dysfunction. In some embodiments, the subject is at risk for severe RSV disease (e.g., LRTI or pneumonia).

[0355] The immunogenic compositions provided herein can be used for vaccination of a fetus. During pregnancy, administration of certain inactivated vaccines, such as tetanus toxoid, attenuated diphtheria toxoid, and acellular pertussis (Tdap) vaccine and influenza vaccine, is recommended to induce immunity in the fetus. Thus, in some embodiments, provided herein is a method of generating an immune response in a fetus, comprising administering an effective amount of an immunogenic composition provided herein to the mother of the fetus. The immunogenic composition can be administered at any suitable time during pregnancy, for example, in the last trimester of pregnancy.

[0356] The immunogenic compositions provided herein may be administered in combination with other treatments, such as other vaccines. Thus, in some embodiments, a subject treated according to the methods provided herein may also be administered one or more seasonal or pandemic vaccines, such as an influenza vaccine or a SARS-Cov2 vaccine. In some embodiments, a subject treated according to the methods provided herein may also be administered a pneumococcal vaccine, a recombinant Shingles vaccine, or a Tdap vaccine. One, two, or more vaccines may be administered in combination with the immunogenic compositions provided herein. "Concomitant administration" includes both simultaneous and subsequent administration. For example, one, two, or more vaccines and the immunogenic compositions provided herein may be administered on the same day. In some embodiments, one, two, or more vaccines and the immunogenic compositions provided herein are administered within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, or 12 hours of each other.

[0357] In another aspect, provided herein is a method of treating a subject suffering from RSV or hMPV infection. As used herein, "treat" or "treating" includes, but is not limited to, achieving one or more of: (a) reducing viral titer in a subject; (b) limiting the increase in viral titer in a subject; (c) reducing the severity of viral infection; (d) limiting or preventing the onset of symptoms after viral infection; (e) inhibiting the worsening of symptoms of viral infection; (f) limiting or preventing the recurrence of symptoms of viral infection in a subject who previously had symptoms of viral infection; and / or (e) increasing survival rate. In some embodiments, the vaccination method reduces the risk of a subject becoming infected with a virus (e.g., RSV and / or hMPV). In some embodiments, the vaccination method limits the onset of viral infection. In some embodiments, the vaccination method reduces the severity of symptoms of viral infection. In a preferred embodiment, the infection is a lower respiratory tract infection (LRTI).

[0358] In some embodiments, the methods provided herein can be used to prevent RSV infection or disease (eg, pneumonia or acute respiratory disease) in a subject.

[0359] When the method includes limiting viral infection, the immunogenic composition is administered prophylactically to a subject who is not known to be infected but may be at risk of being exposed to a virus (e.g., RSV or hMPV). As used herein, "limiting onset" includes, but is not limited to, achieving one or more of: (a) generating an immune response (antibody and / or cell-based, e.g., CD4 T cells, memory B cells, and / or CD8 T cells) against the virus in the subject; (b) generating neutralizing antibodies against the virus or viral proteins in the subject; (b) limiting the accumulation of viral titers in the subject after exposure to the virus; and / or (c) limiting or preventing the onset of symptoms after viral infection. Exemplary symptoms of viral (e.g., RSV or hMPV) infection include, but are not limited to, fever, fatigue, cough, stuffy nose, sneezing, shortness of breath, wheezing, and lower respiratory tract infection.

[0360] The methods provided herein can be used to limit the development of infection with RSV (or hMPV)-A and / or RSV (or hMPV)-B subtypes. Without wishing to be bound by theory, it is believed that due to the high sequence similarity of the F proteins of RSV (or hMPV)-A and RSV (or hMPV)-B, immunization with the F protein of one RSV (or hMPV) subtype provides at least some immunity against the other.

[0361] Additionally, the methods provided herein can be used to limit the onset of infection with original strains of RSV (or hMPV) and / or infection with variant strains of RSV (or hMPV). Examples of variant RSV (or hMPV) strains include, but are not limited to, RSV (or hMPV) ON1, RSV (or hMPV) NA1, RSV (or hMPV) LBA1, RSV (or hMPV) LBA2, RSV (or hMPV) BA, RSV (or hMPV) Long, RAV A2, and others (see, e.g., Pandya et al., Pathogens 2019, 8(2), 67, and Melero and Moore, Curr Top Microbiol Immunol. 2013; 372: 59-82). The pharmaceutical compositions of the present invention can be effective in limiting infection with strains of RSV (or hMPV) that have not yet been reported or discovered.

[0362] In some embodiments, the methods described herein generate an immune response in a subject not known to be infected with RSV (or hMPV) (e.g., RSV(or hMPV)-A and / or RSV(or hMPV)-B) that functions to limit infection and the development of symptoms of RSV (or hMPV) (e.g., RSV(or hMPV)-A and / or RSV(or hMPV)-B) infection. In some embodiments, the immune response includes the generation of neutralizing antibodies and / or cell-based responses against RSV (or hMPV) (e.g., RSV(or hMPV)-A and / or RSV(or hMPV)-B). In some embodiments, the immune response is at least 1x10 3 , at least 1x10 4 , at least 1x10 5 , at least 1x10 6 , at least 1x10 7 , at least 1x10 8 , or at least 1x10 9In a further embodiment, the immune response includes the generation of antibodies to multiple antigenic epitopes or RSV (or hMPV) (e.g., RSV (or hMPV)-A and / or RSV (or hMPV)-B).

[0363] In one embodiment, the methods provided herein can result in an increase in antibody titers in a subject, for example, an increase in RSV (or hMPV)-A specific neutralizing antibodies, RSV (or hMPV)-B specific neutralizing antibodies, RSV (or hMPV) F protein specific IgG antibodies, RSV (or hMPV) F protein specific neutralizing antibodies, core VLP specific IgG antibodies, and / or antibodies to human metapneumovirus. Antibody titers can be determined using any suitable assay known in the art or described herein, including, but not limited to, binding enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunoprecipitation, immunoblotting, and agglutination assays.

[0364] In some embodiments, the methods provided herein provide an improved therapeutic effect of about 1-fold to about 2-fold, about 2-fold to about 3-fold, about 3-fold to about 4-fold, about 4-fold to about 5-fold, about 5-fold to about 6-fold, about 6-fold to about 7-fold, about 7-fold to about 8-fold, about 8-fold to about 9-fold, about 9-fold to about 10-fold, about 10-fold to about 12-fold, about 12-fold to about 15-fold, about 15-fold to about 20-fold, about 20-fold to about 25-fold, about 25-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, or about 70-fold to about 80-fold. fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, or more than about 100-fold increase in antibodies (e.g., RSV (or hMPV)-A-specific neutralizing antibodies, RSV (or hMPV)-B-specific neutralizing antibodies, RSV (or hMPV) F protein-specific IgG antibodies, RSV (or hMPV) F protein-specific neutralizing antibodies, core VLP-specific IgG antibodies, and / or antibodies against human metapneumovirus). In some embodiments, the methods provided herein result in an increase in antibodies (e.g., an increase in RSV (or hMPV)-A-specific neutralizing antibodies, RSV (or hMPV)-B-specific neutralizing antibodies, RSV (or hMPV) F protein-specific IgG antibodies, RSV (or hMPV) F protein-specific neutralizing antibodies, core VLP-specific IgG antibodies, and / or antibodies to human metapneumovirus) of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline.

[0365] "Baseline" refers to an antibody measurement immediately prior to administration of a first dose of an immunogenic composition provided herein. In some embodiments, an increase in antibodies (e.g., RSV (or hMPV)-A-specific neutralizing antibodies, RSV (or hMPV)-B-specific neutralizing antibodies, RSV (or hMPV) F protein-specific IgG antibodies, RSV (or hMPV) F protein-specific neutralizing antibodies, core VLP-specific IgG antibodies, and / or antibodies to human metapneumovirus) compared to baseline is observed at about 3 to about 7 days, about 1 to about 2 weeks, about 3 to about 4 days, about 5 to about 6 days, about 7 to about 8 days, about 9 to about 12 days, about 14 to about 16 days, about 26 to about 28 days, about 32 to about 36 days, about 48 to about 38 days, about 52 to about 36 ... It is detectable within 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 5 weeks to about 6 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 3 months to about 4 months, about 4 months to about 5 months, about 5 months to about 6 months, about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 18 months, about 18 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, or about 5 years to about 10 years. In some embodiments, an increase in antibodies compared to baseline (e.g., an increase in RSV (or hMPV)-A-specific neutralizing antibodies, RSV (or hMPV)-B-specific neutralizing antibodies, RSV (or hMPV) F protein-specific IgG antibodies, RSV (or hMPV) F protein-specific neutralizing antibodies, core VLP-specific IgG antibodies, and / or antibodies to human metapneumovirus) is detectable within about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after administration of the immunogenic composition.

[0366] In another embodiment, the methods provided herein can result in an increase in immune cells in a subject, for example, an increase in RSV (or hMPV) F protein-specific memory B cells and / or RSV (or hMPV) F protein-specific T cells. The memory B cells and / or T cells can be specific for RSV (or hMPV)-AF protein or RSV (or hMPV)-BF protein, or can be reactive to both. The number of immune cells in a subject can be determined using any suitable assay known in the art or described herein, including, but not limited to, FACS and flow cytometry.

[0367] In some embodiments, the methods provided herein provide an effect of increasing or decreasing the cellular metabolism by about 1-fold to about 2-fold, about 2-fold to about 3-fold, about 3-fold to about 4-fold, about 4-fold to about 5-fold, about 5-fold to about 6-fold, about 6-fold to about 7-fold, about 7-fold to about 8-fold, about 8-fold to about 9-fold, about 9-fold to about 10-fold, about 10-fold to about 12-fold, about 12-fold to about 15-fold, about 15-fold to about 20-fold, about 20-fold to about 25-fold, about 25-fold to about 30-fold, about 30-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, about 100-fold to about 120-fold, about 120-fold to about 150-fold, about 150-fold to about 200-fold, about 200-fold to about 250-fold, about 250-fold to about 300-fold, about 300-fold to about 400-fold, about 400-fold to about 500-fold, about 400-fold to about 500-fold, about 400-fold to about 600-fold, about 400-fold to about 700-fold, about 400-fold to about 800-fold, about 400-fold to about 900-fold, about 400-fold to about 1000-fold, about 40 Results in an increase in immune cells (e.g., an increase in RSV (or hMPV) F protein-specific memory B cells and / or RSV (or hMPV) F protein-specific T cells) of 0-fold to about 40-fold, about 40-fold to about 50-fold, about 50-fold to about 60-fold, about 60-fold to about 70-fold, about 70-fold to about 80-fold, about 80-fold to about 90-fold, about 90-fold to about 100-fold, or more than about 100-fold. In some embodiments, the methods provided herein result in an increase in immune cells in a subject, e.g., an increase in RSV (or hMPV) F protein-specific memory B cells and / or RSV (or hMPV) F protein-specific T cells. In some embodiments, the methods provided herein result in an increase in immune cells (e.g., an increase in RSV (or hMPV) F protein-specific memory B cells and / or RSV (or hMPV) F protein-specific T cells) of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline. The memory B cells and / or T cells may be specific for the RSV (or hMPV)-AF protein or the RSV (or hMPV)-BF protein, or may be reactive to both.

