Multivalent vaccines against paramyxovirus and uses thereof

Abstract

Provided herein are compositions or pharmaceutical compositions comprising two or more virus-like particles (VLPs) wherein a first virus-like particle (VLP) comprises a first component comprising an extracellular domain of respiratory syncytial virus (RSV) F protein, or an antigenic variant thereof; and the second virus-like particle (VLP) comprises a first component comprising an extracellular domain of human metapneumovirus (hMPV) F protein or an antigenic variant thereof. Further provided are methods of vaccination using the compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 342,953 filed on May 17, 2022, U.S. Provisional Patent Application No. 63 / 367,109 filed on June 27, 2022, U.S. Provisional Patent Application No. 63 / 378,151 filed on October 3, 2022, and U.S. Provisional Patent Application No. 63 / 387,092 filed on December 12, 2022, each of which is incorporated herein by reference in its entirety.

[0003] Incorporation by reference into the sequence listing

[0004] This application contains a sequence listing that has been submitted by EFS-WEB in .XML format 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 Art

[0005] 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 (subgroups A and B, respectively), which are antigenically related, and infection with either subgroup induces cross-neutralizing antibodies. RSV is seasonally transmitted and is the leading cause of lower respiratory tract infections (LRTIs) in all age groups worldwide. Cumulative data indicate that the burden of disease in adults is considerable, comparable to influenza, with the majority of hospitalizations and deaths occurring in the elderly over the age of 65 years. Epidemiological data indicate that RSV causes >170,000 hospitalizations and ~14,000 deaths per year in the United States alone. Similarly, in Europe, RSV is an important cause of respiratory disease. The incidence and severity of RSV disease are particularly high in the frail elderly and those with cardiopulmonary disease, who are at high risk for complications and hospitalization. RSV is transmitted through respiratory droplets and close contact with infected persons or fomites. In temperate climates, epidemics occur every winter, while in tropical regions, seasonality is less pronounced, but infection is most common during the rainy season. The severity of RSV disease is largely determined by the extent of viral replication following infection. Induction of neutralizing antibodies (NAbs) should be the goal of vaccination as they are associated with protection against disease. However, seropositivity provides only partial protection against infection.

[0006] Human metapneumovirus (hMPV) is a single-stranded, negative-sense RNA virus of the genus Metapneumovirus in the family Pneumoviridae. The most closely related human pathogen is RSV. As with RSV, there are two major genetic lineages, A and B (subgroups A and B, respectively), which are antigenically related, and infection with either subgroup induces cross-neutralizing antibodies. In mouse models, infection with hMPV protects against recent reinfection; macaques infected with hMPV exhibit seroconversion and transient protection against subsequent infection that wanes over months. Similar to RSV, NAbs alone can protect against hMPV disease. Despite protective antibody titers in adults, both healthy and immunocompromised individuals are reinfected with hMPV. In the elderly, hMPV is responsible for a large proportion of severe respiratory infections, with infection rates similar to those of RSV. In the Etiology of Pneumonia in the Community (EPIC) trial, 4% of adults hospitalized for community-acquired pneumonia were diagnosed with hMPV infection, while 3% were diagnosed with RSV infection. There are currently no approved vaccines for either RSV or hMPV.

[0007] Therefore, there is a long-standing and unmet need for multivalent vaccines against paramyxoviruses, such as a bivalent RSV-hMPV vaccine. Summary of the invention

[0008] The present disclosure relates to a multivalent vaccine for paramyxovirus and its use. On the one hand, 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 further components, which are assembled together to form a VLP. 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) may comprise a first component (of this first VLP), which comprises a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof. The second virus-like particle (VLP) may comprise a first component (of this second VLP), which comprises a human metapneumovirus (hMPV) F protein ectodomain or an antigenic variant thereof. Two VLPs can be mixed together to form a composition or pharmaceutical composition, which is then used for vaccination or other purposes.

[0009] On the other hand, the present disclosure provides compositions or pharmaceutical compositions, virus-like particles (VLPs) comprising a plurality of first components. Some first components may comprise a respiratory syncytial virus (RSV) F protein extracellular domain or an antigenic variant thereof, while other first components comprise a human metapneumovirus (hMPV) F protein extracellular domain or an antigenic variant thereof. The VLPs are assembled from a mixture of the first component and optionally one or more further components. These "chimeric" VLPs can be used for vaccination or other purposes.

[0010] The present disclosure provides a composition or pharmaceutical composition comprising two or more virus-like particles (VLPs), wherein a first virus-like particle (VLP) comprises a first component, the first component comprising the extracellular domain of the respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and a second virus-like particle (VLP) comprises a first component, the first component comprising the extracellular domain of the human metapneumovirus (hMPV) F protein or an antigenic variant thereof; and / or a virus-like particle (VLP) comprising multiple first components, some of which comprise the extracellular domain of the respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and some of which comprise the extracellular domain of the human metapneumovirus (hMPV) F protein or an antigenic variant thereof.

[0011] In some embodiments, the VLPs each independently comprise a first component and a second component, wherein the first component comprises a first multimerization domain; and the second component comprises a second multimerization domain.

[0012] 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.

[0013] 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 I53-50AΔCys (SEQ ID NO: 145).

[0014] In some embodiments, relative to SEQ ID NO: 144, 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.

[0015] 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.

[0016] 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).

[0017] In some embodiments, the RSV F protein extracellular 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 RSV F DS-Cav1 (SEQ ID NO: 173).

[0018] In some embodiments, the RSV F protein extracellular domain comprises amino acid substitutions S155C and S290C; and / or amino acid substitutions S190F and V207L.

[0019] 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).

[0020] In some embodiments, the hMPV F protein extracellular 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: 174 or SEQ ID NO: 175.

[0021] In some embodiments, the hMPV F protein extracellular domain comprises amino acid substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G and / or R102G relative to a reference hMPV F protein sequence (SEQ ID NO: 56); or wherein the hMPV F protein extracellular domain comprises amino acid substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, N97G, P98G, R99G, Q100G, H386N, S101G and / or R102G.

[0022] 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.

[0023] 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.

[0024] In some embodiments, the composition comprises one or more pharmaceutically acceptable diluents, adjuvants, or excipients.

[0025] In some embodiments, the composition comprises a stable emulsion. In some embodiments, the vaccine comprises one or more adjuvants. In some embodiments, one or more adjuvants are squalene, alum, SLA, GLA, R848, IMQ, 3M-052, CpG, saponin (QS21) or a combination 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 citric acid buffer containing stabilized nonionic 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.

[0026] The present disclosure provides a unit dose of a composition or pharmaceutical composition as disclosed herein, wherein the unit dose comprises: 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 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 the first VLP;

[0027] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising an extracellular domain of a RSV F protein 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 extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0028] In some embodiments, the unit dose comprises about 100 μg of a first VLP comprising an extracellular domain of a RSV F protein 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 extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0029] In some embodiments, the unit dose comprises about 125 μg of a first VLP comprising an extracellular domain of a RSV F protein 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 extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0030] In some embodiments, the unit dose comprises about 150 μg of a first VLP comprising an extracellular domain of a RSV F protein 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 extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0031] In some embodiments, the unit dose comprises about 175 μg of a first VLP comprising an extracellular domain of a RSV F protein 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 extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0032] In some embodiments, the unit dose comprises about 200 μg of a first VLP comprising an extracellular domain of a RSV F protein 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 extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0033] In some embodiments, the unit dose comprises about 225 μg of a first VLP comprising an extracellular domain of a RSV F protein 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 extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0034] In some embodiments, the unit dose comprises about 250 μg of a first VLP comprising an extracellular domain of a RSV F protein 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 extracellular domain of a hMPV F protein or an antigenic variant thereof.

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

[0036] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising an RSV F protein extracellular domain or an antigenic variant thereof and about 150 μg of a second VLP comprising an hMPV F protein extracellular domain or an antigenic variant thereof.

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

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

[0039] In some embodiments, the unit dose comprises about 225 μg of a first VLP comprising an RSV F protein extracellular domain or an antigenic variant thereof and about 75 μg of a second VLP comprising an hMPV F protein extracellular domain or an antigenic variant thereof.

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

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

[0042] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising an RSV F protein extracellular domain or an antigenic variant thereof, about 150 μg of a second VLP comprising an hMPV F protein extracellular domain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0043] In some embodiments, the unit dose comprises about 75 μg of a first VLP comprising an RSV F protein extracellular domain or an antigenic variant thereof, about 225 μg of a second VLP comprising an hMPV F protein extracellular domain 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 an RSV F protein extracellular domain or an antigenic variant thereof, about 150 μg of a second VLP comprising an hMPV F protein extracellular domain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0045] In some embodiments, the unit dose comprises about 225 μg of a first VLP comprising an RSV F protein extracellular domain or an antigenic variant thereof, about 75 μg of a second VLP comprising an hMPV F protein extracellular domain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0046] In some embodiments, the unit dose comprises about 150 μg of a first VLP comprising an RSV F protein extracellular domain or an antigenic variant thereof, about 75 μg of a second VLP comprising an hMPV F protein extracellular domain or an antigenic variant thereof, and an adjuvant comprising MF59.

[0047] The present disclosure provides a method of vaccinating a subject, comprising administering to the subject an effective amount of a composition according to the present disclosure.

[0048] In some embodiments, a method of 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.

[0049] In some embodiments, a method of preventing infection by a Paramyxovirus comprises administering to a subject an effective amount of a composition according to the present disclosure.

[0050] 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.

[0051] In some embodiments, the paramyxovirus is respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV.

[0052] In some embodiments, the methods of the invention generate protective immunity against respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV.

[0053] In some embodiments, the methods of the invention generate neutralizing antibodies against respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV.

[0054] In some embodiments, the subject is at risk for severe RSV disease and / or at risk for severe hMPV disease.

[0055] 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 the following methods: subcutaneous injection, intramuscular injection, intradermal injection, intranasal, oral, sublingual or buccal.

[0056] 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 and 45.

[0057] 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 the above amounts of the second VLP.

[0058] In some embodiments, the effective amount comprises about 75 μg of a first VLP comprising the extracellular domain of a RSV F protein 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 the extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0059] In some embodiments, the effective amount comprises about 100 μg of a first VLP comprising the RSV F protein extracellular domain 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 the hMPV F protein extracellular domain or an antigenic variant thereof.

[0060] In some embodiments, the effective amount comprises about 125 μg of a first VLP comprising the extracellular domain of a RSV F protein 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 the extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0061] In some embodiments, the effective amount comprises about 150 μg of a first VLP comprising the RSV F protein extracellular domain 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 the hMPV F protein extracellular domain or an antigenic variant thereof.

[0062] In some embodiments, the effective amount comprises about 175 μg of a first VLP comprising the RSV F protein extracellular domain 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 the hMPV F protein extracellular domain or an antigenic variant thereof.

[0063] In some embodiments, the effective amount comprises about 200 μg of a first VLP comprising the RSV F protein extracellular domain 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 the hMPV F protein extracellular domain or an antigenic variant thereof.

[0064] In some embodiments, the effective amount comprises about 225 μg of a first VLP comprising the extracellular domain of a RSV F protein 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 the extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0065] In some embodiments, the effective amount comprises about 250 μg of a first VLP comprising the RSV F protein extracellular domain 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 the hMPV F protein extracellular domain or an antigenic variant thereof.

[0066] 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 above amounts of the second VLP.

[0067] In some embodiments, the effective amount comprises about 75 μg of a first VLP comprising the RSV F protein extracellular domain 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 the hMPV F protein extracellular domain or an antigenic variant thereof.

[0068] In some embodiments, the effective amount comprises about 150 μg of a first VLP comprising an extracellular domain of a RSV F protein 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 an extracellular domain of a hMPV F protein or an antigenic variant thereof.

[0069] In some embodiments, the effective amount comprises about 150 μg of a first VLP comprising an RSV F protein extracellular domain or an antigenic variant thereof; and about 150 μg of a second VLP comprising an hMPV F protein extracellular domain or an antigenic variant thereof.

[0070] In some embodiments, the method comprises administering a second dose of the pharmaceutical composition.

[0071] 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 of the first dose.

[0072] In some embodiments, the method comprises 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.

[0073] 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.

[0074] 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.

[0075] In some embodiments, the method results in the subject producing RSV-A specific neutralizing antibodies.

[0076] In some embodiments, the method results in an increase in RSV-A specific neutralizing antibodies in the subject by 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.

[0077] In some embodiments, the 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, 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.

[0078] In some embodiments, the method results in the subject producing RSV-B specific neutralizing antibodies.

[0079] In some embodiments, the method results in an increase in RSV-B specific neutralizing antibodies in the subject by 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.

[0080] In some embodiments, the increase in RSV-B specific neutralizing antibodies is detectable within about one 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.

[0081] In some embodiments, the method results in the subject producing hMPV-A specific neutralizing antibodies.

[0082] In some embodiments, the method results in an increase in hMPV-A specific neutralizing antibodies in the subject by 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.

[0083] In some embodiments, an increase in hMPV-A specific neutralizing antibodies can be detected within about one 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.

[0084] In some embodiments, the method results in the subject producing hMPV-B specific neutralizing antibodies.

[0085] In some embodiments, the method results in an increase in hMPV-B-specific neutralizing antibodies in the subject by 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.

[0086] In some embodiments, an increase in hMPV-B specific neutralizing antibodies can be detected within about one 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.

[0087] In some embodiments, the methods prevent severe lower respiratory tract infections (LRTIs).

[0088] Pre-filled syringes containing compositions according to the present disclosure are provided.

[0089] Kits comprising a composition according to the disclosure or including a pre-filled syringe of the disclosure are provided.

[0090] The present disclosure provides a kit comprising one or more of the following:

[0091] A composition comprising a virus-like particle (VLP), the virus-like particle comprising a first component, the first component comprising an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof;

[0092] A composition comprising a second virus-like particle (VLP), the second virus-like particle comprising a first component, the first component comprising an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof; and

[0093] A composition comprising a virus-like particle (VLP), wherein the virus-like particle comprises a plurality of first components, some of which comprise the extracellular domain of the F protein of respiratory syncytial virus (RSV) or an antigenic variant thereof, and some of which comprise the extracellular domain of the F protein of human metapneumovirus (hMPV) or an antigenic variant thereof.

[0094] In some embodiments, the kit comprises a composition comprising an adjuvant to be combined with one or more VLP compositions prior to administration to a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1A Exemplary embodiments of protein-based virus-like particles (VLPs) according to the present disclosure are shown.

[0096] Figure 1B Further exemplary embodiments of VLPs and VLP components are shown (F protein not shown).

[0097] Figure 2 Diagram of the secondary structure of the hMPV F protein showing the prefusion conformation (PDB ID: 5WB0; figure generated at www.rcsb.org).

[0098] Figures 3A-3C is a series of graphical representations showing antibody binding profiles of exemplary VLPs of the present disclosure. The antibodies are specific for various forms of the hMPV F protein. MF14 binds to both pre-fusion and post-fusion forms of the hMPV F protein at the site II epitope ( Figure 3A MF16 binds to the pre- and post-fusion forms of the hMPV F protein at the site IV epitope ( Figure 3B MPE8 binds to the prefusion form of hMPV F protein at the site III epitope ( Figure 3C ).

[0099] Figure 4A and 4B The immunogenicity of hMPV F protein mutants expressed on 2-component virus-like particles (VLPs) or as soluble proteins (sol) is shown. The graph shows the immunogenicity of hMPV-A ( Figure 4A ) and hMPV-B( Figure 4B ) of neutralizing antibody titer.

[0100] Figure 5A and 5BThe immunogenicity of hMPV008 VLP and the corresponding soluble protein CompA-hMPV008 at different doses and adjuvant conditions is shown. Also shown is hMPV033 when adjuvanted with squalene emulsion (SE). The graph shows the hMPV-A ( Figure 5A ) and hMPV-B( Figure 5B ) of neutralizing antibody titer.

[0101] Figure 6 RSV neutralization titers from sera of mice immunized with monovalent, bivalent or chimeric vaccine compositions are shown. Mice immunized with only hMPV monovalent vaccine (IVX-241) showed low immune responses to RSV. Monovalent RSV, bivalent RSV / hMPV and chimeric RSV / hMPV groups showed high anti-RSV immune responses.

[0102] Figure 7 hMPV neutralization titers of sera from mice immunized with monovalent or bivalent vaccine compositions are shown. Mice immunized with only RSV monovalent vaccine (IVX-121) showed low immune responses to hMPV. Monovalent hMPV, bivalent RSV / hMPV, and chimeric RSV / hMPV groups showed high anti-hMPV immune responses.

[0103] Figures 8A-8F It was shown that the bivalent RSV-hMPV VLP vaccine induced immunogenicity and protection compared to monovalent RSV or hMPV VLP vaccines in the cotton rat live virus challenge model.

[0104] Fig. 8A Shown are the titers of RSV-A neutralizing antibodies in sera from animals immunized with monovalent RSV ("IVX-121") or bivalent ("IVX-A12") VLPs.

[0105] Figure 8B Shown are the titers of hMPV-A neutralizing antibodies in sera from animals immunized with monovalent hMPV ("IVX-241") or bivalent ("IVX-A12") VLPs.

[0106] Figure 8C Shown are the titers of RSV-A virus in the lungs of animals immunized with monovalent RSV ("IVX-121") or bivalent ("IVX-A12") VLPs and then challenged with RSV virus.

[0107] Fig.8D Shown are the titers of hMPV-A viruses in the lungs of animals immunized with monovalent hMPV ("IVX-241") or bivalent ("IVX-A12") VLPs and then challenged with hMPV viruses.

[0108] Fig. 8E Shown are nasal titers of RSV-A virus in animals immunized with monovalent RSV ("IVX-121") or bivalent ("IVX-A12") VLPs and then challenged with RSV virus.

[0109] Fig.8F Shown are nasal titers of hMPV-A virus in animals immunized with monovalent hMPV ("IVX-241") or bivalent ("IVX-A12") VLPs and then challenged with hMPV virus.

[0110] Fig. 9 is a series of graphs showing the time course of induction of neutralizing antibody titers in mice in response to administration with hMPV F protein VLPs formulated with an adjuvant.

[0111] Fig.10 The study design is a Phase 1 / 1b randomized, observer-blind, placebo-controlled study to evaluate the administration of IVX-121 in young and elderly subjects.

[0112] Fig.11 A summary of safety data is shown. There were no serious adverse events (SAEs), AEs of special interest (AESIs), or adverse events (AEs) leading to study withdrawal.

[0113] Fig.12 A graph of local and systemic adverse events elicited within 7 days of a single dose is shown, with the highest severity ("Alum" = 500 μg / mL aluminum hydroxide). Unadjuvanted IVX-121 reactogenicity was mild in the elderly, with tolerability similar to placebo.

[0114] Fig.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 were comparable in young and elderly subjects.

[0115] Fig.14 A graph of RSV-A neutralizing antibodies (nAB) without and with adjuvant is shown. Geometric mean titers (GMT) are expressed in international units per milliliter (IU / mL). Alum adjuvant had no beneficial effect in young and elderly individuals.

[0116] Fig.15 Tables summarizing neutralizing and binding antibody data are shown.

[0117] Fig.16A graph of unadjuvanted RSV-A neutralizing antibodies (nAB) in young (left) and elderly (right) subjects through Day 180 is shown. Geometric mean titers (GMTs) are expressed in international units per milliliter (IU / mL). Lower limit of quantitation (LloQ) = 9.9; Geometric mean fold increase from baseline (Day 28). Data reflect all subjects available at the time of this six-month analysis; individual numbers may not differ substantially from initial interim readings.

[0118] Fig.17 A graph of unadjuvanted RSV-A neutralizing antibodies (nAB) in young subjects at day 180 is shown. The μg number of total VLPs and the μg number of RSV-A antigen for each treatment dose are shown. The percentage of titers retained between day 180 of the study and day 28 of the study for each treatment dose is shown. The geometric mean titer (GMT) is expressed in international units per milliliter (IU / mL).

[0119] Fig.18 Figures of unadjuvanted RSV-A neutralizing antibodies (nAB) in elderly subjects at day 180 are shown. The μg number of total VLPs and the μg number of RSV-A antigens for each treatment dose are shown. The percentage of titers retained between the 180th day (M6) and the 28th day (M1) of each treatment dose are shown. The geometric mean titer (GMT) is expressed in international units per milliliter (IU / mL).

[0120] Fig.19 A summary of safety data is shown. There were no serious adverse events (SAEs), AEs of special interest (AESIs), or adverse events (AEs) leading to study withdrawal.

[0121] Fig. 20 Shown are graphs comparing unadjuvanted RSV-A neutralizing antibodies (nAB) (left) and unadjuvanted RSV-B neutralizing antibodies (nAB) (right) in elderly subjects at day 28 and day 180. Geometric mean titers (GMTs) are expressed in international units per milliliter (IU / mL).

[0122] Fig.21 It is a research design for a phase 1 randomized, observer-blind, placebo-controlled study to evaluate a single administration of IVX-A12 in elderly subjects (age 60-75 years old). Dosage group and total VLP content (upper left figure). Each dosage includes both RSV and hMPV VLP. The VLP compositions of low, medium and high hMPV dosages and consistent RSV VLP dosage combinations (upper right figure). Mid-term readings include safety and immunogenicity readings (figure below) to D28.

[0123] Fig.22AThe demographics of the trial participants are shown. The demographic characteristics were similar between IVX-A12 and placebo recipients. A total of 141 subjects were enrolled, 140 took medication (safety analysis group), and 17 subjects were excluded due to protocol violations, leaving 123 subjects in the per-protocol analysis group. Fig. 22B A summary of safety data is shown. As of D28, there were no serious adverse events (SAEs), AEs of special interest (AESIs), or adverse events (AEs) leading to withdrawal from the study. A serious spontaneous AE of hypertension (not vaccine related) was observed on Day 28 in a subject with a prior medical history; the AE resolved. Spontaneous AEs are collected until Day 28. Other AEs will be collected until Day 180.

[0124] Fig.23A A graph showing local adverse events elicited within 7 days of a single dose, with the highest severity. Unadjuvanted IVX-A12 reactogenicity was mild in the elderly. Dose groups VLP: 150 μg = 75 μg RSV + 75 μg hMPV; 225 μg = 75 μg RSV + 150 μg hMPV; 300 μg = 75 μg RSV + 225 μg hMPV.

[0125] Fig. 23B A graph showing systemic adverse events induced within 7 days of a single dose is shown. IVX-A12 showed similar tolerability to placebo. Dose group VLP: 150 μg = 75 μg RSV + 75 μg hMPV; 225 μg = 75 μg RSV + 150 μg hMPV; 300 μg = 75 μg RSV + 225 μg hMPV.

[0126] Fig.24A A graph of RSV-A neutralizing antibodies (nAB) is shown. Per-protocol analysis groups of participants. Geometric mean titers (GMTs) are expressed in international units per milliliter (IU / mL). RSV-A LLoQ = 9.4. Proprietary adjuvant from Seqirus Inc. GMFR: Geometric mean fold increase from baseline VLP: 150 μg = 75 μg RSV + 75 μg hMPV; 225 μg = 75 μg RSV + 150 μg hMPV; 300 μg = 75 μg RSV + 225 μg hMPV.

[0127] Fig. 24B A graph of RSV-B neutralizing antibodies (nAB) is shown. Per-protocol analysis groups of participants. Geometric mean titers (GMTs) are expressed in international units per milliliter (IU / mL). RSV-B LLoQ = 8.0. Proprietary adjuvant from Seqirus Inc. GMFR: Geometric mean fold increase from baseline VLP: 150 μg = 75 μg RSV + 75 μg hMPV; 225 μg = 75 μg RSV + 150 μg hMPV; 300 μg = 75 μg RSV + 225 μg hMPV.

[0128] Fig.25A A graph of hMPV-A neutralizing antibodies (nAB) is shown. Geometric mean titers (GMT) are expressed in assay units. Per-protocol analysis groups of participants. hMPV-A and hMPV-B LLoQ = 4.0 log 2. GMFR: GMFR: geometric mean fold rise from baseline. VLPs: 150 μg = 75 μg RSV + 75 μg hMPV; 225 μg = 75 μg RSV + 150 μg hMPV; 300 μg = 75 μg RSV + 225 μg hMPV.

[0129] Fig.25B A graph of hMPV-B neutralizing antibodies (nAB) is shown. Geometric mean titers (GMT) are expressed in assay units. Per-protocol analysis groups of participants. hMPV-A and hMPV-B LLoQ = 4.0 log 2. GMFR: GMFR: geometric mean fold rise from baseline. VLPs: 150 μg = 75 μg RSV + 75 μg hMPV; 225 μg = 75 μg RSV + 150 μg hMPV; 300 μg = 75 μg RSV + 225 μg hMPV.

