Recombinant virus-like particles

Abstract

This disclosure relates to recombinant virus-like particles (VLPs) containing antigens for use as vaccines. In one embodiment, this disclosure relates to recombinant VLPs containing capsid fusion proteins for use as vaccines.

Description

Technical Field

[0001] Related Application Data This application claims priority from U.S. Application No. 63 / 510,970, filed Jun. 29, 2023, entitled “Recombinant virus-like particles”, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application is filed with a sequence listing in electronic form. The entire content of the sequence listing is incorporated herein by reference.

[0003] The present disclosure relates to recombinant virus-like particles (VLPs) containing antigens for use as vaccines. In one aspect, the present disclosure relates to recombinant VLPs containing capsid fusion proteins for use as vaccines.

Background Art

[0004] Respiratory virus infections are a major threat to human health. Infectious diseases such as those caused by influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV) are known to have caused worldwide pandemics and led to the deaths of millions of people around the world. In recent years, SARS-CoV-2 has caused the ongoing worldwide pandemic of coronavirus disease 2019 (COVID-19), which has extremely high infectivity. Moreover, respiratory syncytial virus (RSV) is the most common single cause of hospitalization due to respiratory disease in infants, and reinfection remains common even in later years. Although several vaccines are available for viral infections such as influenza, SARS-CoV-2, and RSV, further improvements can enhance their effectiveness and / or improve treatment strategies.

[0005] Currently, egg-based manufacturing processes are the most common method of producing vaccines. This process requires a considerable amount of time to optimize viral replication within the eggs, and a significant amount of resources (i.e., eggs) are needed to produce a sufficient quantity of vaccine, especially during a pandemic. Furthermore, the long development period means that vaccine strains are selected before the vaccine becomes available, making it difficult to respond to viral mutations. Vaccines are also produced using cell-based manufacturing processes involving cultured mammalian cells (e.g., Madin-Derby canine kidney cells, i.e., MDCK cells) as an alternative to eggs, as well as virus-based platforms involving recombinant viruses (e.g., baculoviruses encoding influenza antigens).

[0006] There remains a demand for the development of specific and efficient viral vaccines that can be produced more rapidly than current egg-based technologies and have broader utility, for the treatment or prevention of respiratory viral infections such as influenza, RSV, and SARS-CoV-2. While nucleic acid vaccines offer clear advantages over current egg-based manufacturing platforms, several challenges remain. For example, the inherently unstable nature of mRNA limits the ability of most RNA-based vaccines to deliver antigens at the doses and durations required to produce a potent and sustained immune response.

[0007] Therefore, it will be apparent to those skilled in the art that there is a need in the art for compositions with broader utility and / or improved efficacy that are suitable for use as vaccines. [Overview of the project]

[0008] This disclosure is based on the inventors' finding that recombinant virus-like particles (VLPs) comprising a capsid fusion protein and a lipid bilayer are suitable for the treatment of diseases, conditions, or infections such as SARS-CoV-2 infection, influenza, or coronavirus infection 2019 (COVID-19). Therefore, these findings by the inventors provide a basis for methods to treat, prevent, or delay the progression of diseases, conditions, or infections such as SARS-CoV-2 infection or COVID-19, as well as their complications, including pneumonia and acute respiratory distress syndrome (ARDS), in subjects. The lipid bilayer present within the VLPs described in this disclosure offers a technical advantage by preventing the immune response in subjects to the capsid protein that forms part of the capsid fusion protein. Furthermore, the VLP structures provided by this disclosure are capable of assembling into VLPs and providing immunogenicity in vaccine form.

[0009] Therefore, this disclosure provides recombinant virus-like particles (VLPs) comprising a capsid fusion protein and a lipid bilayer, wherein the capsid fusion protein is (a) Capsid proteins derived from non-enveloped viruses, (b) Transmembrane (TM) protein domain and (c) Antigen protein, and, The capsid protein is encapsulated within the lipid bilayer.

[0010] This disclosure also provides virus-like particles (VLPs) comprising a capsid fusion protein and a lipid bilayer, wherein the capsid fusion protein is (a) Capsid proteins derived from non-enveloped viruses, (b) an antigen protein, and The capsid protein is encapsulated within the lipid bilayer.

[0011] In one example, the capsid protein derived from a non-enveloped virus is derived from alfalfa mosaic virus (AMV), bacteriophage MS2, or bacteriophage AP205.

[0012] In one example, the capsid protein is derived from AMV. In several examples, the capsid protein contains the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 32.

[0013] In one example, the capsid protein is derived from bacteriophage MS2. In several examples, the capsid protein contains the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 35.

[0014] In some cases, the capsid protein is a dimeric capsid protein derived from bacteriophage MS2. In some cases, the capsid protein contains the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 36. In some cases, the capsid protein contains the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 37.

[0015] In one example, the capsid protein is derived from bacteriophage AP205. In several examples, the capsid protein contains the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 33.