[0368] In some embodiments, the increase in immune cells (e.g., an increase in RSV (or hMPV) F protein-specific memory B cells and / or RSV (or hMPV) F protein-specific T cells) compared to baseline is observed at about 3 to about 7 days, about 1 to about 2 weeks, about 2 to about 3 weeks, about 3 to about 4 weeks, about 4 to about 5 weeks, about 5 to about 6 weeks, about 6 to about 7 weeks, or about 8 to about 9 weeks after administration of the immunogenic composition. weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 3 months to about 4 months, about 4 months to about 5 months, about 5 months to about 6 months, about 6 months to about 9 months, about 9 months to about 12 months, about 12 months to about 18 months, about 18 months to about 24 months, about 2 years to about 3 years, about 3 years to about 4 years, about 4 years to about 5 years, or about 5 years to about 10 years. In some embodiments, an increase in immune cells (e.g., an increase in RSV (or hMPV) F protein-specific memory B cells and / or RSV (or hMPV) F protein-specific T cells) compared to baseline is detectable within about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after administration of the immunogenic composition. The memory B cells and / or T cells may be specific for the RSV (or hMPV)-AF protein or the RSV (or hMPV)-BF protein, or may be reactive to both.

[0369] The polypeptide, virus-like particle, composition, nucleic acid, pharmaceutical composition, or vaccine of any embodiment herein is typically formulated as a pharmaceutical composition, such as those disclosed above, and may be administered by any suitable route, including intranasal, sublingual, oral, parenteral, inhalation spray, rectal, or topical, in a dosage unit formulation containing conventional pharma- ceutically acceptable carriers, adjuvants, and vehicles. As used herein, the term parenteral includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques, or intraperitoneal. The polypeptide composition may be administered via microspheres, liposomes, immune stimulating complexes (ISCOMs), or other microparticulate delivery systems or sustained release formulations that are introduced into suitable tissues, such as blood.

[0370] Dosage regimens can be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). Suitable dosage ranges include, for example, 0.1 μg / kg to 0.5 μg / kg body weight, 0.5 μg / kg to 1 μg body weight, 1 μg / kg to 2 μg / kg body weight, 2 μg / kg to 3 μg / kg body weight, 3 μg / kg to 4 μg / kg body weight, 4 μg / kg to 5 μg / kg body weight, 5 μg / kg to 6 μg / kg body weight, 6 μg / kg to 7 μg / kg body weight, 7 μg / kg to 8 μg / kg body weight, 8 μg / kg to 9 μg / kg body weight, 9 μg / kg to 10 μg / kg body weight, 10 μg / kg to 15 μg / kg body weight, 15 μg / kg to 20 μg / kg body weight, 20 μg / kg to 25μg / kg body weight, 25μg / kg~30μg / kg body weight, 30μg / kg~35μg / kg body weight, 35μg / kg~40μg / kg body weight, 40μg / kg~45μg / kg body weight, 45μg / kg~50μg / kg body weight, 50μg / kg~55μg / kg body weight, 5 5μg / kg~60μg / kg body weight, 60μg / kg~65μg / kg body weight, 65μg / kg~70μg / kg body weight, 70μg / kg~75μg / kg body weight, 75μg / kg~80μg / kg body weight, 80μg / kg~85μg / kg body weight, 85μg / kg~90μg / kg body weight, 90μg / kg~95μg / kg body weight, 95μg / kg~100μg / kg body weight, 100μg / kg~150μg body weight, 150μg / kg~200μg body weight, 200μg / kg~250μg / kg body weight, 250μg / kg~300μg / kg body weight, 30 0μg / kg~350μg / kg body weight, 350μg / kg~400μg / kg body weight, 400μg / kg~450μg / kg body weight, 450μg / kg~500μg body weight, 500μg / kg~550μg body weight, 550μg / kg~600μg body weight, 600μg / kg~6 50μg body weight, 650μg / kg~700μg body weight, 700μg / kg~750μg / kg body weight, 750μg / kg~800μg / kg body weight, 800μg / kg~850μg / kg body weight, 850μg / kg~900μg / kg body weight, 900μg / kg~950μg / kg body weight, 950μg / kg~1mg / kg body weight, 1mg / kg~2mg / kg body weight, 2mg / kg~3mg / kg body weight, 3mg / kg~4mg / kg body weight, 4mg / kg~5mg / kg body weight, 5mg / kg~6mg / kg body weight, 6mg / kg~7mg / kg body weight,7mg / kg~8mg / kg body weight, 8mg / kg~90mg / kg body weight, 90mg / kg~100mg / kg body weight, 100mg / kg~150mg / kg body weight, 150mg / kg~200mg / kg body weight, 200mg / kg~250mg / k g body weight, 250mg / kg~300mg / kg body weight, 300mg / kg~350mg / kg body weight, 350mg / kg~400mg / kg body weight, 400mg / kg~450mg / kg body weight, 450mg / kg~500mg / kg body weight, 500mg The polypeptide or virus-like particle thereof may be in an amount of 1 g / kg to 550 mg / kg body weight, 550 mg / kg to 600 mg / kg body weight, 600 mg / kg to 650 mg / kg body weight, 650 mg / kg to 700 mg / kg body weight, 700 mg / kg to 750 mg / kg body weight, 750 mg / kg to 800 mg / kg body weight, 800 mg / kg to 850 mg / kg body weight, 850 mg / kg to 900 mg / kg body weight, 900 mg / kg to 950 mg / kg body weight, or 950 mg / kg to 1 g / kg.

[0371] The composition may be delivered in a single bolus or may be administered multiple times (e.g., 2, 3, 4, 5, or more times) as determined by the attending medical professional. In some embodiments, the composition may be administered in doses of about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 10 ...25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about In one embodiment, 0 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 425 μg, about 450 μg, about 475 μg, or about 500 μg of the polypeptide or virus-like particle is administered. In some embodiments, the amount of the active ingredient may be from about 5 μg to about 10 μg, from about 10 μg to about 15 μg, from about 15 μg to about 20 μg, from about 20 μg to about 30 μg, from about 30 μg to about 40 μg, from about 40 μg to about 50 μg, from about 50 μg to about 60 μg, from about 60 μg to about 70 μg, from about 70 μg to about 80 μg, from about 80 μg to about 90 μg, from about 90 μg to about 100 μg, from about 100 μg to about 110 μg, from about 110 μg to about 120 μg, or from about 130 μg to about 140 μg. In one embodiment, about 120 μg, about 120 μg to about 130 μg, about 130 μg to about 140 μg, about 140 μg to about 150 μg, about 150 μg to about 200 μg, about 200 μg to about 250 μg, about 250 μg to about 300 μg, about 300 μg to about 350 μg, about 350 μg to about 400 μg, about 400 μg to about 450 μg, or about 450 μg to about 500 μg of the polypeptide or virus-like particle is administered.

[0372] In some embodiments, the immunogenic compositions provided herein are administered as a booster to another RSV (or hMPV) vaccine, e.g., a live attenuated RSV (or hMPV) vaccine, a RSV (or hMPV)-A vaccine, and a RSV (or hMPV)-B vaccine, or a bivalent RSV (or hMPV)-A / B vaccine. In some embodiments, the administration comprises administering a first dose and a second dose of the immunogenic composition, where the second dose is administered about 2 weeks to about 12 weeks, or about 4 weeks to about 12 weeks after the first dose is administered. In various further embodiments, the second dose is administered about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, about 18 months, about 2 years, about 3 years, about 4 years, or about 5 years after the first dose. In another embodiment, three doses may be administered, the second dose being administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, about 18 months, about 2 years, about 3 years, about 4 years, or about 5 years after the first dose, and the third dose being administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, about 18 months, about 2 years, about 3 years, about 4 years, or about 5 years after the second dose. The second dose may be a booster dose of RSV (or hMPV).

[0373] In some embodiments, more than two doses of the immunogenic composition are administered. In some embodiments, the first and second doses of the immunogenic composition are administered within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, or about 12 months of each other, and the third dose is administered about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years after the second dose. In some embodiments, the first and second doses of the immunogenic composition are administered within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, or about 12 months of each other, and the subsequent doses are administered at regular intervals of about 1, 2, 3, 4, or 5 years.

[0374] In some embodiments, the subject has previously been infected with RSV (or hMPV) (e.g., RSV(or hMPV)-A and / or RSV(or hMPV)-B). In another embodiment of the method, the subject is infected with RSV (or hMPV) (e.g., RSV(or hMPV)-A and / or RSV(or hMPV)-B) at the time the pharmaceutical composition provided herein is administered, and the administration induces an immune response in the subject against RSV (or hMPV) (e.g., RSV(or hMPV)-A and / or RSV(or hMPV)-B) that treats the RSV (or hMPV) infection (e.g., RSV(or hMPV)-A infection and / or RSV(or hMPV)-B infection) in the subject. Where the method includes treating a RSV (or hMPV) infection (e.g., a RSV (or hMPV)-A infection and / or a RSV (or hMPV)-B infection), the immunogenic composition is administered to a subject who is already infected with RSV (or hMPV) (e.g., RSV (or hMPV)-A and / or RSV (or hMPV)-B) and / or who is suffering from symptoms (e.g., those described above) indicating that the subject is likely infected with RSV (or hMPV) (e.g., RSV (or hMPV)-A and / or RSV (or hMPV)-B).

[0375] RSV (or hMPV) infection (e.g., RSV (or hMPV)-A infection and / or RSV (or hMPV)-B infection) can be diagnosed using any PCR-based or antigen-based test known in the art. In some embodiments, the subject has antibodies to RSV (or hMPV). Anti-RSV (or hMPV) antibodies (e.g., RSV (or hMPV)-A antibodies and / or RSV (or hMPV)-B antibodies) can be detected using any serological test known in the art. In preferred embodiments, the compositions and methods disclosed herein prevent disease following infection with RSV (or hMPV) subtypes A and B in older adults.