[0130] Figure 26 shows a pre-specified sub-analysis evaluating subjects with the lowest tertile of baseline RSV-A and RSV-B titers. Shown are RSV-A ( Fig.26A ) and RSV-B( Fig.26B ) Neutralizing Antibody (nAB) Data. Data for adjuvanted and unadjuvanted subjects are summarized by dose. Geometric mean titer (GMT) is expressed as international units per milliliter (IU / mL). Per-protocol Analysis Groups of Participants. RSV-A LLoQ = 9.4. RSV-B LLoQ = 8.0. GMFR: geometric mean fold rise from baseline. Dose Group VLP: 150μg = 75μg RSV + 75μg hMPV; 225μg = 75μg RSV + 150μg hMPV; 300μg = 75μg RSV + 225μg hMPV. *Data are summarized within adjuvanted and unadjuvanted groups within dose. DETAILED DESCRIPTION

[0131] Provided herein are compositions or pharmaceutical compositions comprising two or more VLPs of the extracellular domains of the F protein displaying different paramyxoviruses. For example, the disclosure provides compositions comprising two VLPs: VLPs with components comprising the extracellular domain of the F protein of respiratory syncytial virus (RSV) or its antigenic variants; and VLPs with components comprising the extracellular domain of the F protein of human metapneumovirus (hMPV) or its antigenic variants. These compositions can be used for vaccination 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).

[0132] In particular, provided herein are protein-based virus-like particles (VLP) vaccines for respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), wherein the RSV F protein ectodomain and the hMPV F protein ectodomain are each independently connected to a protein-based VLP, such as a designed VLP, such as a symmetric VLP, and are therefore displayed thereon. For example, the vaccine antigen can be an N-terminal fusion of an ectodomain of an RSV F protein or an hMPV F protein with a protein having a multimerization domain of a single-component or two-component de novo designed VLP (such as a two-component icosahedral VLP). Further provided are vaccine compositions, preparation methods, and methods of use, such as immunizing a subject to produce a protective immune response to RSV virus and / or hMPV virus or to enhance an existing immune response to RSV virus and / or hMPV virus.

[0133] In a variation, the disclosure provides "chimeric" VLPs produced by mixing components of RSV and hMPV vaccines and then assembling them into VLPs, such that each VLP contains some components displaying the extracellular domain of the RSV F protein and some components displaying the extracellular domain of the hMPV F protein. Thus, the disclosure further provides virus-like particles (VLPs) comprising a plurality of first components, some of which comprise an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and some of which comprise an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof.

[0134] definition

[0135] All publications, patents and patent applications, including any figures 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, figure or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0136] The term "virus-like particle" or "VLP" refers to a molecular assembly that is similar to a virus but not infectious, and that displays antigenic proteins or antigenic fragments of viral proteins or glycoproteins. "Protein-based VLPs" refer to VLPs that are formed from proteins or glycoproteins and are substantially free of other components (e.g., lipids). Protein-based VLPs may include post-translational modifications and chemical modifications, but are distinguished from micellar VLPs and VLPs formed by extracting viral proteins from live or inactivated viral preparations. The term "designed VLP" refers to a VLP that contains one or more polypeptides generated by computational protein design. Exemplary designed VLPs are those that contain Figure 1B The term "symmetric VLP" refers to a protein-based VLP with a symmetric core, such as Figure 1B These include, but are not limited to, designed VLPs. For example, the protein ferritin has been used to produce symmetrical protein-based VLPs using naturally occurring ferritin sequences. The difference between ferritin-based VLPs and designed VLPs is that, in addition to fusing viral proteins to ferritin molecules, no protein engineering is required to form symmetrical VLPs from ferritin. Protein design methods can be used to produce similar single-component and two-component nanostructures based on template structures (e.g., structures stored in the protein database) or from scratch (i.e., by computationally designing new proteins with desired structures but little or no homology to naturally occurring proteins). Such single-component and two-component nanostructures can then be used as the core of designed VLPs.

[0137] The VLPs of the present disclosure display antigens that are capable of eliciting an immune response to paramyxoviruses, including respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) or other metapneumoviruses. As used herein and throughout the present disclosure, an immune response includes generating an immune response or enhancing an existing immune response. The vaccines of the present disclosure can be used to prevent infection with respiratory syncytial virus and / or human metapneumovirus and / or reduce the severity of infection with respiratory syncytial virus and / or human metapneumovirus.

[0138] The term "icosahedral particle" refers to a designed VLP with an icosahedral symmetric core (e.g., particles labeled I53 and I52 in Table 1). I53 refers to an icosahedral particle composed of a pentamer and a trimer. I52 refers to an icosahedral particle composed of a pentamer and a dimer. T33 refers to a tetrahedral particle composed of two groups of trimers. T32 refers to a tetrahedral particle composed of a trimer and a dimer.

[0139] Antigen can be non-covalently or covalently connected to the core of protein-based VLP, including as fusion protein or by other means disclosed herein. Multimeric antigen can be optionally displayed along the symmetry axis of VLP. Protein and nucleic acid molecules, preparations and methods of use of encoding such proteins are also provided.

[0140] The term "antigen" refers to a polypeptide or polypeptide complex comprising at least one component intended to elicit an immune response. The term antigen as used herein is not limited to the portion of a polypeptide or polypeptide complex that contains an antigenic epitope.

[0141] The term "polypeptide" refers to a series of amino acid residues linked by peptide bonds and optionally one or more post-translational modifications (e.g., glycosylation) and / or other modifications (including but not limited to conjugation of a polypeptide portion for use as a marker, such as a fluorescent label, or an adjuvant).

[0142] The term "infection" refers to both symptomatic and asymptomatic infections.

[0143] The term "extracellular domain" refers to the portion of a transmembrane protein or glycoprotein that, in the native state of the protein, is located outside the cellular or viral membrane.

[0144] The term "variant" refers to a polypeptide that has one or more insertions, deletions, substitutions or amino acid replacements relative to a reference polypeptide, but retains one or more properties of the reference protein.

[0145] The term "antigenic variant" refers to a variant that shares one or more epitopes in common with a reference polypeptide and / or produces the same or similar immune response when administered to a subject as the reference polypeptide.

[0146] The term "functional variant" refers to a variant that exhibits the same or similar functional role as a reference polypeptide. For example, a functional variant of a multimerization domain can promote multimerization to the same degree or to a similar degree as a reference multimerization domain, and / or can multimerize with a homologous multimerization domain that is the same as a reference multimerization domain.

[0147] The term "linker" refers to a chemical linkage (ie, a covalent bond or a series of covalent bonds with intervening chemical moieties) or to polypeptides that are joined by a peptide bond at the N-terminus and C-terminus to produce a fusion protein.

[0148] The term "domain" refers to any portion of a polypeptide that adopts a tertiary structure.

[0149] 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.

[0150] The term "trimerization domain" refers to a multimerization domain that forms trimers.

[0151] The term "VLP-forming domain" refers to a multimerization domain that forms a symmetrical protein complex alone or together with other multimerization domains.

[0152] The term "fragment" refers to a polypeptide having one or more N-terminal or C-terminal truncations compared to a reference polypeptide.

[0153] The term "functional fragment" refers to a functional variant of a fragment.

[0154] The term "substitution" or "replacement" refers to replacing a single amino acid in a sequence with another amino acid residue. Standard abbreviations for amino acid substitutions are used. For example, V94R means that the valine (V) in the reference sequence is replaced by arginine (R). The abbreviation Arg94 refers to any sequence in which the 94th residue relative to the reference sequence is arginine (Arg).

[0155] The term "helix" or "helical" refers to an alpha helical secondary structure known or predicted to exist in a polypeptide. For example, a sequence may be described as helical when computational modeling indicates that it is likely to adopt a helical conformation.

[0156] The term "component" refers to a protein or protein complex (eg, an antigen or polypeptide comprising a multimerization domain) that is capable of assembling into a virus-like particle under appropriate conditions.

[0157] 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.

[0158] The term "pharmaceutically acceptable excipient" means an excipient that is biologically or pharmacologically compatible for use in animals or humans, and can mean an excipient approved by a regulatory agency of the federal or state governments or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals (and particularly humans).

[0159] The term "adjuvant" refers to a pharmaceutically acceptable substance that enhances the immune response to an antigen when co-administered with the antigen or administered before, during, or after administration of the antigen to a subject.

[0160] The term "TLR4 immunostimulator" refers to an adjuvant that stimulates Toll-like receptor 4 (TLR4) in a subject's immune cells to modulate an immune response, such as monophosphoryl lipid A (MPL), glucopyranosyl lipid A (GLA) and / or soluble Leishmania antigen (SLA).

[0161] The term "effective amount" refers to an amount of a formulation according to the invention which, when administered to a patient to treat a condition, disorder or condition, is sufficient to achieve such treatment, or which, when administered to a patient to produce an immune response, is sufficient to produce such an immune response, such as an antibody response. The "effective amount" will vary depending on the active ingredient, the condition, disorder or condition to be treated and its severity, and the age, weight, physical condition and responsiveness of the subject to be treated.

[0162] 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 produce antibodies, the degree of protection desired, the formulation of the vaccine, and other relevant factors.

[0163] As used herein, "effective amount" refers to an amount of the immunogenic composition that is effective in treating and / or limiting 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).

[0164] The term "immune response" refers to the initiation of activity of one or more immune cell types in a subject. Immune responses include, for example, T cell and B cell responses.

[0165] The term "humoral immune response" refers to an immune response that produces plasma or serum antibodies (eg, IgG).

[0166] The term "protective immune response" refers to an immune response that prevents infection by a pathogen and / or reduces the severity of infection by a pathogen when the subject is subsequently challenged with the pathogen, or refers to an immune response that produces a level of immune response associated with protection. For example, if vaccination results in the production of neutralizing antibodies in the plasma or serum of a subject (e.g., a human, pet, or agricultural animal), the neutralizing antibodies protect the subject against subsequent infection, and / or are observed to be present in an amount that provides protection to a test subject (e.g., a New Zealand White (NZW) rabbit), then vaccination can produce a protective immune response.

[0167] The term "polyclonal antibody response" refers to an antibody response that comprises antibodies with more than one specificity and / or variation in the antibody sequence.

[0168] The term "neutralization" (e.g., "neutralizing antibody response") refers to antibodies that prevent infection and / or reduce the level of infection by a pathogen. Neutralizing antibody responses can be measured in an in vitro assay (e.g., infection of cells in culture by a pathogen in the presence of an antibody) or in an in vivo assay (e.g., determining a protective dose of an antibody by administering the antibody to a subject prior to challenge with an infectious dose of the pathogen).

[0169] Antibodies that "bind" to a target (e.g., RSV or hMPV F protein) or are "specific" for a target or "specifically bind" to a target (used interchangeably herein) are terms well known in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if the reaction or association of a molecule with a particular cell or substance is more frequent, faster, longer lasting, and / or with higher affinity than the reaction or association with an alternative cell or substance. For example, an immunoglobulin that specifically or preferentially binds to thymocytes is an immunoglobulin that binds to thymocytes with higher affinity, avidity, more easily, and / or longer lasting than 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. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means specific binding.

[0170] The term "predetermined time" refers to a time interval selected to be appropriate for observing a particular effect. The predetermined time may be selected prior to or during an experiment or procedure.

[0171] 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 in order to elicit an immune response to protect against infection by the pathogen and / or reduce the severity of one or more symptoms of infection by the pathogen.

[0172] 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 performed by intramuscular injection, intravenous injection, intraperitoneal injection, or any other suitable route.

[0173] The term "immunization" and "immunize ... " refer to administering to a subject a composition (e.g., virus-like particles) in an amount sufficient to induce a desired immune response (e.g., a humoral immune response to VLPs) after one or more administration steps. Immunization may include administering (e.g., injecting) the composition between one and ten times or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations. The first administration may not induce a detectable immune response, because typically each subsequent administration will strengthen the immune response generated by the previous administration. As used herein, the term "immunize ... " includes post-exposure prophylaxis.

[0174] The term "subject" refers to a human or non-human animal to which a composition can 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 rabbits, hamsters, gerbils, pigs, cows, sheep, goats, guinea pigs, rats, mice, squirrels, wolves, foxes, horses, zebras, giraffes, elephants, cats, dogs, camels or ferrets.

[0175] The term "preparing" refers to producing a recombinant polypeptide or virus-like particle at any scale, including but not limited to a scale of at least 25-mL, 50-mL, 1-L, 1,000-L, 50,000-L or greater.

[0176] The terms "culture" and "medium" refer to standard cell culture and recombinant protein expression techniques.

[0177] The term "host cell" refers to any cell that can be used to express a recombinant polypeptide.

[0178] The term "secretion" refers to the ability of host cells to secrete a polypeptide into the medium in which they are cultured.

[0179] The term "signal sequence" refers to a polypeptide sequence generally located at the N-terminus of a polypeptide expressed in a host cell that 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 culture medium in which the host cell is cultured. Various signal sequences are known, and selecting an appropriate signal sequence is within the skill of the ordinary technician.

[0180] The term "mixing" refers to bringing two solutions into contact so that the solutions mix.

[0181] The term "purification" refers to the separation of a molecule from other substances present in a composition. The polypeptide can be purified by affinity (e.g., to an antibody or to a tag, such as using a His-tag capture resin), by charge (e.g., ion exchange chromatography), by size (e.g., preparative ultracentrifugation, size exclusion chromatography), or otherwise.

[0182] The terms "polynucleotide" and "nucleic acid" used interchangeably herein refer to a polymeric form of more than about 100 nucleotides of nucleotides, whether ribonucleotides or deoxyribonucleotides. Therefore, this term includes but is not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. "Oligonucleotide" generally refers to a polynucleotide between about 5 and about 100 nucleotides of a single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also referred to as "oligomers" or "oligonucleotides (oligo)", and can be separated from genes, or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include (as applicable to the described embodiments) single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0183] In the context of two or more nucleic acid or polypeptide sequences, the terms "identical", "identity", "percent identity", "sequence identity" or "percent sequence identity" refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence. Methods of sequence alignment for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where the identical nucleotides or amino acid residues are present in the two sequences. The 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 with 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). Gaps in the alignment do not reduce the percent sequence identity as the term is used herein.

[0184] Unless otherwise indicated, 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 (on the World Wide Web at ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al., Nucleic Acids Res. 50(W1):W276-W279 (2022)). In one embodiment, other alignment methods may be used, including but not limited to those described in 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. Math. 48(5) (1988); Computational Molecular Biology (Lesk, AM, ed., 1989); Biocomputing Informatics and Genome Projects (Smith, DW, ed., 1993); Computer Analysis of Sequence Data, Part I (Griffin and Griffin, ed., 1994); Sequence Analysis in Molecular Biology (von Heinje, 2012); Sequence Analysis Primer (Gribskov and Devereux, J., ed., 1993). In one embodiment, sequence identity is calculated using an implementation of the Needleman-Wunsch algorithm provided by the U.S. National Library of Medicine (available on the World Wide Web at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln).

[0185] For example, sequence identity can be determined by the standard method that is usually used for the similarity of two polypeptides or two polynucleotide sequences.Use computer programs such as EMBOSS Needle or BLAST, two polypeptides or two polynucleotide sequences are compared, so that their respective residues are best matched (or along the full length of one or two sequences, or along the predetermined part of one or two sequences).Described program provides the open penalty of acquiescence and the gap penalty of acquiescence and the scoring matrix that can be used in combination with described computer program, such as PAM 250 (standard scoring matrix; See Dayhoff et al., Atlas of Protein Sequence and Structure, Vol. 5, Supplement 3 (1978)).

[0186] The term "treat" means to alleviate, mitigate, delay, reduce, reverse, improve or manage one or more of at least one symptom of a subject's illness. The term "treat" may also mean to prevent, delay the onset of a patient (i.e., the period before the patient presents clinical manifestations) or reduce the risk of a patient developing or worsening one or more.

[0187] The term "a" refers to one or more of the entity, i.e., may refer to plural referents. Thus, the terms "one", "one or more", and "at least one" are used interchangeably herein. Furthermore, reference to an "element" by the indefinite article "a" does not exclude the possibility that more than one element is present, unless the context clearly requires the presence of one and only one element.

[0188] The term "about" or "approximately" means that a particular value determined by one of ordinary skill in the art is within an acceptable error range, which depends in part on how the value is measured or determined, such as limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations. Alternatively, "about" can mean plus or minus a range of, for example, up to 20%, up to 10%, or up to 5%. Unless otherwise indicated, the term "about" when used in conjunction with a range or a series of values ​​applies to the endpoints of the range or each value listed in the series. As used in this application, the terms "about" and "approximately" are used as equivalent expressions.

[0189] Unless otherwise indicated, all weight percentages (ie, "% by weight" and "wt%" and w / w) cited herein are measured relative to the total weight of the pharmaceutical composition.

[0190] As used herein, "substantially" or "essentially" refers to the complete or almost complete range or degree of an action, feature, property, state, structure, material or result. For example, an object that is "substantially" enclosed means that the object is either completely enclosed or almost completely enclosed. In some cases, the exact degree of deviation from absolute completeness may depend on the specific circumstances. However, in general, the proximity of completion will be such that there is an overall result identical to that obtained with absolute and complete completion. When used in a negative sense, the use of "substantially" is also applicable to refer to a complete or almost complete lack of action, feature, property, state, structure, material or result. For example, a composition that is "substantially free of" other activating agents either completely lacks other activating agents or almost completely lacks other activating agents, so that the effect is the same as its complete lack of other activating agents. In other words, a composition that is "substantially free of" a certain ingredient or element or another activating agent can still contain this substance, as long as there is no measurable effect thereof.

[0191] 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 presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0192] Overview

[0193] Vaccination is a therapeutic modality used to prevent or reduce the severity of infection with various infectious agents, including bacteria, viruses, and parasites. The development of new vaccines is of great commercial and public health significance. In particular, Lyme disease, pertussis, herpes viruses, orthomyxoviruses, paramyxoviruses, pneumoviruses, filoviruses, flaviviruses, reoviruses, retroviruses, coronaviruses, and malaria are infectious agents for which vaccines exist, are in development, or are needed.

[0194] 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 proteins of infectious agents that have been observed to elicit natural immune responses after infection, although other components of infectious agents may be used in some cases. Typical antigens for subunit vaccines include proteins expressed on the surface of target infectious agents, such as envelope glycoproteins expressed on the surface of viruses. Preferably, the antigen is the target of neutralizing antibodies. More preferably, the antigen is the target of broadly neutralizing antibodies, so that the immune response to the antigen covers immunity against multiple strains of infectious agents. In some cases, polysaccharides N-linked or O-linked to subunit vaccines may also be important in vaccination, either by contributing to the epitope of the antigen or by guiding the immune response to a specific epitope on the antigen through steric hindrance. The immune response generated in response to vaccination can be directed against the protein itself, against the polysaccharide, or against both the protein and the linked polysaccharide. Subunit vaccines have a variety of advantages, including that they do not contain live pathogens, which eliminates concerns about patients being infected by the vaccine; they can be designed using standard genetic engineering techniques; they are more homogeneous than other forms of vaccines; and they can be prepared in standardized recombinant protein expression production systems using well-characterized expression systems. In some cases, antigens can be genetically engineered to favor the production 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.

[0195] The known limitation of subunit vaccine is that the immune response of initiation may be weaker than the immune response of other types of vaccines (such as whole virus vaccine, live vaccine or attenuated live vaccine) sometimes. The inventor has recognized and disclosed herein the potentiality of design and / or VLP vaccine based on protein to utilize subunit vaccine advantage, and simultaneously increased the effectiveness and breadth of vaccine-induced immune response with the multivalent display of symmetrical ordered array by antigen. In the present disclosure, VLP based on protein is distinguished from nanoparticle vaccine, because the term nanoparticle vaccine has been used in the art to refer to protein-based or glycoprotein-based vaccine (see, for example, US 9,441,019 patent), polymerized liposome (see, for example, US Patent No. 7,285,289), surfactant micelle (see, for example, US Patent Publication No. US 2004 / 0038406 A1) and synthetic biodegradable particles (see, for example, US Patent No. US 8,323,696).

[0196] The protein-based VLPs of the present disclosure are distinguishable from VLPs that display antigens on the surface of micellar particles containing surfactants (e.g., NP-9); or alternatively, made by extracting antigenic proteins from live viruses while retaining the lipid composition of the viral envelope. In contrast, the protein-based VLPs described herein are free of or substantially free of lipids and surfactants. In addition, compared to other VLPs, the symmetric display of RSV and / or hMPV F proteins on protein-based VLPs of some embodiments of the present disclosure can produce a more excellent immune response to F proteins.

[0197] The present disclosure relates in part to combination vaccines for paramyxoviruses (such as RSV and hMPV), such as a bivalent (or multivalent) vaccine having two (or more) VLPs displaying paramyxovirus antigens (such as the extracellular domain of the F protein of each paramyxovirus). In some variations, the VLPs are chimeric VLPs displaying multiple antigens. In further variations, the bivalent display is antigens from two strains of the same paramyxovirus (such as 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).

[0198] In some embodiments, the F protein of the corresponding paramyxovirus can be engineered to preferentially adopt a prefusion conformation.Thus, the present disclosure provides a prefusion stabilized hMPV F protein ectodomain and a prefusion stabilized RSV F protein ectodomain for use in VLPs.

[0199] Metapneumovirus virus-like particles

[0200] A further protein-based virus-like particle (VLP) vaccine directed against human metapneumovirus is described in which the hMPV F protein ectodomain is linked to, and thereby displayed on, a protein-based VLP.

[0201] In one aspect, the present disclosure provides a virus-like particle (VLP) comprising a first component and an optional second component, wherein the first component is a fusion protein comprising the extracellular domain of the human metapneumovirus (hMPV) F protein or an antigenic variant thereof and a first multimerization domain; and wherein the second component (if present) is a protein comprising a second multimerization domain.

[0202] 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 or 145 or a functional variant thereof. In some embodiments, the multimerization domain is I53-50A (SEQ ID NO: 7 or SEQ ID NO: 144) or a functional variant or variant 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 is adapted to drive assembly of icosahedral particles or tetrahedral particles, optionally together with a second multimerization domain.

[0203] 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 a functional variant thereof.

[0204] In some embodiments, the first component and the second component are connected by a linker sequence. In some embodiments, the linker sequence comprises a foldon, wherein the foldon sequence is EKAAKAEEAARK (SEQ ID NO: 125). In some embodiments, the linker sequence comprises GSGGSGSGSGGS (SEQ ID NO: 126).

[0205] In some embodiments, the hMPV F protein extracellular domain contains one or more substitutions or deletions selected from the group consisting of 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 extracellular domain contains one or more substitutions or deletions selected from the group consisting of 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.

[0206] In some embodiments, the hMPV F protein extracellular domain contains two or more substitutions or deletions selected from the group consisting of 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 extracellular domain contains one or more substitutions selected from the group consisting of A185P, Q100R, S101R, T127C, N153C, T365C, V463C, L219K, V231I, G294E, N153C, N97G, P98G, R99G, Q100G, S101G, H368N, and / or R102G.

[0207] In some embodiments, the hMPV F protein extracellular domain contains two or more of A185P, Q100R, S101R, T127C, N153C, T365C, V463C, L219K, V231I, G294E, N153C, N97G, P98G, R99G, Q100G, S101G, H368N and / or R102G. In some embodiments, the hMPV The F protein extracellular domain contains: substitutions of Q100R and S101R; substitutions of A185P, Q100R and S101R; substitutions of A185P, T127C, N153C, Q100R and S101R; substitutions of V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G and R102G; substitutions of A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G and R102G. 01G and R102G; substitutions and deletions of A63C, A140C, A147C, K188C, G106 deletion, N97G, P98G, R99G, Q100G, S101G and R102G; substitutions of A113C, A120C, A339C, Q426C, T160F, I177L, Q100K and S101A; substitutions of V84C, A140C, A147C, A249C, Q100R and S101R; substitutions of 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 A185C, A113C, A339C, Q100R and S101R 39C, T160F, I177L, Q100R and S101R; replace A63C, K188C, N97G, P98G, R99G, Q100G, S101G and R102G; replace A63C, K188C, K450A, S470A, N97G, P98G, R99G, Q100G, S101G and R102G; replace A63C, A140C, A147C, K188C, G294E, N97G, P98G, R99G, Q100G, S101G and R102G;Replaces A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, R102G and G294E; replaces A63C, K188C, N97G, P98G, R99G, Q100G, S101G, R102G and G294E; replaces T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, H368N, Q100R and S101R; replaces A63C, A14 0C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G and R102G; replace V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G and R102G; replace T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, H368N, N97G, P98G, R99G, Q100G, S101G and R102G. ;

[0208] In some embodiments, the hMPV F protein extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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.

[0209] In some embodiments, the hMPV F protein extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 extracellular domain 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 NO: 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 NO: 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.

[0210] In some embodiments, the VLPs are bound to 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 are bound to two or more antibodies selected from MF1, MF2, MF3, MF9, MF12, MF14, MF15, MF11, MF16, MF20, MF17, MF18, MF19, MF10, and MPE8. In some embodiments, the VLPs are bound to three or more antibodies selected from MF1, MF2, MF3, MF9, MF12, MF14, MF15, MF11, MF16, MF20, MF17, MF18, MF19, MF10, and MPE8.

[0211] In some embodiments, the hMPV F protein extracellular domain binds to an antibody that preferentially binds to a pre-fusion form of the hMPV F protein extracellular domain. 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 extracellular domain does not bind or binds poorly to an antibody that preferentially binds to a post-fusion form of the hMPV F protein extracellular domain. 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] In one aspect, the present disclosure provides polynucleotides encoding the VLPs described herein.

[0216] In one aspect, the disclosure provides a host cell comprising a polynucleotide described herein.

[0217] RSV-like particles

[0218] On the one hand, provided herein is a pharmaceutical composition comprising a virus-like particle (VLP) and one or more pharmaceutically acceptable diluents or excipients, wherein the virus-like particle (VLP) comprises a first component and a second component, wherein the first component comprises RSV F protein and a first multimerization domain, and the second component comprises a second multimerization domain. 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.

[0219] 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 the amino acid sequence of 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.

[0220] Implementation

[0221] The present disclosure relates to protein-based VLPs and protein-based VLP vaccines. The VLPs of the present disclosure display antigens that can induce an immune response to paramyxoviruses (e.g., RSV and hMPV). Some vaccines of the present disclosure can be used to prevent paramyxoviruses (e.g., RSV and hMPV) from infecting or reducing the severity of paramyxovirus infections. In particular, antigens of the present disclosure display the extracellular domain of paramyxovirus (e.g., RSV and hMPV) F protein. The extracellular domain can be non-covalently or covalently connected to the core of the VLP, including as a fusion protein or by other means disclosed herein. In some embodiments, the linker connects the extracellular domain to the first polypeptide comprising a multimerization domain. The linker can be any chemical bonding, including but not limited to a polypeptide for forming an N-terminal or C-terminal fusion of an extracellular domain and a first polypeptide. RSV and / or hMPV F protein can be optionally displayed along the axis of symmetry of the VLP. In some embodiments, the F protein is connected to the first polypeptide C-terminus comprising a trimerization domain and optionally an additional trimerization tag (e.g., FoldOn tag). Also provided are proteins and nucleic acid molecules encoding such proteins, formulations, and methods of use.

[0222] Protein-based virus-like particles

[0223] The disclosure relates in part to a protein-based virus-like particle (VLP) containing a first component comprising RSV F protein and a first multimerization domain together with a (VLP) containing a first component comprising hMPV F protein and a first multimerization domain to vaccinate a subject. The protein complex may comprise the F protein of two or more of RSV-A, RSV-B, hMPV-A and hMPV-B. The F protein portion and the first multimerization domain may be connected in any suitable manner, including co-expression as a fusion protein. The protein complex may optionally comprise a second component, which comprises a second multimerization domain. The pharmaceutical composition typically comprises one or more pharmaceutically acceptable diluents or excipients.

[0224] The VLP of the present invention may include a multimeric protein assembly suitable for displaying the extracellular domain of RSV or hMPV F protein or its antigenic fragment. The VLP of the present invention includes at least the first multiple polypeptides. The first multiple polypeptides (also referred to as "the first component") can be derived from a naturally occurring protein sequence by replacing at least one amino acid residue or another one or more residues at the N or C terminus. In some cases, the first component includes a protein sequence determined by a computational method. This first component can form the entire core of the VLP; or the core of the VLP may include one or more additional polypeptides (also referred to as "the second component" or the third, fourth, fifth component, etc.), so that the VLP includes two, three, four, five, six, seven or more multiple polypeptides. In some cases, the first multiple will form a trimer associated by 3-fold rotational symmetry, and the second multiple will form a pentamer associated by 5-fold rotational symmetry. In such a case, VLP forms an "icosahedral particle" with I53 symmetry. These one or more multiple polypeptides can be arranged together so that the members of each multiple polypeptide are related to each other by a symmetry operator. Patent Publication No. US2015 / 0356240 A1 discloses a general computational method for designing self-assembling protein species involving symmetric docking of protein building blocks in a targeted symmetric framework.

[0225] In some cases, VLPs are adapted to display F proteins from two or more different strains of RSV. In a non-limiting example, the same VLP displays a mixed population of protein antigens from different strains of RSV or a mixed heterotrimer of protein antigens. The sequences of the F proteins of various RSV strains are known in the art, see, for example, NCBI accession numbers: 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, APW787 8779.1, APW78768.1, APW78757.1, APW78746.1, APW78735.1, APW78724.1, APW78713.1, APW78702.1, APW78691.1, APW78680.1, APW78669.1, APW78658.1, APW78647.1, APW78636.1, APW78625.1, APW78614.1 and AAR14266.1.

[0226] The "core" of VLP is used herein to describe the central part of VLP, which connects several copies of RSV and / or hMPVF protein extracellular domain or its antigenic fragment displayed by VLP. In one embodiment, the first component comprises: a first polypeptide comprising RSV and / or hMPV F protein extracellular domain or its antigenic variant, a joint and a first polypeptide comprising a multimerization domain. Therefore, "antigen" is a single polypeptide, which can be self-assembled into VLP alone or with a second component (third, fourth or fifth components). The advantage of designing the antigen itself into self-assembly is that the whole VLP then serves as the antigenic component of the vaccine. In other embodiments, the extracellular domain or its antigenic fragment is non-covalently or covalently connected to the first polypeptide comprising the multimerization domain. For example, an antibody or its antigenic fragment can be fused with the first polypeptide and is configured to bind a part of the first polypeptide or a chemical tag on the first polypeptide. Streptavidin-biotin (or neuroavidin-biotin) joints can be used. Alternatively, various chemical joints can be used. The advantage of core design being a universal platform is that one or more multiple polypeptides comprising core can be pre-designed and optimized, and then applied to the extracellular domain continuously to confirm the VLP with excellent efficacy as a vaccine. It will be appreciated that in some cases, the same polypeptide can form a part of "core", then extend outwards, as an adaptor for connecting RSV and / or hMPV F protein extracellular domain, and include extracellular domain (that is, fusion protein). In embodiments of the present disclosure, antigen also includes further polypeptide sequence except RSV and / or hMPV F protein extracellular domain. In certain embodiments, extracellular domain is by native glycosylation, or glycosylation is carried out using alternative oligosaccharides (for example, specific oligosaccharides to host cells for expressing antigens).

[0227] In some cases, self-assembly can be further promoted by the multimerization of ectodomain, although core can independently carry out self-assembly in the absence of ectodomain. RSV and / or hMPV F albumen are detected as trimer (Cseke et al., J.Virol.21 (2): 698-707 (2007)) in its native state. At least in some embodiments, the display of the ectodomain on VLP reduces the equilibrium ratio of the thermodynamic barrier and / or assembly and non-assembly VLP components of assembly. In some cases, the trimer ectodomain arranged along the 3-fold axis of VLP promotes the correct folding and conformational stability of ectodomain, and makes the self-assembly of VLP become a collaborative process, because the ectodomain is correctly trimerized, this part is due to its display on the 3-fold axis of the core of VLP, and VLP is at least partially stable with its assembled form by non-covalent or covalent interactions between trimer units. In some cases, introducing mutations into antigen or into VLP components can optionally further stabilize the assembly, particularly if the cysteine ​​residues are positioned to produce intramolecular disulfide bonds. In some instances, dimer, trimer, tetramer, pentamer or hexamer antigens are displayed on a core designed to have 2-fold, 3-fold, 4-fold, 5-fold or 6-fold symmetry axes of matching, so that the core accommodates the arrangement of multimeric antigens with the symmetry of the native antigen.

[0228] Various non-limiting examples of VLPs

[0229] Figure 1AIn the non-limiting example of the embodiment, it depicts the extracellular domain antigen genetically fused with the component (first multiple polypeptide) of VLP, which is optionally recombinantly expressed in a host cell (e.g., 293F cell or CHO cell); together with a pentamer protein assembly (second multiple polypeptide), which is optionally recombinantly expressed in the same or different host cell (e.g., E. coli cell), these two multiple polypeptides are self-assembled into a VLP displaying 20 antigen trimers around an icosahedral core. In this embodiment, the core has a universal design. As explained below, in other embodiments, the antigen of the present disclosure is mixed with another antigen protein in the same VLP, such as the extracellular domain of the protein from the second virus, or such as the trimeric glycoprotein from another virus. In some embodiments, in addition to RSV and / or hMPV F protein extracellular domain, VLP also includes the trimeric glycoprotein of HIV-1, HIV-2, EBV, CMV, RSV, influenza, Ebola, Marburg, dengue fever, SARS, MERS, Hantan or Zika virus. In some embodiments, VLP comprises a trimeric glycoprotein of a virus related to any of these exemplary viruses (including but not limited to herpesvirus, orthomyxovirus, paramyxovirus, pneumovirus, filovirus, flavivirus, reovirus or retrovirus) in evolution or sequence identity. In one embodiment, VLP comprises an extracellular domain or an antigenic fragment thereof of a transmembrane protein or glycoprotein. In some embodiments, VLP is further connected to a polypeptide or other agents that can serve as an adjuvant. In some embodiments, antigen (first component) and / or second component comprise one or more T cell epitopes, optionally T cell epitopes of heterologous origin.

[0230] The trimeric antigens that can be used together with the VLPs of the present disclosure to form mixed VLPs are, in some cases, but not limited to SARS-CoV-2, respiratory syncytial virus, HIV gp140, influenza HA, dengue fever E protein, or Ebola sGP. The mixed VLPs of the present disclosure include those comprising RSV and / or hMPV F protein ectodomains and antigenic proteins from one or more of vesicular stomatitis virus, herpes simplex virus, baculovirus, togo virus, and Bonaviridae. When other trimeric antigens are used, they can be optionally arranged on the three-fold symmetry axis of the VLP. In some cases, the selected antigen is monomeric, but still arranged on a 3-fold axis. Therefore, Figure 1A The VLP described in can display 20 trimer antigens or 60 monomer antigens. In addition or alternatively, the pentamer complex of VLP is used to display 12 pentamer antigens or 70 monomer antigens. In one embodiment, VLP comprises 20 copies of trimer antigen and 12 copies of pentamer antigen.

[0231] VLP Core

[0232] Figure 1B Other possible arrangements of polypeptides of the present disclosure are shown in . In some embodiments, VLPs are suitable 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 axes of symmetry is not used for antigen display, so in some embodiments, VLPs are suitable 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 antigen is displayed, so VLP is suitable for displaying up to 12,24,60 or 70 monomeric antigens.In some cases, VLP comprises a plurality of polypeptides mixed, makes the other identical polypeptides of the core of VLP display different antigens or do not display antigen.Therefore, according to the ratio of polypeptide, VLP is suitable for displaying 1 and 130 antigens (for example, on I52 particle) in some cases, wherein each in the antigen displayed can be identical, or can be the different members proportional with any ratio of selection of mixed colony.Antigen can be co-expressed in the recombinant expression system, and before purification, self-assembly is carried out.Or, antigen can be expressed separately, then before or after purification from expression host and relevant pollutant, mix together. 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 displayed. Non-limiting example VLPs are provided in the following literature: 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).

[0233] Hybrid VLP

[0234] In some cases, the VLP is adapted to display the same antigen from two or more different strains of hMPV. In a non-limiting example, the same VLP displays a mixed population of homotrimeric protein antigens or a mixed heterotrimer of protein antigens from different strains of hMPV. In one embodiment, the VLP displays the F protein or its antigenic fragment disclosed in any sequence of GenBank found by searching the protein database with the keyword "human metapneumovirus F" alone or in a mixed VLP.

[0235] When preparing mixed VLP, it may be advantageous to ensure that homopolymers are formed in a strain-specific manner rather than allowing heteropolymerization, such as making all strain 1F proteins displayed on a 3-fold axis of T33 particles, and all strain 2F proteins displayed on another 3-fold axis of T33 particles. This can be achieved by using a VLP comprising two or more multiple polypeptides as the core of VLP, wherein each multiple polypeptide is connected to different antigens. Alternatively, VLP can be engineered with one or more mutations that destroy symmetry, such as knob-embedded mutations or intramolecular disulfide mutations, which have the effect of preventing the formation of trimers between different antigens. In this case, VLP displays multimeric antigens from different strains at symmetrical equivalent positions on VLP, but each position on VLP is occupied by homopolymers from the same strain, with only a small proportion of heteromultimeric antigens between strains. In some cases, antigen itself can be genetically engineered to prevent heteromultimerization between strains. In one embodiment, VLPs are engineered to prevent heteropolymerization of two antigenic proteins with conservative structures but different antigenicity (e.g., strain 1 F protein and strain 2 F protein or hMPV F protein and non-hMPV antigenic protein). In addition, when mixed VLPs are prepared and antigens are displayed as fusion proteins, VLPs will contain three or more different proteins because the fusion proteins will share the same (or equivalent) domains for forming the core of VLPs with different antigenic domains, one domain for each antigen displayed on the VLP.

[0236] Connection method (connector)

[0237] VLPs of the present disclosure display antigenic proteins in various ways, including as gene fusions or by other means disclosed herein. As used herein, "linked / attached" means any method 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.

[0238] In some embodiments, connection is realized by genetic engineering to produce one of the N or C-terminal fusions of multiple polypeptides of antigen and composition VLP.Therefore, VLP can be by or basically by displaying one, two, three, four, five, six, seven, eight, nine or ten kinds of multiple antigens, one, two, three, four, five, six, seven, eight, nine or ten kinds of multiple polypeptides, wherein at least one of multiple antigens is genetically fused with at least one of multiple polypeptides.In some cases, VLP is basically composed of a plurality of polypeptides that can self-assemble, and comprises a plurality of antigenic proteins genetically fused with it.In some cases, VLP is mainly composed of: comprising a plurality of antigens, a plurality of polypeptides of the first; and a plurality of polypeptides of the second that can be assembled into two-component VLP together, a plurality of polypeptides connect antigenic proteins to VLP, and another plurality of polypeptides promote the self-assembly of VLP.

[0239] In some embodiments, connection is achieved by post-translational covalent connection between one or more multiple polypeptides and one or more antigenic proteins. In some cases, chemical crosslinking is used for antigen and VLP polypeptide non-specific connection. In some cases, chemical crosslinking is used for antigenic protein and VLP polypeptide (for example, with the first polypeptide or the second polypeptide) specific connection. Various specific and non-specific crosslinking chemical methods are known in the art, such as click chemistry and other methods. In general, any crosslinking chemical method for connecting two proteins can be suitable for VLP disclosed in the present invention. In particular, the chemical method for producing immunoconjugates or antibody drug conjugates can be adopted. In some cases, a cleavable or non-cleavable joint is used to produce VLP. For example, the US2008 / 0145373 A1 patent publication provides a process and method for antigen and carrier conjugation.

[0240] In one embodiment, connection is achieved by non-covalent connection between one or more multiple polypeptides and one or more multiple antigens. In some cases, antigenic protein is engineered to be negatively charged on at least one surface, and core polypeptide is engineered to be positively charged on at least one surface, or positively and negatively charged respectively. This promotes the intermolecular association between antigenic protein and core polypeptide by electrostatic force. In some cases, shape complementation is used to connect antigenic protein to core. Shape complementation can be pre-existing, or rationally designed. In some cases, the protein-protein interface of computational design is used to achieve connection. In one embodiment, antigen is biotin-labeled, and polypeptide comprises streptavidin, and vice versa. In one embodiment, streptavidin is displayed on the 4-fold axis of core as tetramer by gene fusion or otherwise, and biotin-labeled antigen is monomer, dimer or tetramer, so as to allow association with core in a configuration suitable for natural multimerization of antigen. In some cases, protein-based adapters are used to capture antigenic protein. In some cases, polypeptide is fused with a protein that can bind to a complementary protein fused to antigenic protein.

[0241] Can control the immune response to RSV and / or hMPV F by changing the orientation of the ectodomain with respect to core.According to how antigenic protein is connected with the core of VLP, antigenic protein can be displayed with various orientations.In some embodiments, display antigenic protein, make one or more known epi-positions be directed to or towards the far-end of antigenic protein, make these epi-positions preferentially accessible by immune system.In some cases, orientation will summarize the orientation of viral protein with respect to virus.The selection of orientation can be by immune system directed to one or another epi-position.

[0242] In some embodiments, epitope preference is controlled by other means, such as positioning the glycan on the VLP by adding or subtracting the N-linked glycan sequence motif NX-[T / S] at a predetermined position in the amino acid sequence of any polypeptide of the VLP, including the amino acid sequence of an antigenic protein.

[0243] In some cases, epitopes found at intermediate distances from the proximal end to the distal end will be preferred over epitopes located at the more distal end, based on various considerations, including but not limited to the overall geometry of the VLP, surface hydrophobicity, surface charge, and competitive binding of endogenously present proteins in the subject or exogenously provided proteins in the vaccine composition. The present disclosure encompasses all known methods for rationally designing protein structures, and the foregoing is not intended to be limiting.

[0244] VLP polypeptide sequences

[0245] The polypeptide of the present disclosure may have any different amino acid sequences. The US 2015 / 0356240 A1 patent publication describes the various methods of designing protein assemblies. As described in the US 2016 / 0122392 A1 U.S. Patent Publication and the WO2014 / 124301 A1 International Patent Publication, the isolated polypeptides of SEQ ID NO:1-51 are designed to enable them to self-assemble in pairs to form VLPs, such as icosahedral particles. The design relates to suitable interface residues for each member design of the polypeptide pair that can be assembled to form VLPs. The VLP formed in this way includes a non-natural non-covalent polypeptide-polypeptide interface that is symmetrically repeated, and these interfaces direct the first assembly and the second assembly into VLPs, such as VLPs with icosahedral symmetry. Therefore, in one embodiment, the first polypeptide and the second polypeptide (that is, two polypeptides of the core of VLP) are selected from the group consisting of SEQ ID NO:1-51. In each case, the N-terminal methionine residue that is present in the full-length protein but can be removed to form a fusion is not included in the sequence. The identified residues in Table 1 are numbered beginning with 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 (eg, to form a helical extension).

[0246] Table 1

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256] Table 1 provides the amino acid sequences of the first polypeptide and the second polypeptide of the embodiments of the present disclosure. In each case, the sequence pair together forms an I53 polymer with icosahedral symmetry. The right column in Table 1 confirms the number of residues in each exemplary polypeptide that are confirmed to be present at the interface of the resulting assembled virus-like particles (i.e., "confirmed interface residues"). It can be seen that the number of interface residues of the exemplary polypeptides of SEQ ID NO: 1-34 ranges from 4 to 13. In various embodiments, the first polypeptide and the second polypeptide comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the polypeptide selected from the group consisting of SEQ ID NO: 1-34 over its length and is identical to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 confirmed interface positions (depending on the number of interface residues of a given polypeptide). SEQ ID NO:35-51 represents other amino acid sequences of the first polypeptide and the second polypeptide from 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 to the amino acid sequence of the polypeptide selected from the group consisting of SEQ ID NO:1-51 over its length and at least 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the confirmed interface positions.

[0257] As is the case with proteins in general, it is expected that polypeptides will tolerate some variation in the designed sequence without disrupting subsequent assembly into virus-like particles: in particular, when such variation comprises conservative amino acid substitutions. As used herein, "conservative amino acid substitutions" means that: hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) can only be substituted by other hydrophobic amino acids; hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) can only be substituted by other hydrophobic amino acids with bulky side chains; amino acids with positively charged side chains (Arg, His, Lys) can only be substituted by other amino acids with positively charged side chains; amino acids with negatively charged side chains (Asp, Glu) can only be substituted by other amino acids with negatively charged side chains; and amino acids with polar uncharged side chains (Ser, Thr, Asn, Gln) can only be substituted by other amino acids with polar uncharged side chains.

[0258] In some cases, non-conservative amino acid substitutions may be preferred, for example, to eliminate the flexible portion of the secondary structure of the native RSV F protein is desired, for example, by adding cysteine ​​residues (or vice versa). "Non-conservative substitutions" refers to the substitution of an amino acid in one class by an amino acid from another class; for example, Ala is replaced with Asp, Asn, Glu or Gln. Other non-limiting examples of non-conservative substitutions include substitutions of polar (hydrophilic) residues (such as cysteine, glutamine, glutamic acid or lysine) with non-polar (hydrophobic) amino acid residues (such as isoleucine, valine, leucine, alanine, methionine), and / or substitutions of non-polar residues with polar residues. D-Cys substitutions of D-Ala, D-Ser or D-Tyr (or other residues) can be used to remove intramolecular disulfide bonds, which can improve protein stability or expression in some cases. Substitution of D-Cys can be used to produce stable proteins or disulfide bonds that lock proteins into desired conformations.

[0259] In various embodiments of the VLPs of the present invention, the first polypeptide and the second polypeptide (or vice versa) comprise a polypeptide having an amino acid sequence selected from the following pairs or a modified form thereof (i.e., the permissible modifications 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 to the amino acid sequence set forth in SEQ ID NO over its length and / or is identical at least at one confirmed interface position):

[0260] SEQ ID NO: 1 and SEQ ID NO: 2 (I53-34A and I53-34B);

[0261] SEQ ID NO:3 and SEQ ID NO:4 (I53-40A and I53-40B);

[0262] SEQ ID NO:3 and SEQ ID NO:24 (I53-40A and I53-40B.1);

[0263] SEQ ID NO:23 and SEQ ID NO:4 (I53-40A.1 and I53-40B);

[0264] SEQ ID NO:35 and SEQ ID NO:36 (genus I53-40A and genus I53-40B);

[0265] SEQ ID NO:5 and SEQ ID NO:6 (I53-47A and I53-47B);

[0266] SEQ ID NO: 5 and SEQ ID NO: 27 (I53-47A and I53-47B.1);

[0267] SEQ ID NO: 5 and SEQ ID NO: 28 (I53-47A and I53-47B.1NegT2);

[0268] SEQ ID NO:25 and SEQ ID NO:6 (I53-47A.1 and I53-47B);

[0269] SEQ ID NO:25 and SEQ ID NO:27 (I53-47A.1 and I53-47B.1);

[0270] SEQ ID NO:25 and SEQ ID NO:28 (I53-47A.1 and I53-47B.1NegT2);

[0271] SEQ ID NO:26 and SEQ ID NO:6 (I53-47A.1NegT2 and I53-47B);

[0272] SEQ ID NO:26 and SEQ ID NO:27 (I53-47A.1NegT2 and I53-47B.1);

[0273] SEQ ID NO:26 and SEQ ID NO:28 (I53-47A.1NegT2 and I53-47B.1NegT2);

[0274] SEQ ID NO:37 and SEQ ID NO:38 (genus I53-47A and genus I53-47B);

[0275] SEQ ID NO:7 and SEQ ID NO:8 (I53-50A and I53-50B);

[0276] SEQ ID NO:7 and SEQ ID NO:32 (I53-50A and I53-50B.1);

[0277] SEQ ID NO:7 and SEQ ID NO:33 (I53-50A and I53-50B.1NegT2);

[0278] SEQ ID NO:7 and SEQ ID NO:34 (I53-50A and I53-50B.4PosT1);

[0279] SEQ ID NO:29 and SEQ ID NO:8 (I53-50A.1 and I53-50B);

[0280] SEQ ID NO:29 and SEQ ID NO:32 (I53-50A.1 and I53-50B.1);

[0281] SEQ ID NO:29 and SEQ ID NO:33 (I53-50A.1 and I53-50B.1NegT2);

[0282] SEQ ID NO:29 and SEQ ID NO:34 (I53-50A.1 and I53-50B.4PosT1);

[0283] SEQ ID NO:30 and SEQ ID NO:8 (I53-50A.1NegT2 and I53-50B);

[0284] SEQ ID NO:30 and SEQ ID NO:32 (I53-50A.1NegT2 and I53-50B.1);

[0285] SEQ ID NO:30 and SEQ ID NO:33 (I53-50A.1NegT2 and I53-50B.1NegT2);

[0286] SEQ ID NO:30 and SEQ ID NO:34 (I53-50A.1NegT2 and I53-50B.4PosT1);

[0287] SEQ ID NO:31 and SEQ ID NO:8 (I53-50A.1PosT1 and I53-50B);

[0288] SEQ ID NO:31 and SEQ ID NO:32 (I53-50A.1PosT1 and I53-50B.1);

[0289] SEQ ID NO:31 and SEQ ID NO:33 (I53-50A.1PosT1 and I53-50B.1NegT2);

[0290] SEQ ID NO:31 and SEQ ID NO:34 (I53-50A.1PosT1 and I53-50B.4PosT1);

[0291] SEQ ID NO:39 and SEQ ID NO:40 (genus I53-50A and genus I53-50B);

[0292] SEQ ID NO:9 and SEQ ID NO:10 (I53-51A and I53-51B);

[0293] SEQ ID NO: 11 and SEQ ID NO: 12 (I52-03A and I52-03B);

[0294] SEQ ID NO: 13 and SEQ ID NO: 14 (I52-32A and I52-32B);

[0295] SEQ ID NO: 15 and SEQ ID NO: 16 (I52-33A and I52-33B)

[0296] SEQ ID NO: 17 and SEQ ID NO: 18 (I32-06A and I32-06B);

[0297] SEQ ID NO: 19 and SEQ ID NO: 20 (I32-19A and I32-19B);

[0298] SEQ ID NO:21 and SEQ ID NO:22 (I32-28A and I32-28B);

[0299] SEQ ID NO:23 and SEQ ID NO:24 (I53-40A.1 and I53-40B.1);

[0300] SEQ ID NO:41 and SEQ ID NO:42 (T32-28A and T32-28B);

[0301] SEQ ID NO:43 and SEQ ID NO:44 (T33-09A and T33-09B);

[0302] SEQ ID NO:45 and SEQ ID NO:46 (T33-15A and T33-15B);

[0303] SEQ ID NO:47 and SEQ ID NO:48 (T33-21A and T33-21B);

[0304] SEQ ID NO:49 and SEQ ID NO:50 (T33-28A and T32-28B); and

[0305] SEQ ID NO:51 and SEQ ID NO:44 (T33-31A and T33-09B (also referred to as T33-31B))

[0306] In some embodiments, one or more RSV and / or hMPV F proteins or antigenic fragments thereof are expressed as fusion proteins with a first multimerization domain. In some embodiments, the first multimerization domain and RSV and / or hMPV F protein extracellular domain are connected by a linker sequence. In some embodiments, the linker sequence comprises a foldon, wherein the foldon sequence is EKAAKAEEAARK (SEQ ID NO: 125).