[0376] In some embodiments, the protein conjugates and pharmaceutical compositions of the disclosure provide long-term persistence (persistence) of neutralizing antibodies up to 180 days after vaccination. In some embodiments, the protein conjugates and pharmaceutical compositions of the disclosure provide long-term persistence (persistence) of RSV-A and / or RSV-B neutralizing antibodies up to 20 days, up to 40 days, up to 60 days, up to 80 days, up to 100 days, up to 120 days, up to 140 days, up to 160 days, up to 180 days, up to 200 days, up to 250 days, up to 300 days, up to 350 days, up to 365 days, up to 400 days, up to 450 days, or up to 500 days after vaccination.

[0377] In some embodiments, the protein complexes and pharmaceutical compositions of the present disclosure provide a long-term persistence of about 40%, about 45%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98% of RSV-A and / or RSV-B neutralizing antibody geometric mean titers (GMTs) at day 180 post-vaccination compared to day 28 post-vaccination.

[0378] In some embodiments, the protein complexes and pharmaceutical compositions of the present disclosure provide alum adjuvant-independent persistence of RSV-A and / or RSV-B neutralizing antibodies.

[0379] In some embodiments, the protein conjugates and pharmaceutical compositions of the disclosure provide long-term persistence at day 180 post-vaccination that is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than baseline for RSV-A and / or RSV-B neutralizing antibody geometric mean titers (GMTs) at day 28 post-vaccination. In some embodiments, the protein conjugates and pharmaceutical compositions of the disclosure provide long-term persistence at day 180 post-vaccination that is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than baseline for hMPV-A and / or hMPV-B neutralizing antibody geometric mean titers (GMTs) at day 28 post-vaccination.

[0380] Method of production In one aspect, the disclosure provides a method of producing a vaccine comprising culturing a host cell described herein in a culture medium such that the host cell secretes an antigen into the culture medium; optionally purifying the antigen from the culture medium; mixing the antigen with a second component, where the second component multimerizes with the antigen to form a virus-like particle; and optionally purifying the virus-like particle.

[0381] Kits and Unit Doses The present disclosure further provides kits that can be used to prepare the virus-like particles and compositions of the present disclosure. In some embodiments, the kits provided herein include a first component and a second component disclosed herein, and instructions for use in the methods of the present disclosure. In some embodiments, the kits include one or more unit doses disclosed herein, and instructions for use in the methods of the present disclosure. In some embodiments, the kits include a vial containing a single dose of the pharmaceutical composition provided herein. In some embodiments, the kits include a vial containing multiple doses provided herein. In some embodiments, the kits further include instructions for use of the pharmaceutical composition. In some embodiments, the kits further include a diluent for preparing a dilution of the pharmaceutical composition prior to administration. In some embodiments, the pharmaceutical composition includes an adjuvant. In some embodiments, the kits include a pharmaceutical composition and an adjuvant that need to be mixed prior to administration.

[0382] Also provided herein is a unit dose of the pharmaceutical composition described herein. In some embodiments, the unit dose is about 1 μg to about 5 μg, about 5 μg to about 10 μg, about 10 μg to about 15 μg, about 15 μg to about 20 μg, about 20 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg, about 50 μg to about 60 μg, about 60 μg to about 70 μg, about 70 μg to about 80 μg, about 80 μg to about 90 μg, about 90 μg to about 100 μg, about 100 μg to about 110 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about 190 μg, about 200 μg, about 210 μg, about 220 μg, about 230 μg, about 240 μg, about 250 μg, about 260 μg, about 270 μg, about 280 μg, about 290 μg, about 300 μg, about 310 μg, about 320 μg, about 330 μg, about 340 μg, about 350 μg, about 360 μg, about 370 μg, about 380 μg, about 390 μg, about 40 ... The protein complex may be 0 μg to about 120 μg, about 120 μg to about 130 μg, about 130 μg to about 140 μg, about 140 μg to about 150 μg, about 150 μg to about 175 μg, about 175 μg to about 200 μg, about 200 μg to about 250 μg, about 250 μg to about 300 μg, about 300 μg to about 350 μg, about 350 μg to about 400 μg, about 400 μg to about 450 μg, or about 450 μg to about 500 μg of the protein complex. In some embodiments, a unit dose comprises about 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, about 25 μg, about 50 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375 μg, about 400 μg, or about 500 μg of VLPs or each individual VLP. In some embodiments, a unit dose comprises 25 μg, 50 μg, 75 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 400 μg, or 500 μg of VLPs or individual VLPs. The abbreviation "μg" may be used interchangeably with the abbreviation "mcg," which refers to micrograms of substance.

[0383] In some embodiments, the unit dose comprises about 75 μg of a first VLP (for RSV) and about 75 μg of a second VLP (for hMPV).

[0384] In some embodiments, the unit dose comprises about 75 μg of a first VLP (for RSV) and about 100 μg of a second VLP (for hMPV).

[0385] In some embodiments, the unit dose comprises about 75 μg of a first VLP (for RSV) and about 150 μg of a second VLP (for hMPV).

[0386] In some embodiments, the unit dose comprises about 75 μg of a first VLP (for RSV) and about 200 μg of a second VLP (for hMPV).

[0387] In some embodiments, the unit dose comprises about 75 μg of a first VLP (for RSV) and about 225 μg of a second VLP (for hMPV).

[0388] In some embodiments, the unit dose comprises about 75 μg of a first VLP (for RSV) and about 250 μg of a second VLP (for hMPV).

[0389] In some embodiments, the unit dose comprises about 75 μg of a first VLP (for RSV) and about 300 μg of a second VLP (for hMPV).

[0390] In some embodiments, the unit dose comprises about 100 μg of a first VLP (for RSV) and about 75 μg of a second VLP (for hMPV).

[0391] In some embodiments, the unit dose comprises about 100 μg of a first VLP (for RSV) and about 100 μg of a second VLP (for hMPV).

[0392] In some embodiments, the unit dose comprises about 100 μg of a first VLP (for RSV) and about 150 μg of a second VLP (for hMPV).

[0393] In some embodiments, the unit dose comprises about 100 μg of a first VLP (for RSV) and about 200 μg of a second VLP (for hMPV).

[0394] In some embodiments, the unit dose comprises about 100 μg of a first VLP (for RSV) and about 225 μg of a second VLP (for hMPV).

[0395] In some embodiments, the unit dose comprises about 100 μg of a first VLP (for RSV) and about 250 μg of a second VLP (for hMPV).

[0396] In some embodiments, the unit dose comprises about 100 μg of a first VLP (for RSV) and about 300 μg of a second VLP (for hMPV).

[0397] In some embodiments, the unit dose comprises about 150 μg of a first VLP (for RSV) and about 75 μg of a second VLP (for hMPV).

[0398] In some embodiments, the unit dose comprises about 150 μg of a first VLP (for RSV) and about 100 μg of a second VLP (for hMPV).

[0399] In some embodiments, the unit dose comprises about 150 μg of a first VLP (for RSV) and about 150 μg of a second VLP (for hMPV).

[0400] In some embodiments, the unit dose comprises about 150 μg of a first VLP (for RSV) and about 200 μg of a second VLP (for hMPV).

[0401] In some embodiments, the unit dose comprises about 150 μg of a first VLP (for RSV) and about 225 μg of a second VLP (for hMPV).

[0402] In some embodiments, the unit dose comprises about 150 μg of a first VLP (for RSV) and about 250 μg of a second VLP (for hMPV).

[0403] In some embodiments, the unit dose comprises about 150 μg of a first VLP (for RSV) and about 300 μg of a second VLP (for hMPV).

[0404] In some embodiments, the unit dose comprises about 200 μg of a first VLP (for RSV) and about 75 μg of a second VLP (for hMPV).

[0405] In some embodiments, the unit dose comprises about 200 μg of a first VLP (for RSV) and about 100 μg of a second VLP (for hMPV).

[0406] In some embodiments, the unit dose comprises about 200 μg of a first VLP (for RSV) and about 150 μg of a second VLP (for hMPV).

[0407] In some embodiments, the unit dose comprises about 200 μg of a first VLP (for RSV) and about 200 μg of a second VLP (for hMPV).

[0408] In some embodiments, the unit dose comprises about 200 μg of a first VLP (for RSV) and about 225 μg of a second VLP (for hMPV).

[0409] In some embodiments, the unit dose comprises about 200 μg of a first VLP (for RSV) and about 250 μg of a second VLP (for hMPV).

[0410] In some embodiments, the unit dose comprises about 200 μg of a first VLP (for RSV) and about 300 μg of a second VLP (for hMPV).

[0411] In some embodiments, the unit dose comprises about 225 μg of a first VLP (for RSV) and about 75 μg of a second VLP (for hMPV).

[0412] In some embodiments, the unit dose comprises about 225 μg of a first VLP (for RSV) and about 100 μg of a second VLP (for hMPV).

[0413] In some embodiments, the unit dose comprises about 225 μg of a first VLP (for RSV) and about 150 μg of a second VLP (for hMPV).

[0414] In some embodiments, the unit dose comprises about 225 μg of a first VLP (for RSV) and about 200 μg of a second VLP (for hMPV).

[0415] In some embodiments, the unit dose comprises about 225 μg of a first VLP (for RSV) and about 225 μg of a second VLP (for hMPV).

[0416] In some embodiments, the unit dose comprises about 225 μg of a first VLP (for RSV) and about 250 μg of a second VLP (for hMPV).

[0417] In some embodiments, the unit dose comprises about 225 μg of a first VLP (for RSV) and about 300 μg of a second VLP (for hMPV).

[0418] In some embodiments, the unit dose comprises about 250 μg of a first VLP (for RSV) and about 75 μg of a second VLP (for hMPV).

[0419] In some embodiments, the unit dose comprises about 250 μg of a first VLP (for RSV) and about 100 μg of a second VLP (for hMPV).

[0420] In some embodiments, the unit dose comprises about 250 μg of a first VLP (for RSV) and about 150 μg of a second VLP (for hMPV).

[0421] In some embodiments, the unit dose comprises about 250 μg of a first VLP (for RSV) and about 200 μg of a second VLP (for hMPV).

[0422] In some embodiments, the unit dose comprises about 250 μg of a first VLP (for RSV) and about 225 μg of a second VLP (for hMPV).

[0423] In some embodiments, the unit dose comprises about 250 μg of a first VLP (for RSV) and about 250 μg of a second VLP (for hMPV).