[0307] Non-limiting examples of protein complexes that can be used for the design of protein-based VLPs of the present disclosure include those disclosed in U.S. Pat. No. 9,630,994; International Patent Publication No. WO2018187325A1; U.S. Pat. Publication No. 2018 / 0137234A1; U.S. Pat. Publication No. 2019 / 0155988A2, each of which is incorporated herein in its entirety.

[0308] In various embodiments of the VLPs disclosed herein, the multimerization domain is a polypeptide having an amino acid sequence selected from the following pairs or a modified form thereof (i.e., the 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 length and / or identical at least at one confirmed interface position to the amino acid sequence set forth in SEQ ID NO):

[0309] SEQ ID NO: 135 and SEQ ID NO: 136 (T33_dn2A and T33_dn2B);

[0310] SEQ ID NO: 137 and SEQ ID NO: 138 (T33_dn5A and T33_dn5B);

[0311] SEQ ID NO: 139 and SEQ ID NO: 140 (T33_dn10A and T33_dn10B); or

[0312] SEQ ID NO: 141 and SEQ ID NO: 142 (I53_dn5A and I53_dn5B).

[0313] Antigenic protein

[0314] The present disclosure provides protein-based VLP vaccines for RSV and / or hMPV in humans or animals (particularly pets, agricultural animals, and any animals associated with the spread of the disease). The present disclosure relates to incorporating any antigenic fragment of the hMPV F protein, such as an extracellular domain or an antigenic fragment thereof, into a VLP vaccine. Guidance can be obtained in particular based on studies of immune responses to infection or vaccination, such as separation of binding or neutralizing antibodies, genetic analysis of F protein sequences, structural studies of antigenic proteins and antibodies, and most particularly clinical and veterinary experience with subunit vaccines. By adopting the presentation format provided above, subunit vaccines for RSV or hMPV may be suitable for use with the VLPs of the present disclosure. The present disclosure relates to the extracellular domain of RSV or hMPV F, but it will be understood that portions of the transmembrane domain may be included.

[0315] The term "antigenic fragment" refers to any fragment of a protein that produces an immune response (humoral or T cell response) to a protein in vivo. Antigenic fragments can be linear epitopes, discontinuous epitopes, or conformational epitopes (e.g., folded domains). Antigenic fragments can retain the secondary, tertiary, and / or quaternary structures of the full-length protein. In some embodiments, the antigenic fragment comprises a neutralizing epitope. In such a case, VLPs can produce a neutralizing antibody response. Antigenic fragments can be designed computationally, such as by predicting secondary structure and rationally removing N or C-terminal unstructured regions or internal loops or entire structural elements (alpha helices and / or beta folds). Figure 2 An exemplary secondary structure diagram is provided. In some embodiments, the extracellular domain comprises a truncation or deletion of some or all of the N-terminal signal peptide (eg, MSWKVVIIFSLLITPQHGL (SEQ ID NO: 133) or MSWKVMIIISLLITPQHGL (SEQ ID NO: 134).

[0316] In some embodiments, the hMPV F protein extracellular domain is C-terminally truncated. In some embodiments, the hMPV F protein extracellular domain 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.

[0317] The native sequence of the A strain hMPV F protein (GenBank AY145297) is shown below, with the signal sequence underlined and italicized, and the transmembrane and intracellular portions underlined (SEQ ID NO: 56):

[0318]

[0319] When the protein is expressed, the native signal sequence is post-translationally cleaved. The native signal sequence can be replaced by another signal sequence for expressing the extracellular domain, or in some embodiments, no signal sequence is used. Thus, in some embodiments, the hMPV F protein extracellular domain is SEQ ID NO: 57 or a variant thereof:

[0320]

[0321] In some embodiments, the hMPV F protein extracellular 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: 57 or an antigenic fragment thereof.

[0322] Further hMPV F protein sequences are provided in Table 2. In some embodiments, the hMPV F protein extracellular 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 the extracellular domains in Table 2 or antigenic fragments thereof.

[0323] A variety of signal sequences can be used, paired with native extracellular domain sequences or exchanged 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 the signal sequence in Table 2.

[0324] The hMPV F protein may contain one or more substitutions.

[0325] Relative to SEQ ID NO:57, 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. Thus, in various embodiments, the hMPV F protein extracellular domain comprises one or more substitutions selected from this list.

[0326] Relative to SEQ ID NO: 57, 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 extracellular domain comprises one or more substitutions selected from this list.

[0327] 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 extracellular domain comprises two or more substitutions selected from this list.

[0328] 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 extracellular domain comprises one or more substitutions selected from this list.

[0329] 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 extracellular domain comprises two or more substitutions selected from this list.

[0330] In some embodiments, the hMPV F protein extracellular domain comprises substitutions Q100R and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A185P, Q100R and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A185P, T127C, N153C, Q100R and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G and R102G. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein extracellular domain comprises substitutions and deletions A63C, A140C, A147C, K188C, G106 deletion, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A113C, A120C, A339C, Q426C, T160F, I177L, Q100K, and S101A. In some embodiments, the hMPV F protein extracellular domain comprises substitutions V84C, A140C, A147C, A249C, Q100R, and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A63C, A140C, A147C, K188C, K450C, S470C, Q100R, and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions and deletions A63C, A140C, A147C, K188C, G106 deletion, Q100R, and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A113C, A120C, A339C, Q426C, T160F, I177L, Q100R, and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A185P, A113C, A339C, Q100R, and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A185P, T160F, I177L, Q100R, and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A185P, A113C, A339C, T160F, I177L, Q100R, and S101R.In some embodiments, the hMPV F protein extracellular domain comprises substitutions A63C, K188C, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A63C, K188C, K450A, S470A, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A63C, A140C, A147C, K188C, G294E, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, R102G, and G294E. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A63C, K188C, N97G, P98G, R99G, Q100G, S101G, R102G, and G294E. In some embodiments, the hMPV F protein extracellular domain comprises substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, H368N, Q100R, and S101R. In some embodiments, the hMPV F protein extracellular domain comprises substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein extracellular domain comprises substitutions V84C, A140C, A147C, A249C, N97G, P98G, R99G, Q100G, S101G, and R102G. In some embodiments, the hMPV F protein extracellular domain comprises substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, N97G, P98G, R99G, Q100G, S101G, H368N, and R102G.

[0331] In some embodiments, the substitution has an expected effect on the VLP, it being understood that the expected effect is not achieved in every embodiment of the VLP with the substitution. In some embodiments, the expected effect is increased expression in the production cell line (Mas et al., PLoS Pathog. 12: e1005859 (2016)). In some embodiments, the expected effect is to stabilize the post-fusion or pre-fusion form of the extracellular domain of the hMPV F protein (Battles et al., Nat Commun. 8: 1528 (2017)). In some embodiments, the expected effect is to bind neutralizing antibodies against the hMPV F protein. The antigen may further include a leucine at the N-terminus, as shown in SEQ ID NO: 146-178.

[0332] Table 2. Exemplary hMPV protein F extracellular domain antigenic variants

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346] Thus, in some embodiments, the hMPV F protein extracellular 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 NOs: 58-90, or an antigenic fragment thereof.

[0347] In some embodiments, the hMPV F protein comprises one or more furin cleavage sites, optionally one or more copies of the Arg-XX-Arg motif (SEQ ID NO: 52), such as the native sequence RQSR (SEQ ID NO: 54). In the case where the furin cleavage site is present in the hMPV extracellular domain, in some cases, the expression yield can be increased by co-expressing furin or a functional variant thereof in the host cell from a transfected plasmid or from a stably integrated polynucleotide sequence. 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 replacing the Gly linker or by making other amino acid substitutions.

[0348] hMPV neutralizing activity is mediated by antibodies that recognize pre- and post-fusion F protein conformations, and antibodies generated in the in vivo immune response can selectively recognize pre- or post-fusion specific sites, or can recognize 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 accessible antigenic sites in pre- and post-fusion forms of the F protein and are located in 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 to epitopes on the F protein spanning the DI, DII, and DIII subunits, forming contacts that depend on the pre-fusion F conformation. Wen et al., Nat Microbiol. 2: 16272 (2017). The respiratory syncytial virus (RSV) F protein shares several antigenic epitopes, such as site III and site IV epitopes, Huang et al., Front Immunol. 10: 2778 (2019). These are examples of epitopes described for neutralizing antibodies against the hMPV F protein, but not an exhaustive list.

[0349] Neutralizing epitopes are usually conformational epitopes, so peptides corresponding to these epitopes will not bind antibodies and therefore will not induce neutralizing titers. Neutralizing antibodies can be isolated from healthy donors who are seropositive for hMPV, Battles et al., or can be generated by immunization in animal models, Gabriella et al., J Virol. 81(2):698-707 (2007).

[0350] In some embodiments, the VLPs of the present disclosure comprising the extracellular domain of the hMPV F protein may bind to neutralizing antibodies against the hMPV F protein, also referred to as anti-hMPV F protein antibodies. In some embodiments, the VLPs described herein may 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, MF10 and MPE8 antibodies. In some embodiments, the VLPs described herein 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, MF1, MF2, MF3, MF11, MF17, MF18, and MF19 antibodies. In some embodiments, the VLPs described herein may 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 the 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 are bound to one or more anti-hMPV F protein antibodies. In some embodiments, the VLPs described herein are bound to two or more anti-hMPV F protein antibodies.

[0351] Multimerization domains and linkers

[0352] In some embodiments, VLP comprises the trimer assembly of antigen, and the trimer assembly of described antigen comprises: the first polypeptide comprising the first multimerization domain and the first polypeptide comprising the second multimerization domain.The first multimerization domain comprises three copies of inducing the first polypeptide to self-associate to form the protein-protein interface of trimer building block.In VLP with two or more components, each copy of the first multimerization domain further comprises a surface exposed interface, and this surface exposed interface interacts with the complementary surface exposed interface on the second multimerization domain.Similarly speaking, the second multimerization domain is adapted to multimerize with the first multimerization domain (of the first polypeptide of the first component). As described in 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 multimerization domain and the second multimerization domain drives the trimer assembly domain and multiple copies of the second assembly domain to assemble into target VLP. In some embodiments, each copy of the trimer assembly domain of VLP carries an antigenic protein or its antigenic fragment (e.g., as a genetic fusion) connected thereto; These VLPs display proteins in full price. In other embodiments, VLPs of the present disclosure include one or more copies of the first multimerization domain with an antigenic protein or its antigenic fragment (e.g., as a genetic fusion) and one or more first multimerization domains without antigenic protein; These VLPs display G proteins in partial valence. The first multimerization domain can be any polypeptide sequence that forms a trimer and interacts with the second multimerization domain to drive assembly as a target VLP. In some embodiments, VLP includes a first polypeptide and a second polypeptide selected from those disclosed in US 20130274441A1, US 2015 / 0356240 A1, US 2016 / 0122392 A1, WO 2018 / 187325 A1, each of which is incorporated herein by reference in its entirety.

[0353] In some embodiments of VLP of the present disclosure, the core of antigenic protein and VLP can be genetically fused so that they are all present in a single polypeptide, also referred to as a single-chain polypeptide. The linkage between protein and core allows antigenic protein or its antigenic fragment to be displayed outside of VLP. Therefore, the connection point with the core should be outside the core of the formed virus-like particle. Many polypeptide sequences can be used to connect the core of protein or its antigenic fragment and virus-like particle. In some cases, the joint comprises the polypeptide sequence. Any suitable joint polypeptide can be used. In some embodiments, the joint imposes the strict relative orientation of antigenic protein (e.g., extracellular domain) or its antigenic fragment on the core. In some embodiments, the joint connects antigenic protein (e.g., extracellular domain) or its antigenic fragment to the core flexibly. In some embodiments, the joint includes another trimerization domain (e.g., the folding subdomain of T4 fibroin) to assist in stabilizing the trimer form of F protein—e.g., GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:91) or its functional variant.

[0354] In some embodiments, the linker may include a Gly-Ser linker (i.e., a linker consisting of glycine and serine residues) of any suitable length. In some embodiments, the length of 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 some embodiments, the Gly-Ser linker may include or consist of an amino acid sequence of GSGGSGSGSGGSGSG (SEQ ID NO: 127), GGSGGSGS (SEQ ID NO: 128), or GSGGSGSG (SEQ ID NO: 129). In some embodiments, the linker includes the sequence GSGGSGSSG (SEQ ID NO: 130). In some embodiments, the linker includes the sequence GSGSGSGSGSGSGSG (SEQ ID NO: 131). In some embodiments, the linker includes the sequence GSGGSGSGSGGS (SEQ ID NO: 126).

[0355] In some embodiments of the VLPs of the present disclosure, the first component may optionally contain a poly-His tag HHHHHH (SEQ ID NO: 143).

[0356] Table 3A and Table 3B show the exemplary examples of VLP sequences as herein described.These sequences comprise multimerization domain, RSV or hMPV F protein extracellular domain, joint, poly-His tag and signal sequence.Signal and poly-His tag sequence are shown with underline, and are optionally included in the sequence.

[0357] Table 3A

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373] In some embodiments, the RSV protein extracellular domain comprises the substitutions 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, or any combination thereof. In some embodiments, the RSV protein extracellular domain comprises substitutions Q98C and Q361C, A149C and Y458C, N183GC and N428C, N88C and N254C, E92C and N254C, and / or S238C and Q279C, or any combination thereof.

[0374] Table 3B. Exemplary amino acid sequences of VLPs of the present disclosure

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388] Assembly of VLPs

[0389] In some embodiments, single component self-assembles into VLP.In some embodiments, one or more purified samples of the first and second components for forming VLP are mixed (for example, I53-50A / B icosahedral VLP) with approximately equimolar molar ratio under aqueous conditions.The first and second components (by multimerization domain and optionally by extracellular domain) interact with each other to drive the assembling of target VLP.The successful assembling of target VLP can be confirmed by analyzing the in vitro assembly reaction, and the mode is carried out by the common biochemistry or biophysical method for assessing the physical size of protein or protein assembly, including but not limited to size exclusion chromatography, natural (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 VLP of other substances or molecules present in the in vitro assembly reaction can be purified by the preparation technology that is usually used to separate proteins according to the physical size of protein, and these preparation technologies include but are not limited to size exclusion chromatography, preparation ultracentrifugation, tangential flow filtration or preparation gel electrophoresis. The presence of antigenic proteins in VLPs can be assessed by techniques commonly used to determine the properties 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 proteins on the outside of the particles and their conformation or antigenicity can be assessed 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.

[0390] In various embodiments, VLPs of the present disclosure comprise two or more different first polypeptides with different antigenic proteins as genetic fusions; These VLPs co-display a variety of different proteins on the same VLP. These multi-antigen VLPs are produced by assembling in vitro with a mixture of two or more antigens each comprising a multimerization domain. The fraction of each antigen in the mixture determines the average valence of each antigenic protein in the resulting VLP. The presence and average valence of each antigen in a given sample can be assessed by quantitative analysis using the above-mentioned techniques for assessing the presence of antigenic proteins in full-valence VLPs.

[0391] In various embodiments, the VLP has a diameter between about 20 nanometers (nm) and about 40 nm, an internal cavity between about 15 nm and about 32 nm, and a pore size in the longest dimension of the protein shell between about 1 nm and about 14 nm.

[0392] In some embodiments, VLP has icosahedral symmetry. In such embodiments, VLP may include 60 copies of the first component and 60 copies of the second component. In such an 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, VLP includes twelve first assemblies and twenty second assemblies; in such embodiments, each first assembly may, for example, include five copies of the same first component, and each second assembly may, for example, include three copies of the same second component. In other embodiments, VLP includes twelve first assemblies and thirty second assemblies; in such embodiments, each first assembly may, for example, include five copies of the same first component, and each second assembly may, for example, include two copies of the same second component. In further embodiments, VLP includes twenty first assemblies and thirty second assemblies; in this embodiment, each first assembly may, for example, include three copies of the same first component, and each second assembly may, for example, include two copies of the same second component. All of these embodiments are capable of forming protein-based VLPs with regular icosahedral symmetry.

[0393] In various further embodiments, the oligomeric state of the first and second multimerization domains is as follows:

[0394] I53-34A: trimer + I53-34B: pentamer;

[0395] I53-40A: pentamer + I53-40B: trimer;

[0396] I53-47A: trimer + I53-47B: pentamer;

[0397] I53-50A: trimer + I53-50B: pentamer;

[0398] I53-51A: trimer + I53-51B: pentamer;

[0399] I32-06A: dimer + I32-06B: trimer;

[0400] I32-19A: trimer + I32-19B: dimer;

[0401] I32-28A: trimer + I32-28B: dimer;

[0402] I52-03A: pentamer + I52-03B: dimer;

[0403] I52-32A: dimer + I52-32B: pentamer; and

[0404] I52-33A: pentamer + I52-33B: dimer

[0405] 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:

[0406] I53-50A

[0407]

[0408] 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.

[0409] The I53-50A protein sequence has two intra-monomer disulfide bonds. In some embodiments, the cysteine ​​residues are mutated to residues (e.g., alanine or serine) that do not contain thiol groups. Removing thiol groups can promote correct protein folding without weakening 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 here:

[0410] I53-50A

[0411]

[0412] In some embodiments, the multimerization domain of the first polypeptide comprises an amino acid substitution of one or more of C74A, C98A, C163A, and C201A relative to SEQ ID NO: 132. In some embodiments, the multimerization domain of the first polypeptide comprises SEQ ID NO: 132 or a variant thereof.

[0413] I53-50A-Δcys (without disulfide form)

[0414]

[0415] 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 substitution can be C for A, T, S, L, I, or any amino acid other than C.

[0416] Nucleic Acids

[0417] On the other hand, the present disclosure provides isolated nucleic acids encoding the antigens, first components and / or second components of the present disclosure. The isolated nucleic acid sequence may comprise RNA or DNA. As used herein, "isolated nucleic acids" are nucleic acids that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences. Such isolated nucleic acid sequences may comprise additional sequences that can be used to promote expression and / or purification of the encoded protein, including but not limited to poly A sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals and secretion signals, nuclear localization signals, and plasma membrane localization signals. Based on the teachings herein, it will be apparent to those skilled in the art what nucleic acid sequence will encode the protein of the present disclosure.

[0418] 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 connected to a suitable control sequence." recombinant expression vector " includes a vector to which a nucleic acid coding region or gene is operably connected to any control sequence that can affect the expression of a gene product. A "control sequence" operably connected to a nucleic acid sequence of the present disclosure is a nucleic acid sequence that can affect the expression of a nucleic acid molecule. The control sequence does not need to be adjacent to the nucleic acid sequence, as long as they serve to direct its expression. Therefore, for example, there may be an intervening untranslated but transcribed sequence between the promoter sequence and the nucleic acid sequence, and the promoter sequence may still be considered to be "operably connected" 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 may be any type known in the art, including but not limited to plasmids and virus-based expression vectors. The control sequences used to drive expression of the disclosed nucleic acid sequences 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 variety of inducible promoters, including but not limited to tetracycline, ecdysone, steroid responsive promoters). The construction of expression vectors for transfecting prokaryotic cells is also well known in the art and can therefore be achieved via standard techniques. (See, for example, Sambrook, Fritsch and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, EJ Murray, ed., The Humana Press Inc., Clifton, NJ) and Ambion 1998 Catalog (Ambion, Austin, TX). The expression vector must be replicable in the host organism, either as an episome or by integration into the host chromosomal DNA. In a preferred embodiment, the expression vector comprises a plasmid. However, the disclosure is intended to include other expression vectors, such as viral vectors, that serve equivalent functions.

[0419] On the other hand, the present disclosure provides a host cell transfected or transduced with a recombinant expression vector disclosed herein, wherein the host cell can be a prokaryotic cell or a eukaryotic cell. The cell can be transiently or stably transfected or transduced. This transfection or transduction of the expression vector into prokaryotic cells and eukaryotic cells can be achieved via any technique known in the art, including but not limited to standard bacterial transformation, calcium phosphate coprecipitation, electroporation, or liposome-mediated, DEAE dextran-mediated, polycation-mediated or virus-mediated transfection. (See, e.g., Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition (RI Freshney. 1987. Liss, Inc. New York, NY).

[0420] In another aspect, the present disclosure provides methods of producing antigens, components or VLPs according to the present disclosure. In some embodiments, the method comprises the steps of: (a) culturing a host according to this aspect of the present disclosure under conditions conducive to the expression of the polypeptide, and (b) optionally recovering the expressed polypeptide.

[0421] In some embodiments, the present disclosure provides a method for preparing a vaccine, comprising culturing a host cell comprising a polynucleotide comprising a sequence encoding an antigen of the present 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, wherein the second component polymerizes with the antigen to form a VLP; and optionally purifying the VLP.

[0422] In some embodiments, the present disclosure provides a method for preparing a vaccine, comprising culturing a host cell comprising one or more polynucleotides comprising sequences encoding two components of the VLP of any one of the present 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.

[0423] Exemplary host cells include E. coli cells, 293 and 293F cells, HEK293 cells, Sf9 cells, Chinese hamster ovary (CHO) cells, and any other cell line used for production of recombinant proteins.

[0424] 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 preparation 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% of the expression level of RSV and / or hMPV F protein (optionally the same extracellular domain as in VLP) in the same or similar expression system. In various embodiments, the first component expresses 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 expression level of RSV and / or hMPV F protein (optionally the same extracellular domain as in VLP) in the same or similar expression system.

[0425] In some embodiments, the RSV and / or hMPV F protein ectodomain of the first component is in a pre-fusion conformation, or most of the RSV and / or hMPV F protein ectodomain is in a pre-fusion 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 pre-fusion conformation. In some embodiments, the RSV and / or hMPV F protein ectodomain of the VLP is in a pre-fusion conformation, or most of the RSV and / or hMPV F protein ectodomain is in a pre-fusion 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 pre-fusion conformation.

[0426] In some embodiments, the fraction of RSV and / or hMPV F protein extracellular domains in the prefusion state is determined by binding to conformation-specific antibodies (e.g., D1-25 and 1112-1). In some embodiments, the fraction of the hMPV F protein extracellular domain 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 fraction in the reference component or the reference protein not connected to the multimerization domain. In some embodiments, the fraction of the hMPV F protein extracellular domain 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 fraction in the VLP (e.g., micellar VLP).

[0427] Vaccines and administration

[0428] The disclosure also provides vaccines comprising VLPs as described herein. Such compositions can be used to produce antibodies in mammals (e.g., humans). Vaccine compositions of the present disclosure generally include a pharmaceutically acceptable carrier, and an in-depth discussion about such carriers can be obtained in Remington: The Science and Practice of Pharmacy.

[0429] The pH of the composition is typically between about 4.5 and about 11, such as between about 5 and about 11, about 5.5 and about 11, about 6 and about 11, about 5 and about 10.5, about 5.5 and about 10.5, about 6 and about 10.5, about 5 and about 10, about 5.5 and about 10, about 6 and about 10, about 5 and about 9.5, about 5.5 and about 9.5, about 6 and about 9.5, about 5 and about 9, about 5.5 and about 9, about 6 and about 9, about 5 and about 8.5, about 5.5 and about 8.5, about 6 and about 8.5, about 5.5 and about 8, about 6 and 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, and the like. A stable pH may be maintained by the use of a buffer such as a Tris buffer, a citrate buffer, a phosphate buffer or a histidine buffer. The composition will therefore typically include a buffer.

[0430] In some embodiments, the pH of the formulation is between about pH 6.2 and 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 can also be within a numerical range. Therefore, in some embodiments, the pH is between about 6.2 and about 8.0, between about 6.2 and 7.8, between about 6.2 and 7.6, between about 6.2 and 7.4, between about 6.2 and 7.2, between about 6.2 and 7.0, between about 6.2 and 6.8, between about 6.2 and about 6.6, or between about 6.2 and 6.4. In other embodiments, the pH is between 6.4 and about 8.0, between about 6.4 and 7.8, between about 6.4 and 7.6, between about 6.4 and 7.4, between about 6.4 and 7.2, between about 6.4 and 7.0, between about 6.4 and 6.8, or between about 6.4 and about 6.6. In still other embodiments, the pH is between about 6.6 and about 8.0, between about 6.6 and 7.8, between about 6.6 and 7.6, between about 6.6 and 7.4, between about 6.6 and 7.2, between about 6.6 and 7.0, or between about 6.6 and 6.8. In still other embodiments, it is between about 6.8 and about 8.0, between about 6.8 and 7.8, between about 6.8 and 7.6, between about 6.8 and 7.4, between about 6.8 and 7.2, or between about 6.8 and 7.0. In still other embodiments, it is between about 7.0 and about 8.0, between about 7.0 and 7.8, between about 7.0 and 7.6, between about 7.0 and 7.4, between about 7.0 and 7.2, between about 7.2 and 8.0, between about 7.2 and 7.8, between about 7.2 and about 7.6, between about 7.2 and 7.4, between about 7.4 and about 8.0, between about 7.4 and about 7.6, or between about 7.6 and about 8.0.