[0424] In some embodiments, the unit dose comprises about 250 μg of a first VLP (for RSV) and about 300 μg of a second VLP (for hMPV).

[0425] In some embodiments, the unit dose comprises about 300 μg of a first VLP (for RSV) and about 75 μg of a second VLP (for hMPV).

[0426] In some embodiments, the unit dose comprises about 300 μg of a first VLP (for RSV) and about 100 μg of a second VLP (for hMPV).

[0427] In some embodiments, the unit dose comprises about 300 μg of a first VLP (for RSV) and about 150 μg of a second VLP (for hMPV).

[0428] In some embodiments, the unit dose comprises about 300 μg of a first VLP (for RSV) and about 200 μg of a second VLP (for hMPV).

[0429] In some embodiments, the unit dose comprises about 300 μg of a first VLP (for RSV) and about 225 μg of a second VLP (for hMPV).

[0430] In some embodiments, the unit dose comprises about 300 μg of a first VLP (for RSV) and about 250 μg of a second VLP (for hMPV).

[0431] In some embodiments, the unit dose comprises about 300 μg of a first VLP (for RSV) and about 300 μg of a second VLP (for hMPV).

[0432] In some embodiments, the unit dose comprises about 5 μg to about 100 μg of the first VLP, about 10 μg to about 90 μg of the first VLP, about 20 μg to about 80 μg of the first VLP, about 30 μg to about 70 μg of the first VLP, about 40 μg to about 60 μg of the first VLP, or about 50 μg to about 80 μg of the first VLP (for RSV).

[0433] In some embodiments, the unit dose comprises about 5 μg to about 100 μg of the first VLP, about 10 μg to about 90 μg of the first VLP, about 20 μg to about 80 μg of the first VLP, about 30 μg to about 70 μg of the first VLP, about 40 μg to about 60 μg of the first VLP, and / or about 50 μg to about 80 μg of the second VLP (for hMPV).

[0434] In some embodiments, the unit dose comprises about 50 μg to about 180 μg of the first VLP, about 60 μg to about 170 μg of the first VLP, about 80 μg to about 150 μg of the first VLP, about 70 μg to about 155 μg of the first VLP, about 90 μg to about 160 μg of the first VLP, and / or about 100 μg to about 170 μg of the second VLP (for hMPV).

[0435] In some embodiments, the unit dose comprises about 130 μg to about 300 μg of the first VLP, about 150 μg to about 280 μg of the first VLP, about 170 μg to about 260 μg of the first VLP, about 190 μg to about 240 μg of the first VLP, about 200 μg to about 230 μg of the first VLP, and / or about 210 μg to about 225 μg of the second VLP (for hMPV).

[0436] In some embodiments, the unit dose comprises about 130 μg to about 300 μg of the first VLP, about 150 μg to about 280 μg of the first VLP, about 170 μg to about 260 μg of the first VLP, about 190 μg to about 240 μg of the first VLP, about 200 μg to about 230 μg of the first VLP, and about 130 μg to about 300 μg of the second VLP, about 150 μg to about 280 μg of the second VLP, about 170 μg to about 260 μg of the second VLP, about 190 μg to about 240 μg of the second VLP, or about 200 μg to about 230 μg of the second VLP.

[0437] In some embodiments, the unit dose comprises about 75 μg of the first VLP (for RSV) and 75 μg of the second VLP (for hMPV). In some embodiments, the unit dose comprises about 75 μg of the first VLP (for RSV) and 225 μg of the second VLP (for hMPV). In some embodiments, the unit dose comprises about 225 μg of the first VLP (for RSV) and 75 μg of the second VLP (for hMPV). In some embodiments, the unit dose comprises about 75 μg of the first VLP (for RSV) and 150 μg of the second VLP (for hMPV). In some embodiments, the unit dose comprises about 150 μg of the first VLP (for RSV) and 75 μg of the second VLP (for hMPV). In some embodiments, the unit dose comprises about 150 μg of a first VLP (for RSV) and 150 μg of a second VLP (for hMPV).

[0438] In some embodiments, the unit dose comprises about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 175 μg, or about 200 μg of the first VLP (for RSV).

[0439] In some embodiments, the unit dose comprises about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 175 μg, or about 200 μg of the second VLP (for hMPV).

[0440] In some embodiments, the unit dose comprises about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 175 μg, or about 200 μg of the first VL. P (for RSV) and about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 175 μg, or about 200 μg of a second VLP (for hMPV).

[0441] In some embodiments, the unit dose comprises about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, or about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg of the first VLP (for RSV).

[0442] In some embodiments, the unit dose comprises about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, or about 300 μg of a second VLP (for hMPV).

[0443] In some embodiments, the unit dose is about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg of the first VL. P (for RSV) and about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, or about 300 μg of a second VLP (for hMPV). EXAMPLES

[0444] Example 1 This example describes the production of a two-component icosahedral VLP intended for use as a vaccine against the hMPV virus. The vaccine uses as the first component A a polypeptide antigen composed of the ectodomain of the F protein of hMPV fused at its C-terminus to the I53-50A protein (SEQ ID NO: 144). The VLP uses the I53-50B protein as component B. As shown in Figures 1A and 1B, I53-50A and I53-50B are known to spontaneously assemble in a 3:5 ratio to form VLPs with icosahedral (I53) symmetry.

[0445] The expression levels of hMPV F protein fused to CompA were quantified. Human codon-optimized polynucleotide sequences were generated by gene synthesis and cloned into expression vectors. Each expression vector was expressed individually by transient transfection in Expi293 cells. Supernatants were collected 4 days after transfection.

[0446] To determine whether recombinantly expressed fusion proteins were secreted into the supernatant, Western blots using anti-His6 monoclonal antibodies were performed. Conditioned media was diluted a minimum of 10-fold in 2X Laemmli loading buffer (Bio-Rad) containing 5% (v / v) β-mercaptoethanol, heated at 95°C for 10 min, and 10 μl was run on the gel. Reference samples of recombinant His-tagged human serum albumin (HSA) were prepared by initial dilution to 0.01 mg / mL followed by a two-fold dilution series to generate a standard curve from 6.25 to 100 ng in a loading volume of 10 μL in 2X Laemmli loading buffer. Samples were loaded onto a NuPAGE 4-12% Bis-Tris protein gel and run at 150 V for 45 min. Proteins were transferred to Immuno-Blot PDVF membranes (Bio-Rad). After transfer, the blots were blocked with 3% BSA (w / v) in TBST for 1 h at room temperature with shaking. Anti-His HRP conjugated antibody (R&D Systems MAB050H) was diluted 1:8,000 in 3% BSA (w / v) in TBST and incubated with the membrane for 1 h at room temperature. The membrane was then washed 3 times for 5 min with TBST, and then His-tagged proteins were detected using luminol chemiluminescence substrate (VisiGlo, VWR) and captured using a 10–20 s exposure time in a UVP Chemstudio (Analytik Jena).

[0447] Table 4 shows the expression levels of hMPV F protein fused to CompA. Expression levels were assessed by visual inspection of Western blots and designated as no expression (X) or low expression (*) to high expression (****). hMPV008, hMPV009, hMPV024, hMPV026, hMPV027, hMPV033, and hMPV034 CompA fusion proteins showed the highest expression levels in this system. [Table 4-1] [Table 4-2] [Table 4-3]

[0448] Example 2 In this example, conditioned medium containing hMPV F-CompA fusion proteins was characterized using antibody binding activity to determine the conformation of the hMPV F protein. Fusion proteins hMPV005, hMPV008, hMPV021, hMPV024, hMPV026, hMPV027, hMPV027C, and hMPV033 were evaluated (Table 5). Constructs hMPV026 and hMPV027 contain a furin cleavage site that was not cleaved in Expi293 transient transfection. Upon cotransfection of human furin, the expressed protein was appropriately cleaved. This is referred to as hMPV026C or hMPV027C to represent the cleaved form.

[0449] To characterize the conformation of recombinantly expressed hMPV F protein fused to CompA, ELISA was performed using 15 monoclonal antibodies that bind to specific forms of the hMPV F protein ectodomain.

[0450] Wells of a 96-well plate were coated overnight at 4° C. with 200 ng of each antibody, or 50 ng for MPE8, in 100 μL of 50 mM sodium carbonate-bicarbonate buffer, pH 9.6. Plates were washed three times with 300 μl of PBS containing 0.05% Tween 20. After blocking with 150 μl of PBS containing 1% BSA for 1 h at room temperature, plates were emptied by tapping on an absorbent pad. The expected EC of antigen was determined between antigen concentrations. 50 Conditioned media supernatants from Expi293 transfections of each construct were diluted based on semi-quantitative Western blots to contain 100 μL of each antibody (according to Mas data). Three dilutions for each construct were plated at 100 μL per well for each antibody, and the plates were incubated for 1 hour at room temperature. The plates were washed six times with 300 μl of PBS containing 0.05% Tween 20. Anti-His monoclonal antibody (R&D Systems MAB050H) was diluted 1:15,000, 100 μL was added to each well, and the plates were incubated for 1 hour at room temperature. After washing the plates six times with 300 μl of PBS containing 0.05% Tween 20, 100 μl of TMB substrate was added and the plates were developed for 10 minutes in the dark. The reaction was monitored with 100 μl of 0.6NH 2 SO 4 The reaction was stopped by the addition of 0.05% and the absorbance at 450 nm was measured using a plate reader.

[0451] The post-fusion specific antibody bound to hMPV021, suggesting that this construct has a post-fusion conformation. The other constructs did not bind to the post-fusion specific antibody, but bound to MPE8 along with several antibodies that recognize both the pre-fusion and post-fusion conformations. This data suggests that these fusion proteins have characteristics of the pre-fusion conformation. [Example 3] Binding activity observed with purified hMPV F-CompA fusion proteins. Figures 3A-3C show binding of known concentrations of purified component A (CompA) fusions, i.e., hMPV F protein ectodomain fused at the N-terminus to I53-50A (SEQ ID NO: 144) or I53-50AΔCys (SEQ ID NO: 145). MF14, MPE8, and MF16 recognize antigenic sites II, III, and IV, respectively, which are the major sites recognized by neutralizing antibodies. Furthermore, MPE8 preferentially recognizes the prefusion form of hMPV F protein. Wells of a 96-well plate were coated overnight at 4°C with 50 ng of MPE8 and 100 ng of MF14 and MF16 in 100 μL of 50 mM sodium carbonate-bicarbonate buffer, pH 9.6. Plates were washed three times with 300 μl of PBS containing 0.05% Tween 20. After blocking with 150 μl of PBS containing 1% BSA for 1 hour at room temperature, the plate was emptied by tapping on an absorbent pad. Purified samples of each construct were diluted to 40 μg / mL (hMPV008, 021, 026, and 027) or 4 μg / mL (hMPV026C and 033), followed by serial 3- or 4-fold dilutions in PBS containing 1% BSA, respectively, plated at 100 μL per well, and the plate was incubated for 1 hour at room temperature. The plate was washed six times with 300 μl of PBS containing 0.05% Tween 20. Anti-His monoclonal antibody (R&D Systems MAB050H) was diluted 1:15,000, 100 μL was added to each well, and the plate was incubated for 1 hour at room temperature. After washing the plate six times with 300 μl of PBS containing 0.05% Tween 20, 100 μl of TMB substrate was added and the plate was developed for 10 min in the dark. 2 SO 4 The binding was stopped by the addition of MF14 (site II, Fig. 3A) and MF16 (site IV, Fig. 3B) was similar, whereas the binding of hMPV026, hMPV027, and hMPV033 by MPE8 (site III, Fig. 3C) had a higher EC 50(Table 6) This suggests that site III, the recognition site for neutralizing antibodies, is more conserved in hMPV026, hMPV027, and hMPV033, and that these constructs preferentially adopt the prefusion conformation over hMPV008 and hMPV021. [Table 6]

[0452] Example 4 In this example, assembled hMPV008 VLPs or hMPV008 CompA alone were injected intramuscularly into both hind legs of female BALB / c mice on days 0 and 21. The amounts and formulations for each group are listed in Table 7. Blood was collected on days 0, 24, and 35 and processed into serum. Day 0 serum samples were pooled by group. All serum samples were evaluated in an hMPV neutralizing antibody titer assay (Figure 9). hMPV008 formulations with the adjuvants Alhydrogel (Alum) or Addavax (Squalene oil-in-water emulsion) induced measurable neutralizing titers at all doses on days 24 and 35 that were comparable to those of healthy adult human serum (Figure 9). These results show that hMPV008 VLPs induce robust neutralizing titers comparable to healthy human control serum. [Table 7] [Table 5]

[0453] Example 5 A study was conducted to investigate immunogenic hMPV F protein variants displayed on bicomponent virus-like particles (VLPs) in naive BALB / c mice. VLPs containing one of four different hMPV F protein variants, hMPV008, hMPV024, hMPV026C, and hMPV033, or soluble protein corresponding to hMPV026C were tested. Each group consisted of eight female BALB / c mice. Mice were immunized on days 0 and 21. All test articles were formulated with Addavax, an oil-in-water adjuvant. Two dose levels (1 μg and 0.25 μg) or equivalent antigen content of soluble protein were administered (Table 8). Neutralizing antibody titers against hMPV-A and hMPV-B were determined from sera on day 35 (Figures 4A and 4B, respectively). These results demonstrated that robust neutralization titers were induced by all VLPs against both strains of hMPV. The soluble proteins also induced titers against both hMPV strains at lower levels than the VLPs. [Table 8]

[0454] Example 6 To investigate the immunogenicity of hMPV008 VLP and the corresponding soluble protein, CompA-hMPV008, studies were performed in naive mice. hMPV008 VLP and CompA-hMPV008 were formulated in aqueous buffer, squalene emulsion (SE), or Alhydrogel at two dose levels (VLP 1 μg and 0.1 μg, as well as an equivalent antigen dose for CompA-hMPV008). In addition, one group was included with hMPV033 VLP (0.1 μg) formulated with SE (Table 9). Each group consisted of eight female BALB / c mice. Mice were immunized on days 0 and 21. Neutralizing antibody titers against hMPV-A and hMPV-B were determined from sera on day 35. Results showed that hMPV008 VLP formulated with SE resulted in higher titers than Alhydrogel or aqueous formulations (Figures 5A and 5B). VLPs induced higher neutralization titers than the soluble protein CompA-hMPV008 in all formulations (Figures 5A and 5B). [Table 9]

[0455] Example 7: Bivalent non-interfering immunogenicity study using hMPV F protein (IVX-241) and RSV F protein (IVX-121) VLPs A study was conducted in naive BALB / c mice to investigate the immunogenicity of VLPs displaying the hMPV F protein variant IVX-241 and the RSV F protein IVX-121 when administered as monovalent or bivalent formulations. The purpose of the study was to evaluate various ratios of IVX-241 to IVX-121 and determine neutralization titers against RSV-A, RSV-B, hMPV-A, and hMPV-B. Mice were immunized on days 0 and 21 with IVX-121, IVX-241, or IVX-121+IVX-241 at the dose levels and formulations shown in Table 10. IVX-121 was administered at a dose level of 1 μg and IVX-241 was administered at dose levels of 4, 2, 1, and 0.5 μg. The study included VLPs (mosaic) displaying both components incorporating the RSV and hMPV F antigen variants present in IVX-121 and IVX-241. The mosaic particles were administered at a dose level of 2 μg.

[0456] Blood samples were collected on days 0, 21, and 35, processed into serum, and the day 0 and day 35 samples were tested in RSV-A, RSV-B, hMPV-A, and hMPV-B specific neutralization assays. Neutralizing antibody titer data were plotted and geometric means with geometric SDs were shown. Group statistical analysis was performed using the non-parametric Mann-Whitney test (GraphPad Prism).

[0457] All animals were awake and responsive throughout the study, and there were no clinical observations related to the test article or adjuvant.

[0458] Sera from animals immunized with IVX-121 showed a significant increase in RSV-A and RSV-B neutralizing antibodies compared to pre-immune sera (Figure 6) (day 0 titers were less than 2 log2 in all groups). The mean RSV-B titers were approximately 2.5 log2 lower than the RSV-A titers. Animals immunized with IVX-241 showed little to no neutralizing titers against RSV-A or RSV-B (Figure 6). RSV-A neutralizing titers from animals administered IVX-121 and IVX-241 were slightly lower than animals dosed with IVX-121 alone, with statistical significance only seen between the bivalent group administered 4 μg IVX-241 and the mosaic VLPs (Figure 6). IVX-121 / IVX-241 mosaic VLPs yielded titers of RSV-A and RSV-B that tended to be lower than IVX-121 alone, but were not statistically significant. Convalescent human serum samples used in neutralizing titer assays for RSV-A showed lower titers than the average of IVX-121 or IVX-121+IVX-241 immunized animals. However, single sera used in duplicate for RSV-B had higher titers than the IVX-121 group.

[0459] Sera from animals immunized with IVX-241 showed a significant increase in hMPV-A and hMPV-B neutralizing antibodies compared to pre-immune sera (Figure 7) (day 0 titers were less than 2 log2 in all groups). Responses to all four doses of IVX-241 yielded similar titers. Animals immunized with IVX-121 showed hMPV-A and hMPV-B neutralization titers similar to pre-immune sera (Figure 7). hMPV-A and hMPV-B neutralization titers in animals administered IVX-121 and IVX-241 were approximately 1-2 log2 lower than animals dosed with IVX-241 alone (Figure 7). IVX-121 / IVX-241 mosaic VLPs yielded hMPV-A and hMPV-B titers that tended to be higher than IVX-241 (1 μg) alone, but were not statistically significant. Convalescent human serum samples used in hMPV-A and hMPV-B neutralization titer assays showed titers below the average of IVX-241 or IVX121+IVX-241 immunized animals. [Table 10]

[0460] Example 8: Protective immunity produced by monovalent and bivalent hMPV / RSV combination vaccines A study was conducted to screen and evaluate the ability of IVX-121, IVX-241, and IVX-121+IVX-241 candidate vaccines to protect against hMPV and RSV infection in cotton rats. The vaccine candidates were formulated with Addavax, an oil-in-water emulsion.

[0461] The study included 64 female cotton rats, distributed into 8 groups of 8 animals per group. The immunogens, dose levels, formulation details, and challenge viruses are shown in Table 11.

[0462] Three control groups were administered saline and these groups were subsequently either unchallenged or challenged with RSV-A or hMPV-A2. Groups were administered intramuscularly with IVX-121 or IVX-241 candidate vaccines formulated in Addavax at a dose level of 1.0 μg on days 0 and 21 and then challenged with RSV-A or hMPV-A2, respectively, on day 35. Two groups were administered intramuscularly with IVX-121+IVX-241 at a dose level of 1.0 μg of each VLP formulated in Addavax on days 0 and 21 and then challenged with RSV or hMPV, respectively, on day 35. One group was vaccinated with formalin-inactivated (FI) hMPV and then challenged with hMPV-A2 on day 35. All animals were sacrificed on day 40. Lung and nasal homogenates were used for viral titer screening of RSV-A and hMPV-A2. Serum samples were obtained on days 0, 21, 35, and 40 to measure neutralizing antibody titers for RSV-A and hMPV-A using virus neutralization assays. Clinical observations were performed daily and animals were weighed weekly.

[0463] Animals vaccinated with IVX-121, IVX-241, or IVX-121+IVX-241 showed significantly increased neutralizing titers against RSV and hMPV compared to pre-immune sera at day 35. Titers observed with IVX-121 or IVX-241 were comparable to those observed with the IVX-121+IVX241 formulation (Figures 8A-8B).

[0464] 10 5 Animals were challenged with either plaque-forming units (PFU) of RSV-A2 or hMPV-A, and lung tissue samples were tested for viral replication 5 days after challenge. Control animals not challenged with RSV-A or hMPV showed viral replication below the limit of detection (2.2 log 2 (Figures 8C-8D). Unvaccinated cotton rats challenged with RSV or hMPV produced substantial viral titers in the lungs. Monovalent or bivalent formulations blocked RSV and hMPV pulmonary viral replication below the lower limit of quantification.

[0465] Control animals not challenged with RSV-A had a mean serum concentration below the limit of detection (2.0 log 2 ) whereas unvaccinated animals challenged with RSV-A had substantial virus titers in the nose (Figure 8E). Mice immunized with either IVX-121 or IVX-A12 and challenged with RSV-A showed significantly reduced intranasal virus titers, indicating suppression of virus replication in the upper respiratory tract (Figure 8E).