[0431] In some embodiments, preparation can include one or more salts, such as sodium chloride, sodium phosphate or its combination.In general, every kind of salt is present in preparation with about 10mM to about 200mM.Therefore, in some embodiments, any salt present is present with about 10mM to about 200mM, about 20mM to about 200mM, about 25mM to about 200mM, about 30mM to about 200mM, about 40mM to about 200mM, about 50mM to about 200mM, about 75mM to about 200mM, about 100mM to about 200mM, about 125mM to about 200mM, about 150mM to about 200mM or about 175mM to about 200mM. 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 yet other embodiments, any salt present is present at about 10mM to about 150mM, about 20mM to about 150mM, about 25mM to about 150mM, about 30mM to about 150mM, about 40mM to about 150mM, about 50mM to about 150mM, about 75mM to about 150mM, about 100mM to about 150mM, or about 125mM to about 150mM. In yet other embodiments, any salt present is present at about 10mM to about 125mM, about 20mM to about 125mM, about 25mM to about 125mM, about 30mM to about 125mM, about 40mM to about 125mM, about 50mM to about 125mM, about 75mM to about 125mM, or about 100mM to about 125mM. In some embodiments, any salt present is present at about 10mM to about 100mM, about 20mM to about 100mM, about 25mM to about 100mM, about 30mM to about 100mM, about 40mM to about 100mM, about 50mM to about 100mM, or about 75mM to about 100mM. In other embodiments, any salt present is present at about 10mM to about 75mM, about 20mM to about 75mM, about 25mM to about 75mM, about 30mM to about 75mM, about 40mM to about 75mM, or about 50mM to about 75mM. In other embodiments, any salt present is present at about 10mM to about 50mM, about 20mM to about 50mM, about 25mM to about 50mM, about 30mM to about 50mM, or about 40mM to about 50mM.In other embodiments, any salt present is present at about 10mM to about 40mM, about 20mM to about 40mM, about 25mM to about 40mM, about 30mM to about 40mM, about 10mM to about 30mM, about 20mM to about 30, about 25mM to about 30mM, about 10mM to about 25mM, about 20mM to about 25mM, or about 10mM to about 20mM. In some embodiments, sodium chloride is present in the formulation at about 100mM. In some embodiments, sodium phosphate is present in the formulation at about 25mM.

[0432] The composition may be sterile and / or pyrogen-free. The composition may be isotonic to humans.

[0433] Vaccine compositions may include immune adjuvants. Exemplary adjuvants include the following: mineral-containing compositions; oil emulsions; squalene emulsions; saponin preparations; virosomes and virus-like particles; bacterial or microbial derivatives; bioadhesives and mucoadhesives; liposomes; polyoxyethylene ether and polyoxyethylene ester preparations; polyphosphazenes (PCPP); muramyl peptides; imidazoquinolone compounds; thiosemicarbazone compounds; tryptanthrin compounds; human immunomodulators; lipopeptides; benzonaphthyridines; microparticles; immunostimulatory polynucleotides (such as RNA or DNA; for example, oligonucleotides containing CpG).

[0434] For example, the composition may include an aluminum salt adjuvant, an oil-in-water emulsion (e.g., an oil-in-water emulsion containing squalene, such as or AS03), TLR7 agonists (such as imidazoquinoline or imiquimod) or combinations thereof. Suitable aluminum salts include hydroxides (such as hydroxide oxides), phosphates (such as hydroxyphosphates, orthophosphates), (for example, see Chapters 8 and 9 of Vaccine Design. (1995), edited by Powell & Newman, ISBN: 030644867X, Plenum) or mixtures thereof. The salt may take any suitable form (such as gel, crystal, amorphous, etc.), where the antigen is adsorbed onto the salt as an example. Al in the composition for administration to a patient +++ The concentration may be less than 5 mg / ml, for example <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. The preferred range is between 0.3 and 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.

[0435] Exemplary adjuvants that can be used in the pharmaceutical compositions provided herein include, but are not limited to, 3M-052, Adju-Phos TM 、Alhydrogel TM Adjumer TM, albumin-heparin microparticles, algae dextran, Algammulin, alum, antigen preparation, AS-2 adjuvant, ASO1, ASO3, autologous dendritic cells, autologous PBMC, Avridine TM , B7-2, BAK, BAY R1005, BECC TLR-4 agonist, bupivacaine, bupivacaine-HCl, BWZL, calcitriol, calcium phosphate gel, CCR5 peptide, CFA, cholera toxin (CT) and cholera toxin B subunit (CTB), cholera toxin A1-subunit-protein A D-fragment fusion protein, CpG, CPG-1018, CPG-1018 plus aluminum salt, CRL1005, cytokine-containing liposomes, D-murapalmitine, 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-α, IFA, IFN-γ in pcDNA3, IL-12DNA, 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 TM 、ImmTher TM , Immunoliposomes containing antibodies against co-stimulatory molecules, Interferon-γ, Interleukin-1β, Interleukin-12, Interleukin-2, Interleukin-7, ISCOM(s) TM 、Iscoprep7.0.3 TM , keyhole limpet hemocyanin, lipid-based adjuvant, liposomes, loxoribine, LT(R192G), LT-OA or LT oral adjuvant, LT-R192G, LTK63, LTK72, Matrix-M TM Adjuvant, MF59, MONTANIDE ISA 51, MONTANIDE ISA720, MPL. TM ., MPL-SE, MTP-PE, MTP-PE liposomes, Murametide, Magnolia alkaloids, NAGO, nCT natural cholera toxin, nonionic surfactant vesicles, non-toxic mutant E112K of cholera toxin mCT-E112K, methyl paraben, pCIL-10, pCIL12, pCMVmCAT1, pCMVN, peptidomer-NP, Pleuran, PLG, PLGA, PGA and PLA, Pluronic L121, PMMA, PODDS TM, Poly-rA: Poly-rU, Polysorbate 80, Protein Helix, QS-21, Quadri A saponin, Quil-A, Rehydragel HPA, Rehydragel LV, RIBI, Ribi-like adjuvant system (MPL, TMD, CWS), S-28463, SAF-1, Sclavo peptide, Sendai virus proteoliposome, Sendai virus-containing lipid matrix, Span 85, Specol, Squalane 1, Squalene 2, Stearoyltyrosine, SWE, Tetanus toxoid (TT), Theramide TM , threonylmuramyl dipeptide (TMDP), Ty particles and Walter Reed liposomes.

[0436] In a preferred embodiment, the adjuvant is aluminum hydroxide gel (e.g., Alhydrogel TM ). In a preferred embodiment, the adjuvant is SWE. In a preferred embodiment, the adjuvant is MF59.

[0437] MF59 is an oil-in-water emulsion containing squalene (4.3%) in a citric acid buffer containing the stabilizing nonionic surfactants Tween 80 (0.5%) and Span 85 (0.5%). MF59 has been shown to be well tolerated in humans and is 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).

[0438] For example, the composition may include an aluminum salt adjuvant, an oil-in-water emulsion (e.g., an oil-in-water emulsion containing squalene, such as MF59, SWE or AS03), a TL4 agonist, a TLR9 agonist (e.g., a CpG oligodeoxynucleotide), a TLR7 agonist (e.g., 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., hydroxide oxides), phosphates (e.g., hydroxyphosphates, orthophosphates), (e.g., see Chapters 8 and 9 of Vaccine Design. (1995), edited by Powell & Newman, ISBN: 030644867X, Plenum) or mixtures thereof. The salt may take any suitable form (e.g., gel, crystal, amorphous, etc.), with the adsorption of antigens onto the salt being an example. The concentration of Al+++ in the composition for administration to the patient may be less than 5 mg / ml, for example <4 mg / ml, <3 mg / ml, <2 mg / ml, <1 mg / ml, etc. The preferred range is between 0.3 and 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 a preferred embodiment, the pharmaceutical composition provided herein comprises aluminum hydroxide as an adjuvant. In some embodiments, the pharmaceutical composition provided herein comprises 500 μg of aluminum hydroxide.

[0439] On the one hand, the present disclosure provides a vaccine comprising VLPs as described herein, wherein the vaccine optionally comprises one or more pharmaceutically 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, one or more adjuvants are squalene, SLA, GLA, R848, IMQ, 3M-052, CpG, saponin (QS21) or a combination thereof. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is an emulsion based on squalene. In some embodiments, the emulsion based on squalene is MF59. The choice of adjuvant depends on the subject to be treated. Preferably, a pharmaceutically acceptable adjuvant is used.

[0440] In some embodiments, the adjuvant is squalene emulsion.

[0441] In some embodiments, the adjuvant is a TLR4 immunostimulator (e.g., SLA, GLA), for example as described in Van Hoeven et al., PLoS One. 11(2):e0149610 (2016).

[0442] In some embodiments, the adjuvant is a TLR7 / 8 immunostimulator (e.g., R848, IMQ, 3M-052), e.g., as described in Dowling D. Immuno Horizons (6): 185-197 (2018).

[0443] In some embodiments, the adjuvant is a TLR9 immunostimulator (CpG), for example as described in Bode et al., Expert Rev Vaccines. 10(4):499-511 (2011).

[0444] In some embodiments, the adjuvant is a saponin (QS21), for example as described in Zhu et al., Nat Prod Chem Res. 3(4):e113 (2016).

[0445] In some embodiments, the vaccine comprises a combination of two or more adjuvants (eg, squalene emulsion and alum or a TLR4 immunostimulator).

[0446] One suitable immunoadjuvant comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined in WO2011 / 027222 adsorbed onto an aluminium salt.A number of further adjuvants may be used, including any of the adjuvants disclosed in Powell & Newman (1995).

[0447] The composition may include an antimicrobial agent, particularly when packaged in multi-dose form. Antimicrobials such as thimerosal and 2-phenoxyethanol are commonly found in vaccines, but it may sometimes be desirable to use a mercury-free preservative or no preservative at all.

[0448] The composition may comprise a detergent, for example a polysorbate, such as polysorbate 80. Detergents are typically present at low levels, for example < 0.01%.

[0449] The composition may include a sodium salt (eg sodium chloride) to provide tonicity. A concentration of 10±2 mg / ml NaCl is typical, for example about 9 mg / ml.

[0450] 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 can also include a freeze-drying protective agent, such as sucrose, sorbitol or trehalose. In certain embodiments, the composition includes a preservative, such as benzalkonium chloride, phenylethylammonium, chlorhexidine, phenol, metacresol, 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 other embodiments, the composition includes a surfactant, such as 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 a combination thereof. The composition may also include a tension modifier, such as a compound that makes the formulation substantially isotonic or isotonic with human blood. Exemplary tension modifiers include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the composition further includes a stabilizer, such as a molecule that substantially prevents or reduces the chemical and / or physical instability of the VLP in a lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0451] In some embodiments, the present disclosure provides vaccines (immunogenic compositions) comprising one or more pharmaceutically acceptable excipients.

[0452] In some embodiments, the vaccine (immunogenic composition) is a stable emulsion.

[0453] In some embodiments, the present disclosure provides vaccines (immunogenic compositions) comprising one or more adjuvants. In some embodiments, the one or more adjuvants include TLR4 immunostimulants, such as monophosphoryl lipid A (MPL), glucopyranose lipid A (GLA) and / or soluble Leishmania antigen (SLA).

[0454] In another aspect, the present disclosure provides a method of 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 as described herein.

[0455] In certain embodiments, the immune response includes the production of neutralizing antibodies against the infectious agent. In certain embodiments, the neutralizing antibodies are complement independent.

[0456] The immune response may include a humoral immune response, a cell-mediated immune response, or both. In some embodiments, an immune response is induced for each antigenic protein delivered. The cell-mediated 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 antibody is a neutralizing antibody. Neutralizing antibodies prevent viral infection of cells. Viral infection of epithelial cells also includes fibroblasts. In some embodiments, the immune response reduces or prevents infection of two 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-neutralization; that is, the antibodies produced for the composition administered neutralize the viruses of strains other than the strains used in the composition.

[0457] A useful measure of antibody efficacy in the art is the "50% neutralization titer". To determine the 50% neutralization titer, serum from an immunized animal is diluted to assess how the diluted serum still retains the ability to block 50% of the virus from entering the cell. For example, a titer of 700 means that the serum retains the ability to neutralize 50% of the virus after being diluted 700 times. Therefore, the higher the titer, the stronger the neutralizing antibody response. In some embodiments, this 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 upper limit of the 50% neutralization titer range can be 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 12,000, about 13,000, about 14,000, about 15,000, about 16,000, about 17,000, about 18,000, about 19,000, about 20,000, about 21,000, about 22,000, about 23,000, about 24,000, about 25,000, about 26,000, about 27,000, about 28,000, about 29,000, or about 30,000. For example, a 50% neutralization titer can be about 3,000 to about 25,000. "About" means plus or minus 10% of the stated value.

[0458] In some embodiments, the virus-like particles of the present disclosure produce 0.5x10 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 3 IU / mL, 10x10 3 IU / mL, 25x10 3 IU / mL, 50x10 3 IU / mL, 100x10 3 IU / mL or greater immune response. In some embodiments, the virus-like particles of the present disclosure produce 0.5x10 3 Immune response of IU / mL or greater

[0459] The compositions of the present disclosure will generally be administered directly to the subject. Direct delivery may be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or into the interstitial space of a tissue), oral, intranasal, or by any other suitable route. For example, intramuscular administration may be employed, for example, to the thigh or upper arm. Injection may be performed via a needle (e.g., a hypodermic needle), but needle-free injection may alternatively be employed. A typical intramuscular dosage volume is 0.5 ml.

[0460] Can be administered by single dose regimen or multiple dose regimen.Multiple doses can be used for primary immunization regimen and / or booster immunization regimen.In multiple dose regimens, each dosage can be given by the same or different approach, such as parenteral primary immunization and mucosal reinforcement, mucosal primary immunization and parenteral reinforcement, etc.Multiple doses will usually be used at least 1 week apart (for example, 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 can be used at least 1 month apart (for example, 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.).The second or subsequent dose can be used in a longer interval, for example, in about 1 year, about 2 years, about 3 years, about 4 years or about 5 years after the previous dose, such as in about 3-5 years after the previous dose.

[0461] The subject can be a child (e.g., a toddler or infant), an adolescent, or an adult. Vaccines intended for use in children can also be administered to adults, for example to assess safety, dosage, immunogenicity, etc.

[0462] Vaccines of the present disclosure may be prophylactic (i.e., prevent a disease) or therapeutic (i.e., reduce or eliminate the symptoms of a disease). The term prophylactic may be considered to reduce the severity of a particular illness or prevent the onset of a particular illness. For the avoidance of doubt, the term prophylactic vaccine may also refer to a vaccine that mitigates the effects of a future infection, for example by reducing the severity of such infection or shortening the duration of such infection.

[0463] The VLPs separated and / or purified as described herein can be administered alone or as a primary immunization or boost in a mixed mode scheme, such as RNA primary immunization or DNA primary immunization, followed by protein boosting. For example, adenovirus vectors can be used in combination with the vaccine composition of the present disclosure as a primary immunization. Compared with the protein primary immunization / protein boosting strategy, the benefits of the RNA primary immunization / protein boosting strategy include, for example, increased antibody titers, more balanced distribution of IgG1:IgG2a subtypes, induction of TH1 type CD4+T cell-mediated immune responses similar to viral particles, and reduced production of non-neutralizing antibodies. RNA primary immunization can increase the immunogenicity of the composition, regardless of whether they contain adjuvants.

[0464] In the RNA primary immunity / protein boost strategy, RNA and protein are directed to the same target antigen. Examples of suitable modes of delivering RNA include virus-like replicon particles (VRP), alphavirus RNA, replicons encapsulated in lipid nanoparticles (LNPs) or prepared RNA, such as replicons prepared with cationic nanoemulsions (CNEs). Suitable cationic oil-in-water nanoemulsions are disclosed in WO2012 / 006380, for example, comprising an oil core (e.g., comprising squalene) and a cationic lipid (e.g., DOTAP, DMTAP, DSTAP, DC-cholesterol, etc.).

[0465] Alternatively, two doses of VLP can be administered at predetermined intervals to achieve a prime-boost effect. The predetermined interval can 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 predetermined interval can be 1, 2, 3, 4, or 6 weeks; or 2-3 weeks, 3-4 weeks, or 5-6 weeks, etc. The predetermined interval can be 1, 2, 3, or 4 months or 1, 2, 3, or 4 years.

[0466] In some embodiments, the RNA molecules are encapsulated in, associated with, or adsorbed on cationic lipids, liposomes, spirochetes, virosomes, immunostimulatory complexes, microparticles, microspheres, nanospheres, unilamellar vesicles, multilamellar vesicles, oil-in-water emulsions, water-in-oil emulsions, creamsomes, polycationic peptides, cationic nanoemulsions, or a combination thereof.

[0467] The present disclosure further provides a combination vaccine. Vaccines of the present disclosure include vaccines comprising RSV and / or hMPV VLPs and vaccines against one or more of the following: coronaviruses (such as beta coronaviruses, e.g., SARS-CoV-2, respiratory syncytial virus, rabies, pneumococcal virus, typhoid, hepatitis A, polio, influenza, hepatitis B, yellow fever, Japanese encephalitis, parvovirus, canine distemper, adenovirus, parainfluenza virus, influenza, measles, Lyme disease, coronavirus, vesicular stomatitis virus, herpes simplex virus, baculovirus, togotovirus, and Bornaviridae.

[0468] Also provided herein are kits and instructions for use for administering the nucleic acids (eg, RNA), purified proteins, and purified VLPs described herein. The present disclosure also provides a delivery device prefilled with a composition or vaccine disclosed herein.

[0469] Pharmaceutical compositions as described herein can be administered in combination with one or more additional therapeutic agents (such as antiviral agents, such as palivizumab). The additional therapeutic agents may include, but are not limited to, antibiotics or antibacterial agents, antiemetics, antifungals, anti-inflammatory agents, antivirals, immunomodulators, cytokines, antidepressants, hormones, alkylating agents, antimetabolites, antitumor antibiotics, antimitotic agents, topoisomerase inhibitors, cytostatics, anti-invasive agents, anti-angiogenic agents, growth factor function inhibitors, viral replication inhibitors, viral enzyme inhibitors, anticancer agents, alpha-interferon, beta-interferon, ribavirin, hormones and other Toll-like receptor modulators, immunoglobulins (Ig) and antibodies regulating Ig functions (such as anti-IgE (omalizumab)).

[0470] In certain embodiments, the compositions disclosed herein can be used as a medicament, for example, for inducing or enhancing an immune response in a subject (eg, a mammal) in need thereof.

[0471] In certain embodiments, the compositions disclosed herein can be used in the preparation of a medicament for inducing or enhancing an immune response in a subject (eg, a mammal) in need thereof.

[0472] One method of checking the efficacy of therapeutic treatment involves monitoring infection with infectious agents after administration of a composition or vaccine disclosed herein. One method of checking the efficacy of preventive treatment involves monitoring systemic immune responses (such as monitoring the levels of IgG1 and IgG2a production) and / or mucosal immune responses (such as monitoring the levels of IgA production) to the antigen. Typically, antigen-specific serum antibody responses are measured after immunization but before challenge, while antigen-specific mucosal antibody responses are measured after immunization and after challenge.

[0473] Treatment

[0474] In one aspect, the present disclosure provides a method for immunizing a subject against infection by human metapneumovirus (hMPV), the method 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, wherein the vaccine is administered by intramuscular injection. In some embodiments, wherein the vaccine is administered by intradermal injection. In some embodiments, wherein the vaccine is administered intranasally. In one aspect, the present disclosure provides a pre-filled syringe comprising a vaccine as described herein. In one aspect, the present disclosure provides a kit comprising a vaccine as described herein or a pre-filled syringe as described herein.

[0475] 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.

[0476] 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.

[0477] On the other hand, provided herein is a method for vaccinating a subject, comprising administering an effective amount of a pharmaceutical composition provided herein to the subject. On the other hand, provided herein is a method for producing an immune response in a subject, comprising administering an effective amount of a pharmaceutical composition provided herein to the subject. On the other hand, provided herein is a method for preventing RSV disease in a subject, comprising administering an effective amount of a pharmaceutical composition provided herein to the subject. In some embodiments, the subject is at risk of suffering from severe RSV disease. On the other hand, provided herein is a method for preventing hMPV disease in a subject, comprising administering an effective amount of a pharmaceutical composition provided herein to the subject. In some embodiments, the subject is at risk of suffering from severe hMPV disease. In some embodiments, the subject is an adult over the age of 60. In some embodiments, the subject is a healthy adult aged 18-45 years.

[0478] In some embodiments, a composition comprising 25 μg of a first VLP and 125 μg of a second VLP is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof. In some embodiments, a composition comprising 25 μg of a first VLP, 125 μg of a second VLP, and 9.75 mg of an oil-in-water emulsion is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof, and the oil-in-water emulsion comprises squalene, such as

[0479] In some embodiments, a composition comprising 75 μg of a first VLP and 75 μg of a second VLP is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof. In some embodiments, a composition comprising 75 μg of a first VLP, 75 μg of a second VLP, and 9.75 mg of an oil-in-water emulsion is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof, and the oil-in-water emulsion comprises squalene, such as

[0480] In some embodiments, a composition comprising 75 μg of a first VLP and 150 μg of a second VLP is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof. In some embodiments, a composition comprising 75 μg of a first VLP, 150 μg of a second VLP, and 9.75 mg of an oil-in-water emulsion is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof, and the oil-in-water emulsion comprises squalene, such as

[0481] In some embodiments, a composition comprising 75 μg of a first VLP and 225 μg of a second VLP is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof. In some embodiments, a composition comprising 75 μg of a first VLP, 225 μg of a second VLP, and 9.75 mg of an oil-in-water emulsion is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof, and the oil-in-water emulsion comprises squalene, such as

[0482] In some embodiments, a composition comprising 112.5 μg of a first VLP and 112.5 μg of a second VLP is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof. In some embodiments, a composition comprising 112.5 μg of a first VLP, 112.5 μg of a second VLP, and 9.75 mg of an oil-in-water emulsion is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof, and the oil-in-water emulsion comprises squalene, such as

[0483] In some embodiments, a composition comprising 150 μg of a first VLP and 150 μg of a second VLP is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof. In some embodiments, a composition comprising 150 μg of a first VLP, 150 μg of a second VLP, and 9.75 mg of an oil-in-water emulsion is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof, and the oil-in-water emulsion comprises squalene, such as

[0484] In some embodiments, a composition comprising 225 μg of a first VLP and 150 μg of a second VLP is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof. In some embodiments, a composition comprising 225 μg of a first VLP, 150 μg of a second VLP, and 9.75 mg of an oil-in-water emulsion is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof, and the oil-in-water emulsion comprises squalene, such as

[0485] In some embodiments, a composition comprising 150 μg of a first VLP and 75 μg of a second VLP is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof. In some embodiments, a composition comprising 150 μg of a first VLP, 75 μg of a second VLP, and 9.75 mg of an oil-in-water emulsion is administered intramuscularly to a subject, wherein the first VLP comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and the second VLP comprises an extracellular domain of a human metapneumovirus (hMPV) F protein or an antigenic variant thereof, and the oil-in-water emulsion comprises squalene, such as

[0486] In another aspect, provided herein is a method of generating an immune response in an unborn baby, the method comprising administering an effective amount of a pharmaceutical composition provided herein to the mother of the unborn baby. In some embodiments, the pharmaceutical composition is administered to the mother in the last three months of pregnancy.

[0487] In some embodiments, a first dose of the pharmaceutical composition is administered to a woman during pregnancy, and a second dose of the composition is administered to an infant born from pregnancy. Non-limiting examples of generating an immune response in an infant include those disclosed in International Patent Publication No. WO2012103361A1, which is incorporated herein in its entirety.

[0488] In some embodiments, an effective amount of a 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.

[0489] In some embodiments, an effective amount of a 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.

[0490] In some embodiments, the method provided herein further includes administering a second dose of a 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 of the first dose. In some embodiments, the method provided herein further includes administering a 3rd dose of a pharmaceutical composition provided herein. In some embodiments, the 3rd 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 includes administering subsequent doses at regular intervals of about 1, 2, 3, 4 or 5 years.

[0491] In some embodiments, the method provided herein limits the development of RSV infection of the subject.In some embodiments, the method causes the subject to produce RSV-A specific neutralizing antibodies.In some embodiments, the method causes the RSV-A specific neutralizing antibodies in the subject to increase by 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 with baseline.In some embodiments, in about one week of applying the pharmaceutical composition, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks or in about 12 weeks, the increase of RSV-A specific neutralizing antibodies can be detected.In some embodiments, the method causes the subject to produce RSV-B specific neutralizing antibodies. In some embodiments, the method results in an increase in RSV-B-specific neutralizing antibodies in the subject by 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, an increase in RSV-B-specific neutralizing antibodies can be detected within about one 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 administering the pharmaceutical composition.

[0492] In some embodiments, the method causes the subject to produce RSV F protein-specific IgG antibodies. In some embodiments, the method causes the RSV F protein-specific neutralizing antibodies in the subject to increase by 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. In some embodiments, the increase of RSV F protein-specific neutralizing antibodies can be detected in about one week of applying the pharmaceutical composition, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks or in about 12 weeks.