[0466] Control animals not challenged with hMPV-A2 showed a phenotype below the limit of detection (2.0 log 2 ) whereas unvaccinated animals challenged with hMPV-A2 had substantial virus titers in the nose (Figure 8F). Mice immunized with either IVX-241 or IVX-A12 and challenged with hMPV-A showed intranasal titers of 1.0 log , the same level as observed in the control unchallenged group (2.0 log ). 2) indicating complete inhibition of viral replication (Figure 8F). The FI hMPV vaccinated group had a moderate reduction in intranasal viral titers compared to the challenge control group (Figure 8F). [Table 11]

[0467] Example 9: Summary of clinical evaluation of bivalent RSV-hMPV vaccine These examples describe experiments to demonstrate the safety and efficacy of a RSV / hMPV mixed VLP candidate (IVX-A12). The vaccine is a mixture of two monovalent protein-based virus-like particles (VLPs). Both VLPs are bicomponent icosahedral VLPs that display the ectodomain of the RSV F protein or the hMPV F protein, respectively. The vaccine uses a polypeptide antigen composed of the ectodomain of the RSV or hMPV F protein fused C-terminally to the I53-50A protein (SEQ ID NO: 144) as the first component A. The VLP uses the I53-50B protein as component B. As shown in Figures 1A and 1B, I53-50A and I53-50B are known to spontaneously assemble in a 3:5 ratio to form VLPs with icosahedral (I53) symmetry. The RSV and hMPV components are each engineered to stabilize the pre-fusion conformation of their respective F proteins.

[0468] The immunogenicity of the bivalent vaccine candidate is being evaluated in a Phase 1 First-in-Human (FIH) study in healthy young and older adults by measuring changes in RSV and hMPV nAb levels compared to baseline antibody levels. Various combinations of RSV and hMPV are being screened for potential immune interference due to the addition of hMPV VLPs to the RSV VLP vaccine candidate. Subject to favorable safety outcomes, demonstration of immunogenicity, and determination of the optimal RSV-hMPV dose combination, the efficacy of our RSV-hMPV combined vaccine candidate will be screened. Efficacy will be screened by measuring the incidence of lower respiratory tract infections (LRIs) due to either RSV or hMPV in patients receiving IVX-A12 compared to those receiving placebo. Various formulations of IVX-A12, including with and without adjuvants, may be screened.

[0469] Phase 1 study of IVX-A12 A Phase 1 study of IVX-A12 is planned. The goal of the Phase 1 study of IVX-A12 is to screen the safety and immunogenicity of various doses of IVX-A12 with or without adjuvant in older adults aged 60-75 years. IVX-A12 will be given with a fixed dose of IVX-121 and one of three dose levels of IVX-241 VLP, formulated with and without adjuvant. This design will allow for evaluation of immune responses to both individual components of IVX-A12 and to determine whether the combination of VLPs results in increased reactogenicity or immune interference (i.e., disproportionate immune responses to the component VLPs). All subjects in the Phase 1 study will be evaluated for safety and antibody responses for 12 months after administration of IVX-A12 or placebo. Interim data from the Phase 1 study will be evaluated to determine the need for an adjuvant and to select a dose regimen for evaluation in a Phase 2 dose confirmation study.

[0470] Phase 2 dose confirmation study of IVX-A12 Following completion of the Phase 1 clinical trial of IVX-A12, a Phase 2 dose-confirmatory clinical trial will be initiated in healthy older adults aged 60-75 years. The formulations and dose regimens to be evaluated will be selected in the Phase 2 clinical trial based on data from the Phase 1 trial of IVX-A12. The planned Phase 2 clinical trial will evaluate different combinations of hMPV and RSV VLPs at various concentrations in healthy older adults to screen the safety and immunogenicity of the various concentrations of hMPV and RSV VLPs to guide the selection of the final dose in the subsequent PoC Phase 2b trial.

[0471] Phase 2 extension study of IVX-A12 Older adult subjects who complete the Phase 2 study will be enrolled in a Phase 2 extension study to assess antibody durability and long-term safety over multiple years.

[0472] Phase 2b Proof-of-Concept (PoC) Study of IVX-A12 A global, Phase 2b, randomized, observer-blinded, placebo-controlled, PoC efficacy study will be conducted to evaluate selected formulations of IVX-A12 from the Phase 2 dose-confirmation study. Planned PoC objectives for the Phase 2b study include examination of safety, immunogenicity, and efficacy against LRIs caused by either RSV or hMPV. Study populations will include adults aged 60 years and older, including nested cohorts of frail and high-risk elderly subjects, as well as healthy subjects aged 85 years and older.

[0473] Example 10: A Phase 1 Randomized, Observer-Blinded, Placebo-Controlled, Multicenter Study to Evaluate the Safety and Immunogenicity of IVX-A12, a Bivalent Combined Respiratory Syncytial Virus and Human Metapneumovirus Protein Subunit Vaccine, in Healthy Adults The candidate vaccine, IVX-A12, is a bivalent combination formulation containing IVX-121 and IVX-241 virus-like particles (VLPs), computationally designed recombinant protein subunit vaccines against RSV and hMPV, respectively. Each VLP is composed of two recombinant proteins, component A (CompA) and component B (CompB-01), that are designed to assemble in concert to form an icosahedral structure. Component A is a fusion protein specific for each vaccine candidate (either CompA-RSV-02 or CompA-hMPV-01 for RSV and hMPV, respectively), and is expressed together with the pre-fusion F protein from each virus, which has been shown to induce robust neutralizing antibody responses in preclinical models. CompB-01 is a component common to both VLPs and provides the structural elements that support the multimeric display of CompA-RSV-02 or CompA-hMPV-01. When the two components are combined, they self-assemble into a VLP that exhibits improved immunogenicity compared to either the soluble DS-Cav1 or the hMPV pre-fusion antigen trimer. The soluble DS-Cav1 RSV pre-fusion F protein has recently been clinically tested and shown to be well tolerated and of sustained immunogenicity (Ruckwardt et al., 2021).

[0474] IVX-121 and IVX-241 VLPs display 20 copies of each pre-fusion F protein trimer on their surface. Candidate IVX-121 and IVX-241 VLPs are produced independently and combined to create a bivalent formulation (called IVX-A12) containing both VLPs. The proposed mechanism of action of IVX-A12 is to increase the proportion of RSV-specific and hMPV-specific neutralizing antibodies associated with protection compared to non-neutralizing antibodies (Ngwuta et al., 2015; Falloon et al., 2017). IVX-A12 is intended for active immunization of the target population of older adults, who are at highest risk of disease following infection with RSV and hMPV.

[0475] The candidate vaccine, IVX-A12, is expected to be a single-dose liquid formulation (0.5 mL) for intramuscular (IM) use in the target population, adults aged 60-75 years. The IVX-A12 candidate vaccine will be formulated and vialed as an aqueous vaccine and diluted 1:1 (V / V) with either an oil-in-water emulsion as an adjuvant or a diluent.

[0476] The proposed Phase 1 clinical trial is a first-in-human (FIH) dose-ranging evaluation of the IVX-A12 candidate vaccine in healthy older adults aged 60-75 years. The objective of this Phase 1 study is to evaluate the clinical safety and immunogenicity of one dose of bivalent IVX-A12 candidate vaccine, composed of a fixed dose (volume) level of IVX-121 RSV VLPs and various concentrations (defined as low, medium, and high dose levels) of IVX-241 hMPV VLPs, with and without MF59®, compared to placebo. A placebo (diluent) will be used, as there are no licensed RSV or hMPV vaccines. Three different amounts of selected antigens will be evaluated in three different cohorts in a stepwise fashion. -Low dose level: RSV 75μg / hMPV 75μg, -Medium dose level: 75 μg RSV / 150 μg hMPV, and -High dosage level: RSV 75μg / hMPV 225μg.

[0477] Cohort 1 will evaluate the safety and immunogenicity of a single intramuscular dose of the unadjuvanted IVX-A12 candidate vaccine (IVX-A12a) at a low dosage level (75 μg RSV / 75 μg hMPV) compared to placebo in healthy adults aged 60-75 years.

[0478] Cohort 2 will evaluate the safety and immunogenicity of a single intramuscular dose of (i) the IVX-A12 candidate vaccine at a low dose level (75 μg RSV / 75 μg hMPV) adjuvanted with MF59® (IVX-A12d) and (ii) the unadjuvanted IVX-A12 candidate vaccine at a medium dose level (75 μg RSV / 150 μg hMPV) (IVX-A12a) compared to placebo in healthy adults aged 60-75 years.

[0479] Cohort 3 will evaluate the safety and immunogenicity of a single intramuscular dose of (i) the mid-dose level (75 μg RSV / 150 μg hMPV) of the IVX-A12 candidate vaccine adjuvanted with MF59® (IVX-A12d) and (ii) the high-dose level (75 μg RSV / 225 μg hMPV) of the unadjuvanted IVX-A12 candidate vaccine (IVX-A12a) compared to placebo in healthy adults aged 60-75 years. The high-dose level (75 μg RSV / 225 μg hMPV) of the IVX-A12 candidate vaccine will not be evaluated with MF59® in this clinical trial.

[0480] The study will be conducted in accordance with the International Council on Harmonisation (ICH) protocol, Good Clinical Practice (GCP) guidelines, and applicable regulatory requirements.

[0481] Main purpose: To evaluate the safety and immunogenicity of three dose levels (low, medium, and high) of the bivalent combined RSV / hMPV VLP candidate vaccine (IVX-A12) administered as a single dose regimen to healthy older adults aged 60-75 years compared to placebo by reviewing:

[0482] - Non-spontaneously reported local reactions and systemic AEs during the first 7 consecutive days (Days 0-6) after dosing, and spontaneously reported AEs through 28 days after dosing (Day 28),

[0483] - RSV and hMPV neutralizing antibody (NAb) titers measured by live virus assay, and RSV-specific and hMPV-specific pre-fusion F protein IgG antibody titers measured by enzyme-linked immunosorbent assay (ELISA) on day 28;

[0484] - Geometric mean fold increase (GMFR) on day 28 relative to day 0 for RSV-A-specific and RSV-B-specific NAb titers, hMPV-A-specific and hMPV-B-specific NAb titers, and RSV and hMPV IgG pre-fusion F protein specific antibody titers.

[0485] Secondary Objectives: To further examine the safety of IVX-A12 compared to placebo by the incidence of:

[0486] - Serious adverse events (SAEs), medically-acquired adverse events (MAAEs), and AEs leading to study withdrawal during the study period up to 365 days;

[0487] - AEs of special interest (AESIs) through Day 365;

[0488] - moderate to severe LRTI (clinical events of special interest, CESI) through day 365;

[0489] - Clinical safety laboratory parameters from Day 0 dosing through Day 7.

[0490] - To further evaluate the immunogenicity of IVX-A12 compared to placebo through Day 365 by assessing:

[0491] - RSV and hMPV NAb titers measured by live virus assay;

[0492] - RSV and hMPV pre-fusion F protein specific IgG antibody titers measured by ELISA;

[0493] - the ratio of fold increase in IgG titers compared to fold increase in NAb titers against RSV and hMPV;

[0494] - GMFR relative to baseline (day 0) for RSV-A-specific and RSV-B-specific NAb titers, hMPV-A-specific and hMPV-B-specific NAb titers, and RSV and hMPV IgG pre-fusion F protein specific antibody titers.

[0495] - GMFR relative to baseline (day 0) for hMPV-A-specific and hMPV-B-specific NAb titers.

[0496] Exploratory purpose: To further explore the immunogenicity of the IVX-A12 vaccine compared to placebo by examining:

[0497] Epitope specificity of IgG against pre-fusion F proteins of RSV and hMPV

[0498] VLP core-specific IgG titers.

[0499] Clinical Trial Design: The Phase 1 study of IVX-A12 is a randomized, observer-blinded, placebo-controlled, multicenter, dose- and dose-escalation study to examine the safety and immunogenicity of single doses of two IVX-A12 formulations, aqueous (no adjuvant) and MF59®-adjuvanted (IVX-A12a and IVX-A12d, respectively). The study will use a total of eight treatment arms (five active treatment arms and three placebo arms). The active treatment arms receiving the IVX-A12 formulation will be tested at three dose levels of IVX-A12 (low, medium, and high total VLP content). The low and medium dose levels will be tested with and without MF59®. The high dose level will be tested only without MF59®. The clinical trial will be conducted in three cohorts. The three different dose levels selected (low dose level: 75 μg RSV / 75 μg hMPV, medium dose level: 75 μg RSV / 150 μg hMPV, and high dose level: 75 μg RSV / 225 μg hMPV) will be evaluated in three different cohorts in a stepwise fashion. In addition, a placebo will be administered as a control. A placebo treatment group will be included in each cohort. The three cohorts are shown in Table 12.

[0500] [Table 12]

[0501] Test procedure: Approximately 120 healthy adult subjects (ages 60-75) will be enrolled in one of three cohorts in a stepwise dose escalation (lowest to highest) fashion. Each cohort will enroll adults ages 60-75. Within each cohort, subjects will be randomly assigned in a 5:1 ratio to receive either the investigational drug (IMP; IVX-A12a or IVX-A12d) or placebo (diluent). Cohort 1 will include approximately 24 subjects, with 20 subjects in a single active treatment group and 4 subjects in a placebo group. Cohort 2 will include approximately 48 subjects, with 20 subjects in each of the two active treatment groups and 8 subjects in a corresponding placebo group. Cohort 3 will also include approximately 48 subjects, with 20 subjects in each of the two active treatment groups and 8 subjects in a corresponding placebo group. All subjects will receive a single 0.5 mL intramuscular injection of IVX-A12 or placebo.

[0502] Vaccination will be staged within each cohort to first vaccinate sentinels, with staging within each cohort shown in Table 13.

[0503] In cohort 1, two sentinel subjects will receive IVX-A12a and one sentinel subject will receive placebo (from the placebo group in the corresponding cohort) with a minimum observation period of 36 hours. If no stopping rules apply, the remaining 21 subjects (18 from the active group (group A) and 3 from the corresponding placebo group (cohort 1)) will be enrolled.

[0504] In cohorts 2 and 3, 4 sentinel subjects will receive IVX-A12 (2 subjects in group IVX-A12d [same dose level tested in the previous cohort without adjuvant] and 2 subjects in group IVX-A12a [next dose level without adjuvant]) and 2 sentinel subjects will receive placebo (placebo group within the corresponding cohort) with a minimum observation period of 36 hours. If there is no reason to apply a stopping rule, the remaining 42 subjects (18 from the active IVX-A12a group, 18 from the active IVX-A12d group and 6 from the placebo group of each dose level cohort) will be vaccinated. If a stopping rule is met, an independent SMC will be responsible for its application. If all subjects in each dose level cohort have no reason to apply the stopping rule within 7 days after dosing and the reactogenicity of the IVX-A12a and IVX-A12d formulations is found to be acceptable per the judgment of the SMC, the study will progress to subsequent dose level cohorts. Prior to progression to and initiation of subsequent dose level cohorts, the SMC will review all available cumulative safety data through day 7 post-vaccination.

[0505] Cohort 1: Evaluation of safety and immunogenicity of the IVX-A12 unadjuvanted candidate vaccine (IVX-A12a) at a low dose level (75μg RSV / 75μg hMPV) compared to placebo. Enrolled subjects (N=24) will be randomized in a 5:1 ratio to receive the low dose of IVX-A12a (Group A) or placebo (Group B). Two sentinel subjects (Stage 1) will receive IVX-A12a (Group A) and one sentinel subject will receive placebo (Group B). After an observation period, the remaining subjects in Group A (N=18) and Group B (N=3) will be vaccinated (Stage 2). The SMC will review the safety data after enrollment of Cohort 1 is complete and before proceeding with enrollment of Cohort 2.

[0506] Cohort 2: Evaluation of the safety and immunogenicity of the IVX-A12 candidate vaccine at a low dose level (75 μg RSV / 75 μg hMPV) with MF59® and the unadjuvanted IVX-A12 candidate vaccine at a medium dose level (75 μg RSV / 150 μg hMPV) compared to placebo. Enrolled subjects (N=48) are randomized in a 5:1 ratio to receive IVX-A12d (low dose level) or IVX-A12a at a medium dose level (Groups C or D, respectively) or placebo (Group E). Four sentinel subjects (Stage 3) receive IVX-A12 (two subjects in Group C [low dose IVX-A12d] and two subjects in Group D [medium dose IVX-A12a]) and two sentinel subjects receive placebo (Group E). After the observation period, the remaining subjects in Groups C, D, and E will be vaccinated (Stage 4). After enrollment of Cohort 2 is complete, the SMC will review the safety data before proceeding with enrollment of Cohort 3.

[0507] Cohort 3: Evaluation of the safety and immunogenicity of the unadjuvanted IVX-A12 candidate vaccine (IVX-A12a) at medium dose level (75 μg RSV / 150 μg hMPV) and high dose level (75 μg RSV / 225 μg hMPV) with MF59® compared to placebo. Enrolled subjects (N=48) are randomized in a 5:1 ratio to receive IVX-A12d (medium dose) or high dose level IVX-A12a (Groups F or G, respectively) or placebo (Group H). Four sentinel subjects (Stage 5) receive IVX-A12 (2 subjects in Group F [medium dose IVX-A12d] and 2 subjects in Group G [high dose IVX-A12a]) and 2 sentinel subjects receive placebo (Group H). Following the observation period, the remaining subjects in Groups F, G, and H will be vaccinated (Stage 6). The SMC will review safety data following enrollment of Cohort 3 as appropriate and will not evaluate additional dose escalation beyond Cohort 3. Additionally, the study will not evaluate the IVX-A12 candidate vaccine at high dose levels (75 μg RSV / 225 μg hMPV) with MF59®. [Table 13]

[0508] †MF59® contains 9.75 mg squalene and surfactant. ‡Placebo is aqueous diluent. Group B corresponds to stages 1 and 2, Group E corresponds to stages 3 and 4, and Group H corresponds to stages 5 and 6. For the purposes of data analysis, treatment groups B, E, and H are combined into a single placebo treatment group.

[0509] examination: A total of seven scheduled visits will be required for each subject: Screening, Days 0, 7, 28, 90, 180, and 365. Safety blood sampling will occur at Screening and Days 0, 7, and 28, and immunogenicity sampling will occur on Days 0, 7, 28, 180, and 365.

[0510] Safety screening for all subjects in cohorts 1, 2, and 3: All non-spontaneously reported local reactions and systemic AEs will be collected for all subjects beginning on day 0 (day of single dose administration) through day 6 (7 days total), spontaneously reported AEs through day 28, and safety blood samples (laboratory assessments at screening, day 0 / baseline, day 7, and day 28). SAEs, MAAEs, AEs leading to study withdrawal, and AESIs will be recorded from the time of randomization through the end of study evaluations approximately 12 months after vaccination. During treatment and follow-up, all subjects will also be actively monitored for CESIs according to the study definition of moderate to severe LRTI using established case definitions and assessment algorithms for acute respiratory illness (ARI) and nasopharyngeal (NP) swabs for detection of RSV, hMPV, and other respiratory viruses. Subjects will be instructed to return to the clinic for evaluation within 7 days (ideally within 72 hours) of onset of ARI to maximize virus detection. Immunogenicity Screening of all subjects in Cohorts 1, 2 and 3: Blood samples will be collected for serology (Day 0 / Baseline, Day 7, Day 28, Day 180, and Day 365). All subjects will be monitored for persistence of antibody responses throughout the duration of the clinical trial (approximately 12 months).

[0511] In the event of local or national health-related closures and travel restrictions (e.g., as a result of the SARS-CoV-2 outbreak), clinics may follow default facility guidelines for continuing subject follow-up screening as needed.

[0512] Stopping rules: Monitoring of safety signals will be performed by an independent SMC throughout the study. Clear evidence of harm or adverse effects will result in stopping rules or conditions to discontinue the clinical trial. The occurrence of any of the following events (or events) following administration of the vaccine may result in stopping the study and require further scrutiny and review:

[0513] The occurrence of the following in test subjects: - Deaths occurring during the study, - Any vaccine-related SAEs during the study, - Any life-threatening (Grade 4) vaccine-related AE during the study requiring medical intervention, including: o Ulceration, abscess, or necrosis at the injection site; - Laryngospasm, bronchospasm, or anaphylaxis within 24 hours after administration of the vaccine, o An allergic or hypersensitivity reaction such as fever above 40°C or generalized urticaria (defined as occurring in three or more body sites) within 72 hours after administration of the vaccine.

[0514] If 2 or more subjects in a single study arm in any of the 3 cohorts experience the same severe (Grade 3) AE (preferred term [PT] in a given MedDRA system organ class [SOC]) within the first 7 days after vaccination that lasts for at least 48 hours and cannot be attributed to another cause: - Severe (grade 3) non-spontaneous reported local reactions (excluding measurable grade erythema and swelling only) or systemic AEs, - Spontaneously reported severe (grade 3) vaccine-related AEs during the study, - Severe (grade 3) vaccine-related abnormalities in vital sign(s), - Severe (grade 3) vaccine-related laboratory abnormalities.