[0493] In some embodiments, described method causes experimenter to produce core-VLP specific IgG antibody.In some embodiments, described method causes the core-VLP specific IgG antibody in experimenter to increase 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 with baseline.In some embodiments, in about one week of applying pharmaceutical composition, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks or in about 12 weeks, the increase of core-VLP specific IgG antibody can be detected.

[0494] In some embodiments, the method causes the subject to produce RSV F protein-specific memory B cells. In some embodiments, the method causes the RSV F protein-specific memory B cells in the subject to increase by 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. In some embodiments, the increase of RSV F protein-specific memory B cells can be detected in about one week of applying the pharmaceutical composition, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks or in about 12 weeks.

[0495] In some embodiments, the method causes the subject to produce RSV F protein-specific T cells. In some embodiments, the method causes the RSV F protein-specific T cells in the subject to increase by 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. In some embodiments, the increase of RSV F protein-specific T cells can be detected in about one week of applying the pharmaceutical composition, 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.

[0496] In some embodiments, the method causes the subject to produce antibodies against human metapneumovirus. In some embodiments, the method causes an increase in antibodies against human metapneumovirus (hMPV) in the subject by 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. In some embodiments, an increase in antibodies against human metapneumovirus can be detected within about one 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 administering the pharmaceutical composition.

[0497] In some embodiments, the method provided herein limits the development of hMPV infection in the subject. In some embodiments, the method causes the subject to produce hMPV-A specific neutralizing antibodies. In some embodiments, the method causes the hMPV-A specific neutralizing antibodies in the subject to increase by 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 the baseline. In some embodiments, the increase of hMPV-A specific neutralizing antibodies can be detected in about one week of applying the pharmaceutical composition, 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. In some embodiments, the method causes the subject to produce hMPV-B specific neutralizing antibodies. In some embodiments, the method results in an increase in hMPV-B specific neutralizing antibodies in the subject by 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-B specific neutralizing antibodies is detectable within about one 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.

[0498] In some embodiments, the method causes the subject to produce hMPV F protein-specific IgG antibodies. In some embodiments, the method causes the hMPV F protein-specific neutralizing antibodies in the subject to increase by 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 the baseline. In some embodiments, the increase in hMPV F protein-specific neutralizing antibodies can be detected within about one 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 administering the pharmaceutical composition.

[0499] In some embodiments, described method causes experimenter to produce core-VLP specific IgG antibody.In some embodiments, described method causes the core-VLP specific IgG antibody in experimenter to increase 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 with baseline.In some embodiments, in about one week of applying pharmaceutical composition, in about 2 weeks, in about 3 weeks, in about 4 weeks, in about 5 weeks, in about 6 weeks, in about 7 weeks, in about 8 weeks, in about 9 weeks, in about 10 weeks, in about 11 weeks or in about 12 weeks, the increase of core-VLP specific IgG antibody can be detected.

[0500] In some embodiments, the method causes the subject to produce hMPV F protein-specific memory B cells. In some embodiments, the method causes the hMPV F protein-specific memory B cells in the subject to increase by 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 the baseline. In some embodiments, the increase of hMPV F protein-specific memory B cells can be detected within about one 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 administering the pharmaceutical composition.

[0501] In some embodiments, the method causes the subject to produce hMPV F protein-specific T cells. In some embodiments, the method causes the hMPV F protein-specific T cells in the subject to increase by 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 the baseline. In some embodiments, the increase of hMPV F protein-specific T cells can be detected within about one 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 administering the pharmaceutical composition. On the other hand, the present disclosure provides a method for vaccinating a subject to resist RSV infection (e.g., RSV-A and / or RSV-B infection) and / or hMPV infection (e.g., hMPV-A and / or hMPV-B infection), including administering to a subject in need thereof a polypeptide, virus-like particle, composition, nucleic acid, pharmaceutical composition or vaccine (referred to as "immunogenic composition") of any embodiment of the present invention in an amount that effectively treats infection or limits the development of infection. In some embodiments, this method prevents diseases after RSV subtype A and / or B infection. In some embodiments, this method prevents the development of RSV-related diseases (e.g., severe diseases), such as pneumonia and / or acute respiratory diseases. The subject can be any suitable mammalian subject, including but not limited to human subjects. In some embodiments, the subject is a human child, such as a child less than 12 months old. In some embodiments, the subject is a human toddler, such as an age of about 1 to about 3 years old or an age of about 1 to about 5 years old. In some embodiments, the subject is an adult over 50 years old or over 60 years old. In some embodiments, the subject is an adult over 65 years old. In a specific embodiment, the subject relies on the help of others or has serious health problems or risks (e.g., frail elderly people). In some embodiments, the subject is a healthy adult aged 18-60 years. In some embodiments, the subject is a healthy adult aged 18-45 years. 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 suffering from chronic potential heart and / or lung disease or suffering from dysfunction. In some embodiments, the subject has the risk of suffering from severe RSV disease (e.g., LRTI or pneumonia).

[0502] The immunogenic compositions provided herein can be used for vaccination of unborn infants. It is suggested to use certain inactivated vaccines during pregnancy to induce immunity in unborn infants, such as tetanus toxoid, attenuated diphtheria toxoid and acellular pertussis (Tdap) vaccine and influenza vaccine. Therefore, in some embodiments, there is provided herein a method for producing an immune response in unborn infants, the method comprising administering an effective amount of the immunogenic compositions provided herein to the mother of the unborn infant. The immunogenic compositions can be administered at any suitable time point in pregnancy, such as in the last three months of pregnancy.

[0503] The immunogenic compositions provided herein can be co-administered with other treatments (such as other vaccines). Therefore, in some embodiments, one or more seasonal or pandemic vaccines, such as influenza vaccines or SARS-Cov2 vaccines, can also be administered to subjects treated according to the methods provided herein. In some embodiments, pneumococcus, recombinant herpes zoster (herpes zoster) or Tdap vaccines can also be administered to subjects treated according to the methods provided herein. One, two or more vaccines can be co-administered with the immunogenic compositions provided herein."Co-administration" includes simultaneous administration and subsequent administration. For example, one, two or more vaccines and the immunogenic compositions provided herein can 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, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 8 hours, within 10 hours or within 12 hours.

[0504] On the other hand, provided herein is a method for treating a subject infected with RSV or hMPV. As used herein, "treatment (treat / treating)" includes but is not limited to achieving one or more of the following: (a) reducing the viral titer of the subject; (b) limiting any increase in the viral titer of the subject; (c) reducing the severity of viral infection; (d) limiting or preventing the development of symptoms after viral infection; (e) suppressing the deterioration of viral infection symptoms; (f) limiting or preventing the recurrence of viral infection symptoms in subjects with previous viral infection symptoms; and / or (e) improving survival rate. In some embodiments, the method of vaccination reduces the risk of subject infection with virus (e.g., RSV and / or hMPV). In some embodiments, the method of vaccination limits the development of viral infection. In some embodiments, the method of vaccination reduces the severity of viral infection symptoms. In preferred embodiments, infection is a lower respiratory tract infection (LRTI).

[0505] 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.

[0506] When the method includes limiting viral infection, the immunogenic composition is prophylactically administered to a subject who is unknown to be infected but may be at risk of exposure to a virus (e.g., RSV or hMPV). As used herein, "limiting development" includes, but is not limited to, achieving one or more of the following: (a) generating an immune response to the virus in a subject (antibody and / or cell-based, e.g., CD4 T cells, memory B cells, and / or CD8 T cells); (b) generating neutralizing antibodies against viruses or viral proteins in a subject; (b) limiting the accumulation of viral titers in a subject after exposure to the virus; and / or (c) limiting or preventing the development of symptoms after viral infection. Exemplary symptoms of viral (e.g., RSV or hMPV) infection include, but are not limited to, fever, fatigue, cough, nasal congestion, sneezing, shortness of breath, wheezing, and lower respiratory tract infection.

[0507] The methods provided herein can be used to limit the development of RSV (or hMPV)-A subtype and / or RSV (or hMPV)-B subtype infection. Without wishing to be bound by theory, it is believed that due to the high sequence similarity between the F proteins of RSV (or hMPV)-A and RSV (or hMPV)-B, immunization with the F protein of one RSV (or hMPV) subtype will produce at least some immunity against another subtype.

[0508] Further, the method provided herein can be used to limit the original strain infection of RSV (or hMPV) and / or the development of RSV (or hMPV) variant strain infection. 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, etc. (see, for example, Pandya et al., Pathogens 2019, 8 (2), 67; and Melero and Moore, Curr Top Microbiol Immunol. 2013; 372: 59-82). The pharmaceutical composition of the present invention can effectively limit the infection of RSV (or hMPV) strains that have not yet been described or found.

[0509] 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), wherein the immune response is used to limit the development of infection and 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 includes the generation of a RSV (or hMPV) F protein-specific (e.g., RSV (or hMPV)-A and / or RSV (or hMPV)-B F protein-specific) response with an average geometric titer of 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 9 In further embodiments, the immune response includes the production of antibodies against multiple antigenic epitopes or RSV (or hMPV) (eg, RSV (or hMPV)-A and / or RSV (or hMPV)-B).

[0510] In one aspect, the methods provided herein can result in an increase in antibody titer in a subject, such as 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. Antibody titer can be determined using any suitable assay known in the art or described herein, including but not limited to enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunoprecipitation, immunoblotting, and agglutination assays.

[0511] In some embodiments, the methods provided herein result in 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 against human metapneumovirus) 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 compared to baseline. times, about 5 times to about 6 times, about 6 times to about 7 times, about 7 times to about 8 times, about 8 times to about 9 times, about 9 times to about 10 times, about 10 times to about 12 times, about 12 times to about 15 times, about 15 times to about 20 times, about 20 times to about 25 times, about 25 times to about 30 times, about 30 times to about 40 times, about 40 times to about 50 times, about 50 times to about 60 times, about 60 times to about 70 times, about 70 times to about 80 times, about 80 times to about 90 times, about 90 times to about 100 times, or more than about 100 times. 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 against human metapneumovirus) by 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.

[0512] By "baseline" is meant the measurement of antibodies immediately prior to administering the first dose of an immunogenic composition provided herein. In some embodiments, compared to the baseline, at about 3 days to about 7 days, about 1 week to about 2 weeks, about 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 1 month ...0 weeks, about 1 month to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 1 month to about 12 months 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 against human metapneumovirus) may be detected from 2 months to about 18 months, from about 18 months to about 24 months, from about 2 years to about 3 years, from about 3 years to about 4 years, from about 4 years to about 5 years, or from about 5 years to about 10 years. In some embodiments, 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 against human metapneumovirus) can be detected 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 of administration of the immunogenic composition compared to baseline.

[0513] On the other hand, the methods provided herein can result in an increase in immune cells in a subject, such as an increase in RSV (or hMPV) F protein-specific memory B cells and / or RSV (or hMPV) F protein-specific T cells. Memory B cells and / or T cells may be specific to RSV (or hMPV)-AF protein or RSV (or hMPV)-BF protein, or they may 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.

[0514] 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) 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, or more than about 100-fold, compared to baseline. In some embodiments, the method provided herein causes the immune cells of the subject to increase, such as RSV (or hMPV) F protein-specific memory B cells and / or RSV (or hMPV) F protein-specific T cells to increase. In some embodiments, compared with baseline, the method provided herein causes immune cells to increase (for example, RSV (or hMPV) F protein-specific memory B cells and / or RSV (or hMPV) F protein-specific T cells to increase) 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. Memory B cells and / or T cells may have specificity to RSV (or hMPV)-AF protein or RSV (or hMPV)-BF protein, or they may have reactivity to both.

[0515] 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) can be detected within about 3 days to about 7 days, about 1 week to about 2 weeks, about 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 of administration of the immunogenic composition compared to baseline. 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) can be detected 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 of administration of the immunogenic composition compared to baseline. Memory B cells and / or T cells may be specific to RSV (or hMPV)-AF protein or RSV (or hMPV)-BF protein, or they may be reactive to both.

[0516] The polypeptide, virus-like particle, composition, nucleic acid, pharmaceutical composition or vaccine of any embodiment herein is generally formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including intranasal, sublingual, oral, parenteral, by inhalation spray, rectal or topical administration with a dosage unit formulation containing conventional pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneal administration. The polypeptide composition can also be administered via a sustained release formulation of microspheres, liposomes, immunostimulatory complexes (ISCOMs) or other microparticle delivery systems or introduced into suitable tissues (such as blood).

[0517] The dosage regimen can be adjusted to provide the best desired response (e.g., treatment or prevention response). Suitable dosage ranges can be, 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 25 μg / kg g body weight, 25μg / kg to 30μg / kg body weight, 30μg / kg to 35μg / kg body weight, 35μg / kg to 40μg / kg body weight, 40μg / kg to 45μg / kg body weight, 45μg / kg to 50μg / kg body weight, 50μg / kg to 55μg / kg body weight, 55μg / kg to 60μg / kg body weight, 60μg / kg to 65μg / kg body weight, 65μg / kg to 70μg / kg body weight, 70μg / kg to 75μg / kg body weight, 75μg / kg to 80μg / kg body weight, 80μg / kg to 85μg / kg body weight, 85μg / kg to 90μg / kg body weight, 90μg / kg to 100μg / kg g / kg to 95μg / kg body weight, 95μg / kg to 100μg / kg body weight, 100μg / kg to 150μg body weight, 150μg / kg to 200μg body weight, 200μg / kg to 250μg / kg body weight, 250μg / kg to 300μg / kg body weight, 300μg / kg to 350μg / kg body weight, 350μg / kg to 400μg / kg body weight, 400μg / kg to 450μg / kg body weight, 450μg / kg to 500μg body weight, 500μg / kg to 550μg body weight, 550μg / kg to 600μg body weight, 600μg / kg to 650μg body weight, 650μg / kg / kg to 700μg / kg body weight, 700μg / kg to 750μg / kg body weight, 750μg / kg to 800μg / kg body weight, 800μg / kg to 850μg / kg body weight, 850μg / kg to 900μg / kg body weight, 900μg / kg to 950μg / kg body weight, 950μg / kg to 1mg / kg body weight, 1mg / kg to 2mg / kg body weight, 2mg / kg to 3mg / kg body weight, 3mg / kg to 4mg / kg body weight, 4mg / kg to 5mg / kg body weight, 5mg / kg to 6mg / kg body weight, 6mg / kg to 7mg / kg body weight, 7mg / kg to 8mg / kg body weight,8mg / kg to 90mg / kg body weight, 90mg / kg to 100mg / kg body weight, 100mg / kg to 150mg / kg body weight, 150mg / kg to 200mg / kg body weight, 200mg / kg to 250mg / kg body weight, 250mg / kg to 300mg / kg body weight, 300mg / kg to 350mg / kg body weight, 350mg / kg to 400mg / kg body weight, 400mg / kg to 450mg / kg body weight, 450mg / kg to 500mg / kg body weight, 500mg / kg to 600mg / kg body weight g / kg to 550mg / kg body weight, 550mg / kg to 600mg / kg body weight, 600mg / kg to 650mg / kg body weight, 650mg / kg to 700mg / kg body weight, 700mg / kg to 750mg / kg body weight, 750mg / kg to 800mg / kg body weight, 800mg / kg to 850mg / kg body weight, 850mg / kg to 900mg / kg body weight, 900mg / kg to 950mg / kg body weight or 950mg / kg to 1g / kg of polypeptide or its virus-like particles. ,

[0518] The composition may be delivered as a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times), as determined by the attending medical professional. In some embodiments, 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 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 polypeptide or virus-like particles. In some embodiments, 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 to about 120 μg, about 130 μg to about 140 μg, about 150 μg to about 160 μg 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 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 a polypeptide or virus-like particle.

[0519] In some embodiments, the immunogenic composition provided herein is used as a booster for another RSV (or hMPV) vaccine, and the another RSV (or hMPV) vaccine is, for example, a live attenuated RSV (or hMPV) vaccine, RSV (or hMPV)-A vaccine and RSV (or hMPV)-B vaccine or a bivalent RSV (or hMPV)-A / B vaccine. In some embodiments, administration includes administering the first and second doses of the immunogenic composition, wherein the second dose is administered about 2 weeks to about 12 weeks or about 4 weeks to about 12 weeks after administering the first dose. 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 can be administered, wherein the second dose is 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 is 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 can be an RSV (or hMPV) booster dose.

[0520] In some embodiments, more than two doses of 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 subsequent doses are administered at regular intervals of about 1, 2, 3, 4, or 5 years.

[0521] 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) when administering a pharmaceutical composition provided herein, wherein the administration induces an immune response against RSV (or hMPV) (e.g., RSV (or hMPV)-A and / or RSV (or hMPV)-B) in the subject, thereby treating the RSV (or hMPV) infection (e.g., RSV (or hMPV)-A infection and / or RSV (or hMPV)-B infection) of the subject. When the method includes treating RSV (or hMPV) infection (e.g., RSV (or hMPV)-A infection and / or RSV (or hMPV)-B infection), the immunogenic composition is administered to a subject who has been infected with RSV (or hMPV) (e.g., RSV (or hMPV)-A and / or RSV (or hMPV)-B) and / or a subject who suffers from symptoms (as described above) indicating that the subject may have been infected with RSV (or hMPV) (e.g., RSV (or hMPV)-A and / or RSV (or hMPV)-B).

[0522] 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 test or antigen-based test known in the art. In some embodiments, the subject has antibodies against 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 diseases following infection of RSV (or hMPV) subtypes A and B in the elderly.

[0523] In some embodiments, the protein complexes and pharmaceutical compositions of the present disclosure provide sustained durability (persistence) of neutralizing antibodies for up to 180 days after vaccination. In some embodiments, the protein complexes and pharmaceutical compositions of the present disclosure provide sustained durability (persistence) of RSV-A and / or RSV-B neutralizing antibodies for 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.

[0524] In some embodiments, the protein complexes and pharmaceutical compositions of the present disclosure provide about 40%, about 45%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98% sustained durability at 180 days after vaccination relative to the geometric mean titer (GMT) of RSV-A and / or RSV-B neutralizing antibodies at 28 days after vaccination.

[0525] In some embodiments, the protein complexes and pharmaceutical compositions of the present disclosure provide for alum adjuvant-independent durability of RSV-A and / or RSV-B neutralizing antibodies.

[0526] In some embodiments, the protein complexes and pharmaceutical compositions of the present disclosure provide a sustained durability 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold higher than the baseline at 180 days after vaccination, relative to the geometric mean titer (GMT) of RSV-A and / or RSV-B neutralizing antibodies 28 days after vaccination. In some embodiments, the protein complexes and pharmaceutical compositions of the present disclosure provide a sustained durability 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold higher than the baseline at 180 days after vaccination, relative to the geometric mean titer (GMT) of hMPV-A and / or hMPV-B neutralizing antibodies 28 days after vaccination.

[0527] Preparation method

[0528] In one aspect, the present disclosure provides a method for preparing a vaccine, comprising culturing the host cells described herein in a culture medium so that the host cells secrete an antigen into the culture medium; optionally purifying the antigen from the culture medium; mixing the antigen with a second component, wherein the second component polymerizes with the antigen to form a virus-like particle; and optionally purifying the virus-like particle.

[0529] Kits and Unit Dosages

[0530] The present disclosure further provides a kit, which can be used to prepare the virus-like particles and compositions of the present disclosure. In a certain embodiment, the kit provided herein comprises the first component and the second component disclosed herein and the instructions for use of the method disclosed herein. In a certain embodiment, the kit comprises one or more unit doses disclosed herein and the instructions for use of the method disclosed herein. In some embodiments, the kit includes a single-dose vial containing a pharmaceutical composition provided herein. In some embodiments, the kit includes a vial containing multiple doses provided herein. In some embodiments, the kit further includes instructions for use of the pharmaceutical composition. In some embodiments, the kit further includes a diluent for preparing a diluent of the pharmaceutical composition before administration. In some embodiments, the pharmaceutical composition includes an adjuvant. In some embodiments, the kit includes a pharmaceutical composition and an adjuvant that must be mixed before administration.

[0531] Also provided herein are unit doses of the pharmaceutical compositions described herein. In some embodiments, a unit dose comprises 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 1 10 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 the VLPs or each VLP individually. In some embodiments, the 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 each VLP individually. The abbreviation "μg" can be used interchangeably with the abbreviation "mcg" to refer to micrograms of a substance.

[0532] In some embodiments, the unit dose comprises about 75 μg of the first VLP (for RSV) and about 75 μg of the second VLP (for hMPV).

[0533] In some embodiments, a unit dose comprises about 75 μg of the first VLP (for RSV) and about 100 μg of the second VLP (for hMPV).

[0534] In some embodiments, a unit dose comprises about 75 μg of the first VLP (for RSV) and about 150 μg of the second VLP (for hMPV).

[0535] In some embodiments, a unit dose comprises about 75 μg of the first VLP (for RSV) and about 200 μg of the second VLP (for hMPV).

[0536] In some embodiments, a unit dose comprises about 75 μg of the first VLP (for RSV) and about 225 μg of the second VLP (for hMPV).

[0537] In some embodiments, a unit dose comprises about 75 μg of the first VLP (for RSV) and about 250 μg of the second VLP (for hMPV).

[0538] In some embodiments, a unit dose comprises about 75 μg of the first VLP (for RSV) and about 300 μg of the second VLP (for hMPV).

[0539] In some embodiments, a unit dose comprises about 100 μg of the first VLP (for RSV) and about 75 μg of the second VLP (for hMPV).

[0540] In some embodiments, a unit dose comprises about 100 μg of a first VLP (for RSV) and about 100 μg of a second VLP (for hMPV).

[0541] In some embodiments, a unit dose comprises about 100 μg of a first VLP (for RSV) and about 150 μg of a second VLP (for hMPV).

[0542] In some embodiments, a unit dose comprises about 100 μg of a first VLP (for RSV) and about 200 μg of a second VLP (for hMPV).

[0543] In some embodiments, a unit dose comprises about 100 μg of the first VLP (for RSV) and about 225 μg of the second VLP (for hMPV).

[0544] In some embodiments, a unit dose comprises about 100 μg of a first VLP (for RSV) and about 250 μg of a second VLP (for hMPV).

[0545] In some embodiments, a unit dose comprises about 100 μg of a first VLP (for RSV) and about 300 μg of a second VLP (for hMPV).

[0546] In some embodiments, a unit dose comprises about 150 μg of the first VLP (for RSV) and about 75 μg of the second VLP (for hMPV).

[0547] In some embodiments, a unit dose comprises about 150 μg of the first VLP (for RSV) and about 100 μg of the second VLP (for hMPV).

[0548] In some embodiments, a unit dose comprises about 150 μg of the first VLP (for RSV) and about 150 μg of the second VLP (for hMPV).

[0549] In some embodiments, a unit dose comprises about 150 μg of the first VLP (for RSV) and about 200 μg of the second VLP (for hMPV).

[0550] In some embodiments, a unit dose comprises about 150 μg of the first VLP (for RSV) and about 225 μg of the second VLP (for hMPV).

[0551] In some embodiments, a unit dose comprises about 150 μg of the first VLP (for RSV) and about 250 μg of the second VLP (for hMPV).

[0552] In some embodiments, a unit dose comprises about 150 μg of the first VLP (for RSV) and about 300 μg of the second VLP (for hMPV).

[0553] In some embodiments, a unit dose comprises about 200 μg of the first VLP (for RSV) and about 75 μg of the second VLP (for hMPV).

[0554] In some embodiments, a unit dose comprises about 200 μg of a first VLP (for RSV) and about 100 μg of a second VLP (for hMPV).

[0555] In some embodiments, a unit dose comprises about 200 μg of the first VLP (for RSV) and about 150 μg of the second VLP (for hMPV).

[0556] In some embodiments, a unit dose comprises about 200 μg of the first VLP (for RSV) and about 200 μg of the second VLP (for hMPV).

[0557] In some embodiments, a unit dose comprises about 200 μg of the first VLP (for RSV) and about 225 μg of the second VLP (for hMPV).

[0558] In some embodiments, a unit dose comprises about 200 μg of the first VLP (for RSV) and about 250 μg of the second VLP (for hMPV).

[0559] In some embodiments, a unit dose comprises about 200 μg of the first VLP (for RSV) and about 300 μg of the second VLP (for hMPV).

[0560] In some embodiments, a unit dose comprises about 225 μg of the first VLP (for RSV) and about 75 μg of the second VLP (for hMPV).

[0561] In some embodiments, a unit dose comprises about 225 μg of the first VLP (for RSV) and about 100 μg of the second VLP (for hMPV).

[0562] In some embodiments, a unit dose comprises about 225 μg of the first VLP (for RSV) and about 150 μg of the second VLP (for hMPV).

[0563] In some embodiments, a unit dose comprises about 225 μg of the first VLP (for RSV) and about 200 μg of the second VLP (for hMPV).

[0564] In some embodiments, a unit dose comprises about 225 μg of the first VLP (for RSV) and about 225 μg of the second VLP (for hMPV).

[0565] In some embodiments, a unit dose comprises about 225 μg of the first VLP (for RSV) and about 250 μg of the second VLP (for hMPV).

[0566] In some embodiments, a unit dose comprises about 225 μg of the first VLP (for RSV) and about 300 μg of the second VLP (for hMPV).

[0567] In some embodiments, a unit dose comprises about 250 μg of the first VLP (for RSV) and about 75 μg of the second VLP (for hMPV).

[0568] In some embodiments, a unit dose comprises about 250 μg of the first VLP (for RSV) and about 100 μg of the second VLP (for hMPV).