[0515] If a predefined safety signal is met in any study arm, subsequent dosing will result in at least a temporary halt of the study to allow for a full evaluation of the reported event(s) and to consult with the SMC, which may recommend temporary or permanent discontinuation or continuation of dosing based on review of the data. After the pause, further safety measures may be introduced.

[0516] Target population: Health Target: Applicable.

[0517] Age range: 60-75 years old.

[0518] Planned number of subjects: Approximately 120 total (24 in Cohort 1, 48 in Cohort 2, and 48 in Cohort 3).

[0519] Planned Number of Study Arms: Eight total treatment arms (five active treatment arms and three placebo arms [one placebo arm for each cohort]). The active treatment arms receiving the IVX-A12 formulation will be tested at three dose levels of IVX-A12 (low, medium, and high total VLP content). The low and medium dose levels will be tested with and without MF59®. The high dose level will be tested only without MF59®.

[0520] Key inclusion criteria: 1. Healthy male or non-pregnant female older adults aged 60...

Claims

1. A composition or pharmaceutical composition, a) Two or more virus-like particles (VLPs), i. The first virus-like particle (VLP) comprises a first component containing the respiratory syncytial virus (RSV) F protein ectodomain or its antigenic variant, ii. The second virus-like particle (VLP) contains a first component which includes the human metapneumovirus (hMPV) F protein ectodomain or its antigenic variant. Virus-like particles (VLPs), and / or b) Virus-like particles (VLPs) comprising multiple first components, wherein some of the first components comprise the respiratory syncytial virus (RSV) F protein ectodomain or its antigenic variant, and some of the first components comprise the human metapneumovirus (hMPV) F protein ectodomain or its antigenic variant. Includes, If necessary, each of the VLPs may independently a) The first component comprising a first multimerization domain, b) A second component containing a second multimerization domain and A composition or pharmaceutical composition containing the following:

2. (i) The first multimerization domain is selected from Sequence IDs 1, 4, 5, 7, 9, 18, 19, 21, 24, 25, 26, 29, 30, 31, 34, 36, 37, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 144, 145, or a functional variant thereof, (ii) The first multimerization domain shares at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50A (SEQ ID NO: 145); and optionally the first multimerization domain includes amino acid substitutions C74A and C98A, amino acid substitutions C163A and C201A, or amino acid substitutions C74A, C98A, C163A, and C201A, compared to SEQ ID NO:

144. The composition or pharmaceutical composition according to claim 1.

3. The composition or pharmaceutical composition according to claim 1, wherein the second multimerizing domain is selected from SEQ ID NOs: 2, 3, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 20, 22, 23, 27, 28, 32, 33, 35, 38, 40, and 41, or functional variants and fragments thereof, and optionally the second multimerizing domain shares at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with I53-50B (SEQ ID NO: 8) or I53-50B.4PosT1 (SEQ ID NO: 34).

4. The RSV F protein ectodomain is RSV F DS-Cav1 (SEQ ID NO: 173): QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKY KNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSA IASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILN KQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITND QKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGS NICLTRTDRGWYCDNAGSVSFFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKY DCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSV GNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL Sharing at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with, The composition or pharmaceutical composition according to claim 1, wherein the RSV F protein ectodomain optionally comprises amino acid substitutions S155C and S290C, and / or amino acid substitutions S190F and V207L.

5. The first component of the first VLP is DS-Cav1-I53-50A (SEQ ID NO: 151): QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPPRFM NYTLNNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPI LNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEV LAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIF NPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINF YDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLGSGGSGSGSGGGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFA GGVHLIEITFTVPDADVIKALSVLKEKGAIIGAGTVTSVEQCRKAVESGAEEFIVSPHLDEEISQFCKEKGVFYMPGVMPTTELLVKAAMKLGHTILKLFPGEVVGPQFVKAAMKGPFPNVKFVPTGGVNLDNVCEWFKAAGVLAVGVGGSALVKGTPDEVREKAKAAFVEKIRGCTE (Sequence ID 151) The composition or pharmaceutical composition according to claim 1, comprising a polypeptide that shares at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the above.

6. The hMPV F protein ectodomain is SEQ ID NO: 174 or SEQ ID NO: 175: KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDCPSLIKTELDLTKSALRELKTVSADQLAREEQIEGGGGGGGFVLGAIALGVATAAAVTAGIAIAKTIRLESEV NAIKGCLKTTNECVSTLGNGVRVLATAVRELKEFVSKNLTSAINKNKCDIADLCMAVS FSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAM VRRKGFGILIGVYGSSVIYMVQLPIFGVIIDTPPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNDKDCETRGDHVFCDTAAGINVAEQSRECNINIRSTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLPKGCSYITNQDADTVTIIDNTVYQLSKVEGEQHVIKGRPVVSSSFSFDPICFPEDQFNVALDQVFESIENCQA (Sequence ID 174), KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEGGGGGGGFVLGAIALGVATAAAVTAGVAIAKCIRLESEV TAIKNALKKTNEAVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVS FSQFNRRFLNVVRQFSDNAGITPAISKDLMTDAELARAISNMPTSAGQIKLMLENRAM VRRKGFGILIGVYGSSVYMVQLPIFGVIIDTPPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINITTNYPCKVSCGRNPISMVALSPLGALVACYKGVSSCSIGSNRVGIIKQLNKGCSYITNQDADTVTINDTVYQLSKVEGEQHVIKGRPVVSSSFSFDPVKFPEDQFNVALDQCFESIENSQA (Sequence ID 175) Sharing at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with, The composition or pharmaceutical composition according to claim 1, wherein the hMPV F protein ectodomain optionally contains amino acid substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, and / or R102G, or the hMPV F protein ectodomain contains amino acid substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, N97G, P98G, R99G, Q100G, H386N, S101G, and / or R102G compared to the reference hMPV F protein sequence (SEQ ID NO: 56).

7. (i) The first component of the second VLP comprises a polypeptide that shares at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 176; or (ii) The first component of the second VLP contains a polypeptide that shares at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 177 The composition or pharmaceutical composition according to claim 1.

8. The composition or pharmaceutical composition according to claim 1, wherein the composition or pharmaceutical composition comprises one or more pharmaceutically acceptable diluents, adjuvants, or excipients.

9. A unit dose of the composition or pharmaceutical composition according to claim 1, a) The first VLP in amounts of approximately 0.5 μg, approximately 1 μg, approximately 20 μg, approximately 25 μg, approximately 70 μg, approximately 75 μg, approximately 100 μg, approximately 125 μg, approximately 150 μg, approximately 200 μg, approximately 225 μg, approximately 250 μg, approximately 300 μg, or approximately 500 μg, b) The second VLP in amounts of approximately 0.5 μg, 1 μg, 20 μg, 25 μg, 70 μg, 75 μg, 100 μg, 125 μg, 150 μg, 200 μg, 225 μg, 250 μg, 300 μg, or 500 μg and including; or a) Approximately 75 μg or approximately 150 μg of the first VLP, b) Approximately 75 μg of the second VLP and including; or a) Approximately 75 μg or approximately 150 μg of the first VLP, b) Approximately 150 μg of the second VLP and including; or a) Approximately 75 μg or approximately 150 μg of the first VLP, b) Approximately 150 μg or approximately 225 μg of the second VLP and including; or a) Approximately 5 μg, approximately 10 μg, approximately 15 μg, approximately 20 μg, approximately 25 μg, approximately 30 μg, approximately 35 μg, approximately 40 μg, approximately 45 μg, approximately 50 μg, approximately 55 μg, approximately 60 μg, approximately 65 μg, approximately 70 μg, approximately 75 μg, approximately 100 μg, approximately 150 μg, approximately 200 μg, approximately 250 μg, or approximately 300 μg of the first VLP, b) The second VLP in amounts of approximately 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 100 μg, 150 μg, 200 μg, 250 μg, or 300 μg, and Unit doses, including.

10. The composition or pharmaceutical composition according to Claim 1, (i) A method of vaccinating a target, comprising administering an effective amount of the composition or pharmaceutical composition to the target; or (ii) A method for inducing an immune response in a subject, comprising administering an effective amount of the composition or pharmaceutical composition to the subject; or (iii) A method for preventing infection by paramyxovirus, comprising administering an effective amount of the composition or pharmaceutical composition to the subject; or (iv) A method for sensitizing a subject to paramyxovirus, comprising administering an effective amount of the composition or pharmaceutical composition to the subject. A composition or pharmaceutical composition for use in [location / area].

11. (i) The method generates protective immunity against respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV; and / or (ii) The method generates neutralizing antibodies against respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV; and / or (iii) The subject is at risk of severe RSV disease and / or severe hMPV disease; or (iv) The method results in the production of an RSV-B specific neutralizing antibody in the subject. The composition or pharmaceutical composition according to claim 10.

12. The effective amount includes approximately 0.5 μg, approximately 1 μg, approximately 20 μg, approximately 25 μg, approximately 70 μg, approximately 75 μg, approximately 100 μg, approximately 125 μg, approximately 150 μg, approximately 200 μg, approximately 225 μg, approximately 250 μg, approximately 300 μg, or approximately 500 μg of the first VLP and / or the second VLP, as needed. The pharmaceutical composition according to claim 10, wherein the effective amount comprises about 75 μg of the first VLP and about 75 μg of the second VLP; or the effective amount comprises about 75 μg of the first VLP and about 150 μg of the second VLP; or the effective amount comprises about 150 μg of the first VLP and about 150 μg of the second VLP.

13. The pharmaceutical composition according to claim 10, wherein the method further comprises administering a second dose of the pharmaceutical composition; the second dose is administered, if necessary, within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 12 months, about 24 months, or about 36 months after the first dose.

14. The pharmaceutical composition according to claim 13, further comprising administering a third dose of the pharmaceutical composition; the third dose being administered, if necessary, about one year, about two years, about three years, about four years, or about five years after the second dose; and the method further comprising administering subsequent doses at regular intervals of about one, two, three, four, or five years, if necessary.

15. (i) the method restricts the development of RSV infection in the subject, and / or the method restricts the development of hMPV infection in the subject; and / or (ii) The method results in the production of an RSV-A specific neutralizing antibody in the subject; and / or (iii) The method results in an increase in the subject of at least about 2 times, about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, or at least about 25 times compared to baseline; and / or (iv) The method results in the production of an hMPV-A specific neutralizing antibody in the subject; and / or (v) The method results in the production of an hMPV-B specific neutralizing antibody in the subject; and / or (vi) The above method prevents severe lower respiratory tract infections (LRTIs), The pharmaceutical composition according to claim 11.

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