[0569] In some embodiments, a unit dose comprises about 250 μg of the first VLP (for RSV) and about 150 μg of the second VLP (for hMPV).

[0570] In some embodiments, a unit dose comprises about 250 μg of the first VLP (for RSV) and about 200 μg of the second VLP (for hMPV).

[0571] In some embodiments, a unit dose comprises about 250 μg of the first VLP (for RSV) and about 225 μg of the second VLP (for hMPV).

[0572] In some embodiments, a unit dose comprises about 250 μg of the first VLP (for RSV) and about 250 μg of the second VLP (for hMPV).

[0573] In some embodiments, a unit dose comprises about 250 μg of the first VLP (for RSV) and about 300 μg of the second VLP (for hMPV).

[0574] In some embodiments, a unit dose comprises about 300 μg of the first VLP (for RSV) and about 75 μg of the second VLP (for hMPV).

[0575] In some embodiments, a unit dose comprises about 300 μg of the first VLP (for RSV) and about 100 μg of the second VLP (for hMPV).

[0576] In some embodiments, a unit dose comprises about 300 μg of the first VLP (for RSV) and about 150 μg of the second VLP (for hMPV).

[0577] In some embodiments, a unit dose comprises about 300 μg of the first VLP (for RSV) and about 200 μg of the second VLP (for hMPV).

[0578] In some embodiments, a unit dose comprises about 300 μg of the first VLP (for RSV) and about 225 μg of the second VLP (for hMPV).

[0579] In some embodiments, a unit dose comprises about 300 μg of the first VLP (for RSV) and about 250 μg of the second VLP (for hMPV).

[0580] In some embodiments, a unit dose comprises about 300 μg of the first VLP (for RSV) and about 300 μg of the second VLP (for hMPV).

[0581] In some embodiments, the unit dose comprises between about 5 μg and about 100 μg of the first VLP, between about 10 μg and about 90 μg of the first VLP, between about 20 μg and about 80 μg of the first VLP, between about 30 μg and about 70 μg of the first VLP, between about 40 μg and about 60 μg of the first VLP, or between about 50 μg and about 80 μg of the first VLP (for RSV).

[0582] In some embodiments, the unit dose comprises between about 5 μg and about 100 μg of the first VLP, between about 20 μg and about 80 μg of the first VLP, between about 30 μg and about 70 μg of the first VLP, between about 40 μg and about 60 μg of the first VLP, and / or between about 50 μg and about 80 μg of the second VLP (for hMPV).

[0583] In some embodiments, the unit dose comprises between about 50 μg and about 180 μg of the first VLP, between about 60 μg and about 170 μg of the first VLP, between about 80 μg and about 150 μg of the first VLP, between about 70 μg and about 155 μg of the first VLP, between about 90 μg and about 160 μg of the first VLP, and / or between about 100 μg and about 170 μg of the second VLP (for hMPV).

[0584] In some embodiments, the unit dose comprises between about 130 μg and about 300 μg of the first VLP, between about 150 μg and about 280 μg of the first VLP, between about 170 μg and about 260 μg of the first VLP, between about 190 μg and about 240 μg of the first VLP, between about 200 μg and about 230 μg of the first VLP, and / or between about 210 μg and about 225 μg of the second VLP (for hMPV).

[0585] In some embodiments, the unit dose comprises between about 130 μg and about 300 μg of the first VLP, between about 150 μg and about 280 μg of the first VLP, between about 170 μg and about 260 μg of the first VLP, between about 190 μg and about 240 μg of the first VLP, between about 200 μg and about 230 μg of the first VLP, and between about 130 μg and about 300 μg of the second VLP, between about 150 μg and about 280 μg of the second VLP, between about 170 μg and about 260 μg of the second VLP, between about 190 μg and about 240 μg of the second VLP, between about 200 μg and about 230 μg of the second VLP.

[0586] 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 the first VLP (for RSV) and 150 μg of the second VLP (for hMPV).

[0587] 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).

[0588] 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).

[0589] In some embodiments, a 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) 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).

[0590] 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).

[0591] 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, about 300 μg of the second VLP (for hMPV).

[0592] In some embodiments, a 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, about 300 μg of the first VLP ( 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, about 300 μg of a second VLP (for hMPV).

[0593] Example

[0594] Example 1

[0595] This example illustrates the production of a two-component, icosahedral VLP intended for use as a vaccine against hMPV virus. As the first component A, the vaccine uses a polypeptide antigen consisting of the extracellular domain of the hMPV F protein fused at the C-terminus to the I53-50A protein (SEQ ID NO: 144). As component B, the VLP uses the I53-50B protein. It is known that I53-50A and I53-50B spontaneously assemble in a ratio of 3:5 to form VLPs with icosahedral (I53) symmetry, such as Figure 1A and 1B shown.

[0596] The expression levels of hMPV F protein fused to CompA were determined. Human codon-optimized polynucleotide sequences were prepared by gene synthesis and cloned into expression vectors. Each expression vector was expressed individually in Expi293 cells by transient transfection. Supernatants were collected four days after transfection.

[0597] To determine whether the recombinantly expressed fusion protein is secreted into the supernatant, Western blotting was performed using an anti-His6 monoclonal antibody. The conditioned medium was diluted at least 10 times in 2X Laemmli loading buffer (Bio-Rad) containing 5% (v / v) β-mercaptoethanol, heated to 95°C for 10 minutes, and 10 μl was run on the gel. The reference sample of recombinant His-tagged human serum albumin (HSA) was prepared by initial dilution to 0.01 mg / mL, followed by a continuous 2-fold dilution to obtain a standard curve from 6.25 to 100 ng in a 10 μL loading volume in 2X Laemmli loading buffer. The sample was loaded onto a NuPAGE 4-12% Bis-Tris protein gel and run at 150V for 45 minutes. The protein was transferred to an Immuno-Blot PDVF membrane (Bio-Rad). After transfer, the blot was blocked with 3% BSA (w / v) in TBST at room temperature for 1 hour. 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 hour at room temperature. The membrane was then washed three times with TBST for five minutes, and His-tagged proteins were detected using luminol chemiluminescent substrate (VisiGlo, VWR) and captured on a UVP Chemstudio (Analytik Jena) using an exposure time of 10-20 seconds.

[0598] Table 4 shows the expression levels of hMPV F proteins 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.

[0599] Table 4. Expression of hMPV F-CompA fusion proteins tested in Expi293 cells.

[0600]

[0601]

[0602]

[0603]

[0604] Example 2

[0605] In this example, antibody binding activity was used to characterize conditioned medium containing hMPV F-CompA fusion protein to determine the conformation of 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. After co-transfection with human furin, the expressed protein was correctly cleaved and was referred to as hMPV026C or hMPV027C to represent the cleavage form.

[0606] To characterize the conformation of the recombinantly expressed hMPV F protein fused to CompA, an ELISA was performed using 15 monoclonal antibodies that bind to specific forms of the extracellular domain of the hMPV F protein.

[0607] Each well of a 96-well plate was coated overnight at 4°C with 200 ng of each antibody or 50 ng of MPE8 in 100 μL of 50 mM sodium carbonate-bicarbonate buffer (pH 9.6). The 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 plates were emptied by tapping on an absorbent pad. The conditioned medium supernatant from the Expi293 transfection of each construct was diluted based on semi-quantitative Western blotting so that the antigen concentration would cover the expected EC of the antigen. 50 (From Mas data). For each antibody, three dilutions of each construct were plated at 100 μL per well and the plates were incubated at room temperature for 1 hour. The plates were washed 6 times with 300 μL of PBS containing 0.05% Tween 20. Anti-His monoclonal antibody (R&D Systems MAB050H) was diluted 1:15,000 and 100 μL was added to each well, and the plates were incubated at room temperature for 1 hour. After the plates were washed 6 times with 300 μL of PBS containing 0.05% Tween 20, 100 μL of TMB substrate was added and the plates were developed in the dark for 10 minutes. The reaction was terminated by adding 100 μL of 0.6NH2SO4 and the absorbance at 450 nm was measured on a plate reader.

[0608] The post-fusion specific antibody binds to hMPV021, indicating that the construct has a post-fusion conformation. Other constructs do not bind to the post-fusion specific Ab, but bind to MPE8 and multiple Abs that recognize the pre-fusion and post-fusion conformations. This data indicates that the fusion protein has pre-fusion conformational characteristics.

[0609] Example 3

[0610] Binding activity observed for purified hMPV F-CompA fusion protein. Figures 3A-3C Binding to a known concentration of purified component A (CompA) fusion (i.e., an hMPV F protein extracellular domain fused to I53-50A (SEQ ID NO: 144) or I53-50AΔCys (SEQ ID NO: 145) at the N-terminus) is shown. MF14, MPE8, MF16 recognize antigenic sites II, III, and IV, respectively, which are the main sites recognized by neutralizing antibodies. In addition, MPE8 preferentially recognizes the pre-fusion form of the hMPV F protein. Each well of a 96-well plate was coated overnight with 50ng MPE8 and 100ng MF14 and MF16 in 100μL 50mM sodium carbonate-sodium bicarbonate buffer (pH 9.6) at 4°C. The plate was washed 3 times with 300μl PBS containing 0.05% Tween 20. After blocking with 150μl PBS containing 1% BSA for 1 hour at room temperature, the plate was tapped on an absorbent pad to empty it. 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-fold or 4-fold dilutions in PBS containing 1% BSA, respectively, and plated at 100 μL per well, and the plates were incubated at room temperature for 1 hour. The plates were washed 6 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 at room temperature for 1 hour. After washing the plates 6 times with 300 μl of PBS containing 0.05% Tween 20, 100 μl of TMB substrate was added, and the plates were developed in the dark for 10 minutes. The reaction was stopped by adding 100 μl 0.6 NH2SO4 and the absorbance was measured at 450 nm on a plate reader. Figure 3A ) and MF16 (site IV, Figure 3B ) binding is similar, but MPE8 (site III, Figure 3C ) combined with hMPV026, hMPV027 and hMPV033 to obtain EC 50 Higher than binding to hMPV008 and hMPV021 (Table 6). This suggests that site III (neutralizing antibody recognition site) is more conserved in hMPV026, hMPV027, and hMPV033, and that these constructs prefer to adopt a prefusion conformation over hMPV008 and hMPV021.

[0611] Table 6. Neutralizing antibody binding EC50 value

[0612]

[0613] Example 4

[0614] 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 samples were collected on days 0, 24, and 35 and processed into serum. Serum samples on day 0 were pooled by group. All serum samples were evaluated by hMPV neutralizing antibody titer assay ( Fig. 9 hMPV008 formulations containing either adjuvants, Alhydrogel (alum) or Addavax (squalene oil-in-water emulsion), induced measurable neutralization titers at all doses on days 24 and 35 that were comparable to healthy adult sera titers ( Fig. 9 ). These results indicate that hMPV008 VLPs induce strong neutralization titers comparable to those of healthy human control sera.

[0615] Table 7. Amount and formulation of VLPs injected

[0616] antigen dose formula hMPV008 VLP 5μg Addavax hMPV008 VLP 5μg Alhydrogel hMPV008 VLP 0.25 μg Alhydrogel hMPV008 CompA 4.08 μg Alhydrogel hMPV008 CompA 0.2 μg Alhydrogel

[0617]

[0618] Example 5

[0619] A study was conducted in treatment-naive BALB / c mice to explore the immunogenicity of hMPV F protein mutants displayed on two-component virus-like particles (VLPs). VLPs containing one of four different hMPV F protein mutants (hMPV008, hMPV024, hMPV026C, and hMPV033) or a 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 the oil-in-water adjuvant Addavax. Two dose levels (1 μg and 0.25 μg) or soluble proteins of equivalent antigenic content were administered (Table 8). Neutralizing antibody titers against hMPV-A and hMPV-B were determined from day 35 sera (1 μg and 0.25 μg, respectively). Figure 4A and Figure 4B The results showed that all VLPs could induce strong neutralization titers against both hMPV strains. The titers induced by soluble proteins against both hMPV strains were also lower than those induced by VLPs.

[0620] Table 8: Amount and formulation of VLPs injected

[0621] antigen dose formula hMPV033 VLP 1 μg Addavax hMPV008 VLP 1 μg Addavax hMPV024 VLP 1 μg Addavax hMPV026C VLP 1 μg Addavax hMPV033 VLP 0.25 μg Addavax hMPV008 VLP 0.25 μg Addavax hMPV024 VLP 0.25 μg Addavax hMPV026C VLP 0.25 μg Addavax Soluble hMPV026C 0.64 μg Addavax Soluble hMPV026C 0.16 μg Addavax No treatment N / A N / A

[0622] Example 6

[0623] A study was conducted in naive mice to explore the immunogenicity of hMPV008 VLPs and the corresponding soluble protein CompA-hMPV008. hMPV008 VLPs and CompA-hMPV008 were formulated in aqueous buffer, squalene emulsion (SE), or Alhydrogel at two dose levels (1 μg and 0.1 μg VLPs and equivalent antigenic doses of CompA-hMPV008). In addition, a group in which hMPV033 VLPs (0.1 μg) were formulated with SE was included (Table 9). Each group consisted of 8 female BALB / c mice. Mice were immunized on days 0 and 21. Neutralizing antibody titers against hMPV-A and hMPV-B were determined from day 35 sera. The results showed that hMPV008 VLPs formulated with SE had higher titers than Alhydrogel or aqueous formulations ( Figure 5A and 5B The neutralization titers induced by VLPs of all preparations were higher than those of the soluble protein CompA-hMPV008 ( Figure 5A and Figure 5B ).

[0624] Table 9: Amount and formulation of VLPs injected

[0625] antigen dose formula hMPV008VLP 1 μg SE hMPV008VLP 1 μg Alhydrogel hMPV08VLP 1 μg Aqueous hMPV008VLP 0.1 μg SE hMPV008VLP 0.1 μg Alhydrogel hMPV008VLP 0.1 μg Aqueous CompA-hMPV008 0.81 μg SE CompA-hMPV008 0.81 μg Alhydrogel CompA-hMPV008 0.81 μg Aqueous CompA-hMPV008 0.081 μg SE CompA-hMPV008 0.081 μg Alhydrogel CompA-hMPV008 0.081 μg Water-based hMPV033 0.1 μg SE

[0626] Example 7: Bivalent non-interfering immunogenicity study using hMPV F protein (IVX-241) and RSV F protein (IVX-121) VLPs

[0627] A study was conducted in BALB / c mice that had not received treatment to explore the immunogenicity of VLPs displaying hMPV F protein mutants IVX-241 and RSV F protein IVX-121 when administered in monovalent or bivalent formulations. The purpose of this study was to evaluate the different ratios of IVX-241 to IVX-121 and determine the neutralization titers against RSV-A, RSV-B, hMPV-A and hMPV-B. Mice were immunized with IVX-121, IVX-241 or IVX-121+IVX-241 on days 0 and 21, with dose levels and formulations as shown in Table 10. The dose level of IVX-121 was 1 μg, while the dose level of IVX-241 was 4, 2, 1 and 0.5 μg. The study included displaying VLPs of two components of RSV and hMPV F antigen mutants present on IVX-121 and IVX-241 (chimeric). The chimeric particles were administered at a dose level of 2 μg.

[0628] Blood samples were collected on days 0, 21 and 35 and processed into serum, and samples on days 0 and 35 were tested in RSV-A, RSV-B, hMPV-A and hMPV-B specific neutralization assays. Neutralizing antibody titer data were plotted, showing geometric mean and geometric SD. Group statistical analysis was performed using the nonparametric Mann-Whitney test (GraphPad Prism).

[0629] All animals remained alert and responsive throughout the study, with no test article or adjuvant-related clinical observations.

[0630] Sera from animals immunized with IVX-121 demonstrated a significant increase in RSV-A and RSV-B neutralizing antibodies compared to pre-immune sera (titers were less than 2 log2 on day 0 in all groups) ( Figure 6 The mean RSV-B titer was approximately 2.5 log2 lower than the RSV-A titer. Animals immunized with IVX-241 showed little or no neutralization titer against either RSV-A or RSV-B ( Figure 6 RSV-A neutralization titers were slightly lower in animals administered IVX-121 and IVX-241 than in animals dosed with IVX-121 alone, and statistical significance was only seen between the bivalent group administered 4 μg IVX-241 and chimeric VLPs ( Figure 6). IVX-121 / IVX-241 chimeric VLPs resulted in RSV-A and RSV-B titers that trended lower than IVX-121 alone, but were not statistically significant. Convalescent human serum samples run in the RSV-A neutralization titer assay showed titers below the mean of IVX-121 or IVX-121+IVX-241 immunized animals. However, RSV-B titers for single sera run in duplicate were higher than the IVX-121 group.

[0631] Sera from animals immunized with IVX-241 demonstrated a significant increase in hMPV-A and hMPV-B neutralizing antibodies compared to pre-immune sera (titers on day 0 were less than 2 log2 in all groups) ( Figure 7 Responses to all four doses of IVX-241 produced similar titers. Animals immunized with IVX-121 demonstrated hMPV-A and hMPV-B neutralization titers similar to those of preimmune sera ( Figure 7 Animals administered IVX-121 and IVX-241 had hMPV-A and hMPV-B neutralization titers that were approximately 1-2 log2 lower than those in animals that received IVX-241 alone ( Figure 7 IVX-121 / IVX-241 chimeric VLPs resulted in hMPV-A and hMPV-B titers that trended higher than IVX-241 alone (1 μg), but were not statistically significant. Convalescent human serum samples run in the hMPV-A and hMPV-B neutralization titer assays exhibited titers equal to or lower than the mean of IVX-241 or IVX121+IVX-241 immunized animals.

[0632] Table 10

[0633] antigen dose formula IVX-121 1ug Addavax IVX-241 4ug Addavax IVX-241 2ug Addavax IVX-241 1ug Addavax IVX-241 0.5ug Addavax IVX-121+IVX-241 1+4ug Addavax IVX-121+IVX-241 1+2ug Addavax IVX-121+IVX-241 1+1ug Addavax IVX-121+IVX-241 1+0.5ug Addavax IVX-121 / IVX-241 chimera 2ug Addavax

[0634] Example 8: Protective Immunity Generated by Monovalent and Bivalent hMPV / RSV Combination Vaccines

[0635] A study was conducted to evaluate and assess the ability of IVX-121, IVX-241, and IVX-121+IVX-241 vaccine candidates to prevent infection with hMPV and RSV in cotton rats. The vaccine candidates were formulated with Addavax, an oil-in-water emulsion.

[0636] Sixty-four female cotton rats were included in the study and divided into 8 groups of 8. The immunogens, dose levels, formulation details and challenge viruses are shown in Table 11.

[0637] Three control groups were administered saline, and these groups were not attacked or attacked by RSV-A or hMPV-A2 subsequently. At the 0th and 21st days, IVX-121 or IVX-241 candidate vaccines were administered intramuscularly to each group at a 1.0 μg dosage level prepared in Addavax, and then RSV-A or hMPV-A2 was used to attack respectively at the 35th day. At the 0th and 21st days, IVX-121+IVX-241 was administered intramuscularly to two groups at a dosage level of each 1.0 μg VLP prepared in Addavax, and then RSV or hMPV was used to attack respectively at the 35th day. One group was vaccinated with the vaccine of formalin inactivated (FI) hMPV, and then attacked with hMPV-A2 at the 35th day. All animals were killed at the 40th day. RSV-A and hMPV-A2 virus titer assessment was performed using lung and nasal homogenate. Serum samples were collected on days 0, 21, 35, and 40 to measure RSV-A and hMPV-A neutralizing antibody titers using a virus neutralization assay. Clinical observations were performed daily and animals were weighed weekly.

[0638] Animals vaccinated with IVX-121, IVX-241, or IVX-121+IVX-241 had significantly increased neutralization titers against RSV and hMPV at day 35 compared to preimmune sera. Titers observed with IVX-121 or IVX-241 were equivalent to those observed with the IVX-121+IVX241 formulation. Figures 8A-8B ).

[0639] 10 with RSV-A2 or hMPV-A 5 Animals were challenged intranasally with 100 plaque forming units (PFU) and lung tissue samples were tested for viral replication 5 days after challenge. Control animals not challenged with RSV-A or hMPV showed lung titers below the limit of detection (2.2 log2). Unvaccinated cotton rats challenged with RSV or hMPV produced large viral titers in the lungs. Monovalent or bivalent formulations blocked viral replication of RSV and hMPV in the lungs to below the lower limit of quantitation (2.2 log2). Figure 8C-8D ).

[0640] Control animals not challenged with RSV-A showed nasal titers below the limit of detection (2.0 log2), whereas unvaccinated animals challenged with RSV-A had substantial viral titers in the nose ( Fig. 8E Mice immunized with IVX-121 or IVX-A12 and challenged with RSV-A showed significantly reduced rhinovirus titers, demonstrating that viral replication in the upper respiratory tract was suppressed ( Fig. 8E ).

[0641] Control animals not challenged with hMPV-A2 showed nasal titers below the limit of detection (2.0 log2), whereas unvaccinated animals challenged with hMPV-A2 had substantial viral titers in the nose ( Fig.8F Mice immunized with IVX-241 or IVX-A12 and challenged with hMPV-A showed rhinovirus titers at levels observed in the control and unchallenged groups (2.0 log2), indicating complete inhibition of viral replication ( Fig.8F ). The FI hMPV vaccinated group had slightly reduced rhinovirus titers compared to the challenged control group ( Fig.8F ).

[0642] Table 11

[0643]

[0644] Example 9: Overview of clinical evaluation of bivalent RSV-hMPV vaccine

[0645] These embodiments describe experiments for demonstrating the safety and efficacy of a RSV / hMPV combination VLP candidate (IVX-A12). The vaccine is a mixture of two monovalent protein-based virus-like particles (VLPs). Both VLPs are two-component icosahedral VLPs that display the extracellular domain of the RSV F protein or hMPV F protein, respectively. As the first component A, the vaccine uses a polypeptide antigen consisting of an extracellular domain of the RSV or hMPV F protein fused to the I53-50A protein (SEQ ID NO: 144) at the C-terminus. As component B, VLP uses the I53-50B protein. It is known that I53-50A and I53-50B spontaneously assemble to form VLPs with icosahedral (I53) symmetry at a ratio of 3:5, such as Figure 1A and Figure 1B As shown in . The RSV and hMPV components were each engineered to stabilize the prefusion conformation of the corresponding F protein.

[0646] The immunogenicity of the bivalent vaccine candidate is being evaluated in a Phase 1 first-in-human (FIH) trial in healthy young and elderly adults by measuring changes in RSV and hMPV nAb levels relative to baseline antibody levels. Different combinations of RSV and hMPV are being evaluated to determine whether the addition of hMPV VLPs to the RSV VLP vaccine candidate will cause potential immune interference. Based on favorable safety results, demonstration of immunogenicity, and determination of the optimal RSV-hMPV dose combination, the efficacy of the RSV-hMPV combination vaccine candidate will be evaluated. The efficacy will be evaluated by measuring the incidence of lower respiratory tract infections (LRIs) caused by RSV or hMPV in patients treated with IVX-A12 versus those treated with placebo. Different formulations of IVX-A12, such as those with or without adjuvants, can be evaluated.

[0647] IVX-A12 Phase 1 trial

[0648] A Phase 1 trial of IVX-A12 will be conducted. The goal of the Phase 1 trial of IVX-A12 will be to evaluate the safety and immunogenicity of different doses of IVX-A12 with and without adjuvants in elderly people aged 60-75 years. IVX-A12 will be administered with a fixed dose of IVX-121 and one of three dose levels of IVX-241VLPs, which are formulated with and without adjuvants. This design will be able to evaluate the immune response to the two separate components of IVX-A12 and see if the combination of VLPs increases reactogenicity or leads to immune interference (i.e., an imbalance in the immune response to the component VLPs). All subjects in the Phase 1 trial will be evaluated for safety and antibody responses within twelve months after administration of IVX-A12 or placebo. The plan is to evaluate the interim data of the Phase 1 trial to determine whether adjuvant therapy is needed and to select a dose regimen to be evaluated in a Phase 2 dose confirmation trial.

[0649] IVX-A12 Phase 2 Dose Confirmation Trial

[0650] After the completion of the IVX-A12 Phase 1 clinical trial, a Phase 2 dose confirmation clinical trial will be initiated in healthy elderly people aged 60-75 years. The formulation and dose regimen for evaluation will be selected in the Phase 2 clinical trial based on the data from the IVX-A12 Phase 1 trial. The planned Phase 2 clinical trial will evaluate different combinations of hMPV and RSV VLPs at different concentrations in healthy elderly people to evaluate the safety and immunogenicity of different concentrations of hMPV and RSV VLPs and guide the final dose selection for the subsequent PoC Phase 2b trial.

[0651] IVX-A12 Phase 2 Extension Trial

[0652] Elderly subjects who completed the Phase 2 trial will be recruited into a Phase 2 extension trial to evaluate the duration of antibody persistence and long-term safety over many years.

[0653] IVX-A12 Phase 2b Proof of Concept (PoC) Trial

[0654] A global, Phase 2b, randomized, observer-blind, placebo-controlled, PoC efficacy trial will be conducted to evaluate the IVX-A12 formulation selected from the Phase 2 dose confirmation trial. Planned PoC objectives for the Phase 2b trial will include evaluation of safety, immunogenicity, and efficacy against LRI caused by RSV or hMPV. The trial population will include adults aged 60 years or older, including nested cohorts of frail and high-risk elderly, and healthy subjects aged 85 years or older.

[0655] Example 10: A Phase 1 Randomized, Observer-Blind, Placebo-Controlled, Multicenter Trial to Evaluate the Safety and Immunogenicity of IVX-A12 (a Bivalent Combination Virus-Like Particle Protein Subunit Vaccine of Respiratory Syncytial Virus and Human Metapneumovirus) in Healthy Adults

[0656] The vaccine candidate IVX-A12 is a bivalent combination formulation containing IVX-121 and IVX-241 virus-like particles (VLPs), which are in silico designed recombinant protein subunit vaccines against RSV and hMPV, respectively. Each VLP consists of two recombinant proteins, component A (CompA) and component B (CompB-01), which are designed to assemble cooperatively to form an icosahedral structure. Component A is a fusion protein specific for each vaccine candidate (CompA-RSV-02 or CompA-hMPV-01 for RSV and hMPV, respectively) and is expressed together with the prefusion F protein of the corresponding virus, which has been shown to induce strong neutralizing antibody responses in nonclinical models. CompB-01 is a common component of both VLPs and provides structural elements that support the multimeric display of CompA-RSV-02 or CompA-hMPV-01. When combined together, the two components self-assemble into VLPs that exhibit enhanced immunogenicity compared to soluble DS-Cav1 or hMPV prefusion antigen trimers. Soluble DS-Cav1 RSV prefusion F protein was recently tested in the clinic and demonstrated tolerability and durable immunogenicity ( Ruckwardt et al., 2021 ).

[0657] IVX-121 and IVX-241 VLPs display 20 copies of each prefusion F protein trimer on their surface. Candidate IVX-121 and IVX-241 VLPs will be manufactured independently and combined to produce a bivalent formulation containing both VLPs, referred to as IVX-A12. The proposed mechanism of action of IVX-A12 is to increase the proportion of RSV- 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 a target population of elderly individuals who are at highest risk of disease following infection with RSV and hMPV.

[0658] The vaccine candidate IVX-A12 is expected to be a single-dose liquid formulation (0.5 mL) for intramuscular (IM) use in the target population of adults aged 60 to 75 years. The IVX-A12 vaccine candidate will be formulated and bottled as an aqueous vaccine for 1:1 (V / V) dilution with an oil-in-water emulsion as an adjuvant or diluent.

[0659] The proposed Phase 1 clinical trial is a first-in-human (FIH) dose-ranging evaluation of the IVX-A12 vaccine candidate in healthy elderly adults aged 60 to 75 years. The purpose of this Phase 1 trial is to evaluate the clinical safety and immunogenicity of a bivalent IVX-A12 vaccine candidate composed of a constant dose (amount) level of IVX-121RSV VLPs in combination with varying concentrations (defined as low, medium, and high dose levels) of IVX-241hMPV VLPs (with and without ). As there are no licensed RSV or hMPV vaccines, a placebo (diluent) will be used. The three different antigen amounts selected will be evaluated in three different cohorts in a stepwise manner.

[0660] - Low dose level: 75 μg RSV / 75 μg hMPV,

[0661] -Medium dose level: 75 μg RSV / 150 μg hMPV, and

[0662] -High dose level: 75 μg RSV / 225 μg hMPV.

[0663] Cohort 1 will evaluate the safety and immunogenicity of one low-dose level (75 μg RSV / 75 μg hMPV) of the unadjuvanted IVX-A12 vaccine candidate (IVX-A12a) administered intramuscularly compared to placebo in healthy adults aged 60 to 75 years.

[0664] Cohort 2 will evaluate the efficacy of a single intramuscular dose of (i) a low dose level (75 μg RSV / 75 μg hMPV) compared to placebo. Safety and immunogenicity of (i) an adjuvanted IVX-A12 vaccine candidate (IVX-A12d) and (ii) an intermediate dose level (75 μg RSV / 150 μg hMPV) of the non-adjuvanted IVX-A12 vaccine candidate (IVX-A12a) in healthy adults aged 60 to 75 years.

[0665] Cohort 3 will evaluate the efficacy of one dose of the (i) intermediate dose level (75 μg RSV / 150 μg hMPV) administered intramuscularly compared to placebo. The safety and immunogenicity of (i) the IVX-A12 vaccine candidate with adjuvant (IVX-A12d) and (ii) the high-dose level (75 μg RSV / 225 μg hMPV) of the unadjuvanted IVX-A12 vaccine candidate (IVX-A12a) in healthy adults aged 60 to 75 years will not be evaluated in this clinical trial. IVX-A12 vaccine candidate.

[0666] The trial will be conducted in accordance with the protocol, the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), Good Clinical Practice (GCP) guidelines, and applicable regulatory requirements.

[0667] Main objectives:

[0668] To evaluate the safety and immunogenicity of three dose levels (low, medium, and high) of a bivalent combination RSV / hMPV VLP vaccine candidate (IVX-A12) compared to placebo when administered as a single dose regimen in healthy older adults aged 60 to 75 years by evaluating:

[0669] - Non-spontaneous local reactions and systemic AEs within 7 consecutive days after dosing (Day 0 to Day 6), and spontaneous adverse reactions up to 28 days after dosing (Day 28);

[0670] - On day 28, RSV and hMPV neutralizing antibody (NAb) titers were measured by live virus assay, and RSV-specific and hMPV-specific prefusion F protein IgG antibody titers were measured by enzyme-linked immunosorbent assay (ELISA);

[0671] - Geometric mean fold increase (GMFR) in RSV-A-specific and RSV-B-specific NAb titers, hMPV-A-specific and hMPV-B-specific NAb titers, and RSV and hMPV IgG prefusion F protein-specific antibody titers on day 28 compared to day 0.

[0672] Secondary Objectives:

[0673] The safety of IVX-A12 compared to placebo was further evaluated by the incidence of:

[0674] - Serious adverse events (SAEs), adverse events requiring physician attention (MAAEs), and AEs leading to withdrawal from the trial that occurred throughout the trial (up to Day 365);

[0675] - AEs of special interest (AESI) up to Day 365;

[0676] - Moderate to severe LRTI (clinical events of special interest, CESI) through Day 365;

[0677] - Clinical safety laboratory parameters for up to 7 days following Day 0 dosing.

[0678] - Further evaluation of the immunogenicity of IVX-A12 compared to placebo through Day 365 by assessing:

[0679] - RSV and hMPV NAb titers measured by live virus assay;

[0680] - RSV and hMPV prefusion F protein-specific IgG antibody titers measured by ELISA;

[0681] -Ratio of fold increase in IgG titer to fold increase in NAb titer for RSV and hMPV;

[0682] - GMFR compared 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 prefusion F protein-specific antibody titers;

[0683] - GMFR of hMPV-A-specific and hMPV-B-specific NAb titers compared to baseline (day 0).

[0684] Exploration objectives:

[0685] The immunogenicity of the IVX-A12 vaccine compared to placebo was further explored by evaluating:

[0686] IgG epitope specificity for RSV and hMPV prefusion F proteins;

[0687] VLP core-specific IgG titers.

[0688] Clinical trial design:

[0689] The IVX-A12 Phase 1 trial is a randomized, observer-blind, placebo-controlled, multicenter, dose-escalation trial designed to evaluate the safety and immunogenicity of a single dose of two IVX-A12 formulations: an aqueous form (without adjuvant) and an adjuvant-added form (without adjuvant). The trial will have a total of 8 treatment groups (5 active treatment groups and 3 placebo groups). The active treatment group receiving the IVX-A12 formulation will be tested at 3 dose levels of IVX-A12 (low, medium, and high total VLP content). Low and medium dose levels were tested in the absence of The clinical trial will be conducted in three cohorts. The three different dose levels selected (low dose level: 75μg RSV / 75μg hMPV, medium dose level: 75μg RSV / 150μg hMPV, and high dose level: 75μg RSV / 225μg hMPV) will be evaluated in a stepwise manner in three different cohorts. In addition, a placebo will be administered as a control. Each cohort will have a placebo treatment group. Table 12 lists the three cohorts.

[0690] Table 12

[0691]

[0692] Total number of placebo recipients N = 20. Total VLP content was 150 μg (Groups A and C), 225 μg (Groups D and F), and 300 μg (Group G).

[0693] Contains 9.75mg squalene and surfactants. Placebo will be aqueous diluent. For data analysis, treatment groups B, E, and H will be combined into a single placebo group.

[0694] Test procedure:

[0695] Approximately 120 healthy adult subjects (60-75 years) will be recruited to one of three cohorts in a stepwise dose escalation (from lowest to highest). Each cohort will enroll adults aged 60 to 75 years. In each cohort, subjects will be randomized in a 5:1 ratio to receive either study product (IMP; IVX-A12a or IVX-A12d) or placebo (diluent). Cohort 1 will have approximately 24 subjects, 20 in a single active group and 4 in a placebo group. In Cohort 2, there will be approximately 48 subjects: 20 in each of the two active treatment groups and 8 in the corresponding placebo group. In Cohort 3, there will also be approximately 48 subjects. 20 in each of the two active treatment groups and 8 in the corresponding placebo group. All subjects will receive one dose of 0.5 mL IVX-A12 or placebo injected intramuscularly.

[0696] Within each cohort, vaccination will be phased so that sentinels are vaccinated first. Table 13 provides the phased approach for each cohort.

[0697] In Cohort 1, two sentinel subjects will receive IVX-A12a and one sentinel subject will receive placebo (from the placebo group within the corresponding cohort) for an observation period of at least 36 hours. If no stopping rules are triggered, the remaining 21 subjects (18 from the active group (Arm A) and 3 from the corresponding placebo group (Cohort 1)) will be enrolled.

[0698] In Cohorts 2 and 3, four sentinel subjects will receive IVX-A12 (two subjects from the IVX-A12d group [the same dose level tested in the absence of adjuvant in the previous cohort] and two subjects from the IVX-A12a group [the next unadjuvanted dose level]) and two sentinel subjects will receive placebo (placebo group within the corresponding cohort) with an observation period of at least 36 hours. If no stopping rules are triggered, the remaining 42 subjects (18 in the active IVX-A12a group, 18 in the active IVX-A12d group, and 6 in the placebo group per dose level cohort) will be vaccinated. If achieved, an independent SMC will be responsible for applying the stopping rules. If no stopping rules are triggered within 7 days of dosing for all subjects in each dose level cohort, and the IVX-A12a and IVX-A12d formulations are found to have acceptable reactogenicity as determined by the SMC, the trial will continue to subsequent dose level cohorts. The SMC will review all available cumulative safety data up to day 7 post-vaccination before proceeding and initiating subsequent dose level cohorts.

[0699] Cohort 1: To evaluate the safety and immunogenicity of the IVX-A12 unadjuvanted vaccine candidate (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 a low dose of IVX-A12a (Group A) or placebo (Group B). Two sentinel subjects (Phase 1) will receive IVX-A12a (Group A) and one sentinel subject will receive placebo (Group B). After the observation period, the remaining subjects in Group A (N=18) and Group B (N=3) will be vaccinated (Phase 2). After the completion of Cohort 1 recruitment, the SMC will review the safety data before continuing with recruitment of Cohort 2.

[0700] Cohort 2: Evaluation of the low dose level (75 μg RSV / 75 μg hMPV) compared with placebo The safety and immunogenicity of the IVX-A12 vaccine candidate at the low dose level and the unadjuvanted IVX-A12 vaccine candidate at the intermediate dose level (75μg RSV / 150μg hMPV) will be evaluated. Enrolled subjects (N=48) will be randomized in a 5:1 ratio to receive IVX-A12d (low dose level) or IVX-A12a at the intermediate dose level (Group C or D, respectively) or placebo (Group E). Four sentinel subjects (Phase 3) will receive IVX-A12 (two subjects from Group C [low dose IVX-A12d] and two subjects from Group D [intermediate dose IVX-A12a]), and two sentinel subjects will receive placebo (Group E). After the observation period, the remaining subjects in Groups C, D, and E will be vaccinated (Phase 4). After the completion of Cohort 2 enrollment, the SMC will review the safety data before continuing with enrollment in Cohort 3.

[0701] Cohort 3: Evaluation of the intermediate dose level (75 μg RSV / 150 μg hMPV) compared with placebo ) and high dose levels (75μg RSV / 225μg hMPV) of the unadjuvanted IVX-A12 vaccine candidate (IVX-A12a). Enrolled subjects (N=48) will be randomized in a 5:1 ratio to receive IVX-A12d (medium dose) or high dose levels of IVX-A12a (Group F or Group G, respectively) or placebo (Group H). Four sentinel subjects (Phase 5) will receive IVX-A12 (two subjects from Group F [medium dose IVX-A12d] and two subjects from Group G [high dose IVX-A12a]), and two sentinel subjects will receive placebo (Group H). After the observation period, the remaining subjects in Groups F, G, and H will be vaccinated (Phase 6). The SMC will review the safety data after recruitment of Cohort 3 accordingly, and no further dose increases will be evaluated beyond Cohort 3. In addition, the high dose level (75 μg RSV / 225 μg hMPV) will not be evaluated in this clinical trial. IVX-A12 vaccine candidate.

[0702] Table 13

[0703]

[0704]

[0705] Contains 9.75mg squalene and surfactants. Placebo will be aqueous diluent. Group B corresponds to Periods 1 and 2, Group E corresponds to Periods 3 and 4; and Group H corresponds to Periods 5 and 6. For data analysis, treatment groups B, E, and H will be combined into a single placebo treatment group.

[0706] Evaluate:

[0707] Each subject will have a total of 7 scheduled clinic visits (at screening, then on Days 0, 7, 28, 90, 180, and 365). Blood sampling for safety will be performed at screening and on Days 0, 7, and 28, while sampling for immunogenicity will be performed on Days 0, 7, 28, 180, and 365.

[0708] Safety assessment of all subjects in Cohorts 1, 2, and 3: Starting from Day 0 (the day of single-dose administration) to Day 6 (7 days in total), all non-spontaneous local reactions and systemic AEs will be collected for all subjects; spontaneous adverse reactions to Day 28; and safety blood samples (clinical laboratory evaluations at screening, Day 0 / baseline, Day 7 and Day 28). SAEs, MAAEs, AEs leading to withdrawal from the trial, and AESI will be obtained from the time of randomization to the end of trial evaluation (approximately 12 months after vaccination). During treatment and...

Claims

1. A composition or pharmaceutical composition comprising: a) two or more virus-like particles (VLPs), wherein i. The first virus-like particle (VLP) comprises a first component, wherein the first component comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof, and ii. the second virus-like particle (VLP) comprises a first component, wherein the first component comprises the extracellular domain of the human metapneumovirus (hMPV) F protein or an antigenic variant thereof; and / or b) virus-like particles (VLPs) comprising a plurality of first components, some of which comprise the extracellular domain of the F protein of respiratory syncytial virus (RSV) or an antigenic variant thereof, and some of which comprise the extracellular domain of the F protein of human metapneumovirus (hMPV) or an antigenic variant thereof.

2. The composition of claim 1, wherein the VLPs each independently comprise: a) the first component, the first component comprising a first multimerization domain; and b) a second component comprising a second multimerization domain.

3. The composition of claim 2, wherein 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.

4. The composition of claim 2, wherein 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: 144) or I53-50AΔCys (SEQ ID NO: 145).

5. The composition of claim 4, wherein relative to SEQ ID NO: 144, 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.

6. A composition as described in any one of claims 1 to 5, wherein 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.

7. A composition as described in claim 6, wherein the second 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 I53-50B (SEQ ID NO:8) or I53-50B.4PosT1 (SEQ ID NO:34).

8. The composition of any one of claims 1 to 7, wherein the RSV F protein ectodomain 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 RSV F DS-Cav1 (SEQ ID NO: 173): QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSA IASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITND QKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKY DCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL.

9. The composition of claim 8, wherein the RSV F protein ectodomain comprises the amino acid substitutions S155C and S290C; and / or the amino acid substitutions S190F and V207L.

10. The composition of any one of claims 1 to 9, wherein 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: 151): (SEQ ID NO: 151).

11. The composition of any one of claims 1 to 10, wherein the hMPV F protein extracellular 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 SEQ ID NO: 174 or SEQ ID NO: 175: KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDCPSLIKTELDLTKSALRELKTVSADQLAREEQIEGGGGGGFVLGAIALGVATAAAVTAGIAIAKTIRLESE VNAIKGCLKTTNECVSTLGNGVRVLATAVRELKEFVSKNLTSAINKNKCDIADLCMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLEN RAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNDKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPC KVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPICFPEDQFNVALDQVFESIENCQA(SEQ ID NO:174); KESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEGGGGGGFVLGAIALGVATAAAVTAGVAIAKCIRLESE VTAIKNALKKTNEAVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISKDLMTDAELARAISNMPTSAGQIKLMLEN RAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPC KVSCGRNISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQCFESIENSQA(SEQ ID NO:175).

12. The composition of claim 11, wherein the hMPV F protein extracellular domain comprises amino acid substitutions A63C, A140C, A147C, K188C, K450C, S470C, N97G, P98G, R99G, Q100G, S101G and / or R102G relative to a reference hMPV F protein sequence (SEQ ID NO: 56); or wherein the hMPV F protein extracellular domain comprises amino acid substitutions T127C, N153C, T365C, V463C, A185P, L219K, V231I, G294E, N97G, P98G, R99G, Q100G, H386N, S101G and / or R102G.

13. A composition as described in any one of claims 1 to 12, wherein the first component of the second VLP comprises a polypeptide that has 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.

14. A composition as described in any one of claims 1 to 12, wherein the first component of the second VLP comprises a polypeptide that has 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.

15. The composition of any one of claims 1 to 14, wherein the composition comprises one or more pharmaceutically acceptable diluents, adjuvants or excipients.

16. The composition of any one of claims 1 to 15, wherein the composition is a stable emulsion.

17. The composition of any one of claims 1 to 16, wherein the vaccine comprises one or more adjuvants.

18. The composition of claim 17, wherein the one or more adjuvants are squalene, alum, SLA, GLA, R848, IMQ, 3M-052, CpG, saponin (QS21), or a combination thereof.

19. The composition of claim 17, wherein the adjuvant is alum.

20. The composition of claim 17, wherein the adjuvant is a squalene-based emulsion, optionally 21. The composition of claim 17, wherein the adjuvant is a squalene-based emulsion and a TLR4 agonist.

22. A unit dose of a composition or pharmaceutical composition according to any one of claims 1 to 21, wherein the unit dose comprises: a) 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, about 300 μg or about 500 μg of said first VLP; and b) 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, about 300 μg or about 500 μg of the second VLP.

23. A unit dose of a composition or pharmaceutical composition according to any one of claims 1 to 21, wherein the unit dose comprises: a) about 75 μg or about 150 μg of said first VLP; and b) about 75 μg of said second VLP.

24. A unit dose of a composition or pharmaceutical composition according to any one of claims 1 to 21, wherein the unit dose comprises: a) about 75 μg or about 150 μg of said first VLP; and b) about 150 μg of said second VLP.

25. A unit dose of a composition or pharmaceutical composition according to any one of claims 1 to 21, wherein the unit dose comprises: a) about 75 μg or about 150 μg of said first VLP; and b) about 150 μg or about 225 μg of said second VLP.

26. A unit dose as described in any one of claims 23-25, wherein the first VLP comprises a first component, the first component comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, and The second VLP comprises a first component, and the first component comprises the extracellular domain of human metapneumovirus (hMPV) F protein or an antigenic variant thereof.

27. A unit dose of a composition or pharmaceutical composition according to any one of claims 1 to 21, wherein the unit dose comprises: a) 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 150 μg, about 200 μg, about 250 μg or about 300 μg of said first VLP; and b) 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 150 μg, about 200 μg, about 250 μg or about 300 μg of the second VLP.

28. A method of vaccinating a subject, comprising administering to the subject an effective amount of a composition according to any one of claims 1-21.

29. A method of generating an immune response in a subject comprising administering to the subject an effective amount of a composition according to any one of claims 1-21.

30. A method for preventing infection by a Paramyxovirus, comprising administering to a subject an effective amount of a composition according to any one of claims 1 to 21.

31. A method of immunizing a subject against a Paramyxovirus, comprising administering to the subject an effective amount of a composition according to any one of claims 1-21.

32. The method of claim 30 or claim 31, wherein the paramyxovirus is respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV.

33. The method of any one of claims 28 to 32, wherein the method produces protective immunity against respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV.

34. The method of any one of claims 28 to 33, wherein the method produces neutralizing antibodies against respiratory syncytial virus (RSV), human metapneumovirus (hMPV), or both RSV and hMPV.

35. The method of any one of claims 28 to 34, wherein the subject is at risk for severe RSV disease and / or is at risk for severe hMPV disease.

36. The method of any one of claims 28 to 35, wherein the vaccine is administered by subcutaneous injection.

37. The method of any one of claims 28 to 35, wherein the vaccine is administered by intramuscular injection.

38. The method of any one of claims 28 to 35, wherein the vaccine is administered by intradermal injection.

39. The method of any one of claims 28 to 35, wherein the vaccine is administered intranasally.

40. The method of any one of claims 28 to 39, wherein the subject is an adult over the age of 60.

41. The method of any one of claims 28 to 39, wherein the subject is a healthy adult aged 18-45 years.

42. The method of any one of claims 28 to 41, wherein 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, about 300 μg, or about 500 μg of the first VLP and / or the second VLP.

43. The method of any one of claims 28 to 42, wherein the effective amount comprises about 75 μg of the first VLP and about 75 μg of the second VLP.

44. The method of any one of claims 28 to 42, wherein the effective amount comprises about 75 μg of the first VLP and about 150 μg of the second VLP.

45. The method of any one of claims 28 to 42, wherein the effective amount comprises about 150 μg of the first VLP and about 150 μg of the second VLP.

46. ​​The method of any one of claims 43 to 45, wherein the first VLP comprises a first component comprising a respiratory syncytial virus (RSV) F protein ectodomain or an antigenic variant thereof, and The second VLP comprises a first component, and the first component comprises the extracellular domain of human metapneumovirus (hMPV) F protein or an antigenic variant thereof.

47. The method of any one of claims 28 to 46, further comprising administering a second dose of the pharmaceutical composition.

48. The method of claim 47, wherein 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, about 12 months, about 24 months, or about 36 months after the first dose.

49. The method of claim 47 or claim 48, further comprising administering a third dose of the pharmaceutical composition.

50. The method of claim 49, wherein 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.

51. The method of claim 49 or claim 50, further comprising administering subsequent doses at regular intervals of about 1, 2, 3, 4, or 5 years.

52. The method of any one of claims 28 to 51, wherein 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.

53. The method of any one of claims 28 to 52, wherein the method results in the subject producing RSV-A specific neutralizing antibodies.

54. The method of claim 53, wherein the method results in an increase in RSV-A specific neutralizing antibodies in the subject by 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.

55. The method of claim 53 or claim 54, wherein an increase in RSV-A specific neutralizing antibodies is detectable within about one 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 administering the pharmaceutical composition.

56. The method of any one of claims 28 to 32, wherein the method results in the subject producing RSV-B specific neutralizing antibodies.

57. methods as described in any one of claims 28 to 56, wherein said method causes the RSV-B specific neutralizing antibodies in the subject to increase by 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.

58. methods as claimed in claim 57, wherein the increase of RSV-B specific neutralizing antibodies can be detected within about one 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 using the pharmaceutical composition.

59. The method of any one of claims 28 to 58, wherein the method results in the subject producing hMPV-A specific neutralizing antibodies.

60. The method of claim 59, wherein the method results in an increase in hMPV-A specific neutralizing antibodies in the subject by 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.

61. The method of claim 59 or claim 60, wherein an increase in hMPV-A specific neutralizing antibodies is detectable within about one 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 administering the pharmaceutical composition.

62. The method of any one of claims 28 to 61, wherein the method results in the subject producing hMPV-B specific neutralizing antibodies.

63. The method of claim 62, wherein the method results in an increase in hMPV-B-specific neutralizing antibodies in the subject by 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.

64. The method of claim 63, wherein an increase in hMPV-B specific neutralizing antibodies is detectable within about one 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 administering the pharmaceutical composition.

65. The method of any one of claims 28 to 64, wherein the method prevents severe lower respiratory tract infection (LRTI).

66. A prefilled syringe comprising a composition according to any one of claims 1-21.

67. A kit comprising a composition according to any one of claims 1-21 or a pre-filled syringe according to claim 66.

68. A kit comprising one or more of the following: a) A composition comprising a virus-like particle (VLP), wherein the virus-like particle comprises a first component, wherein the first component comprises an extracellular domain of a respiratory syncytial virus (RSV) F protein or an antigenic variant thereof; b) a composition comprising a second virus-like particle (VLP), the second virus-like particle comprising a first component, the first component comprising the extracellular domain of the human metapneumovirus (hMPV) F protein or an antigenic variant thereof; and c) A composition comprising a virus-like particle (VLP), wherein the virus-like particle comprises a plurality of first components, some of which comprise the extracellular domain of the F protein of respiratory syncytial virus (RSV) or an antigenic variant thereof, and some of which comprise the extracellular domain of the F protein of human metapneumovirus (hMPV) or an antigenic variant thereof.

69. The kit of claim 68, wherein the kit comprises a composition comprising an adjuvant to be combined with the one or more VLP compositions prior to administration to a subject.

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