[0016] In some cases, the capsid protein is a dimeric capsid protein derived from bacteriophage AP205. In some cases, the capsid protein contains the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 34.

[0017] In one example, the capsid protein of a capsid fusion protein is either a trimer or a dimer.

[0018] In one example, the capsid fusion protein further includes a signal sequence. In several examples, the signal sequence includes the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 21.

[0019] In one example, the capsid fusion protein further comprises a peptide tag, such as a polyhistidine tag. In one embodiment, the peptide tag is His6. In some examples, the polyhistidine tag comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 28.

[0020] In one example, the capsid protein is fused to the TM protein domain by a peptide linker. In this example, the linker is a short protein with a length of up to approximately 30 amino acids, e.g., approximately 5-30 amino acids, approximately 5-25 amino acids, approximately 5-20 amino acids, approximately 10-20 amino acids, approximately 5-15 amino acids, or approximately 10-15 amino acids. In another example, the linker has a length of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Suitable linkers are known in the art and include flexible linkers such as the Gly-Ser linker.

[0021] In some cases, the peptide linker contains one of the amino acid sequences from SEQ ID NOs. 22-27.

[0022] In another example, the capsid protein is fused to the TM protein domain by a hinge.

[0023] In some examples, the capsid protein is disposed at the C-terminus relative to the antigen protein and the TM protein domain. For example, the capsid protein may include, in order from the N-terminus to the C-terminus, an antigen protein, a TM protein domain, and then the capsid protein. Adjacent components of the capsid fusion protein may be fused via a peptide linker. <​​​​​​​​​​​​​​​​​In one example, the TM protein domain and antigen protein are derived from influenza A virus strains. For instance, the TM protein domain and antigen protein are derived from the hemagglutinin (HA) protein, neuraminidase (NA) protein, matrix (M) protein, nucleoprotein (NP), non-structural (NS) protein, or immunogenic fragments or variants thereof of influenza A virus. In one example, the TM protein domain and antigen protein are derived from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 of influenza A hemagglutinin (HA) and / or subtypes N1, N2, N3, N4, N5, N6, N7, N8, or N9 of influenza A neuraminidase (NA) and / or subtypes M1 or M2 of the influenza A matrix (M) protein and / or subtypes NS1 or NS2 of the influenza A non-structural (NS) protein.

[0027] Those skilled in the art will recognize that pandemic strains of influenza viruses are generally influenza A virus strains of the H1, H2, H3, H5, H6, H7, or H9 subtypes. For example, H1N1, H2N2, H3N2, H5N1, H5N3, H6N1, H7N2, H7N3, H7N7, H7N9, and H9N2 strains. Thus, in one example, the TM protein domain and antigen protein are the H1N1 protein derived from the A / Delaware / 55 / 2019 virus strain. In another example, the TM protein domain and antigen protein are the H1 protein derived from the A / Delaware / 55 / 2019 virus strain. In yet another example, the TM protein domain and antigen protein are the H1N1, H2N2, H3N2, H5N1, H5N3, H6N1, H7N2, H7N3, H7N7, H7N9, and H9N2 proteins.

[0028] In another example, the TM protein domain and antigen protein are derived from an influenza B virus strain. Those skilled in the art will recognize that influenza B viruses are not classified into subtypes, but rather into two lineages, namely B / Yamagata and B / Victoria.

[0029] In one example, the TM protein domain and antigen protein are derived from a B / Yamagata influenza B virus strain. For example, the influenza B virus strain is the B / Singapore / INFTT 16 0610 / 16(By) virus strain. In another example, the TM protein domain and antigen protein are derived from a B / Victoria influenza B virus strain. In one example, the TM protein domain and antigen protein are derived from the Hyam protein or Nyam protein of the influenza B virus. For example, the TM protein domain and antigen protein are derived from the Hyam protein of the influenza B virus. In yet another example, the TM protein domain and antigen protein are derived from the Nyam protein of the influenza B virus. In further examples, the TM protein domain and antigen protein are derived from the Hyam protein and Nyam protein of the influenza B virus.

[0030] In one example, the TM protein domain and antigen protein are derived from influenza B. In another example, the TM protein domain and antigen protein are derived from influenza C.

[0031] In one example of this disclosure, recombinant virus-like particles (VLPs) comprising a capsid fusion protein and a lipid bilayer are provided, and the capsid fusion protein is (a) Capsid proteins derived from non-enveloped viruses such as AMV, bacteriophage AP205, or bacteriophage MS2, (b) Transmembrane (TM) protein domain derived from the SARS-CoV-2 spike (S) protein, (c) The antigen protein derived from the spike (S) protein of SARS-CoV-2, The capsid protein is encapsulated within the lipid bilayer.

[0032] In some cases, the capsid protein is derived from the bacteriophage MS2 virus, the TM protein domain is derived from the SARS-CoV-2 spike (S) protein, and the antigen protein is derived from the SARS-CoV-2 spike (S) protein.

[0033] Therefore, in one example, the TM protein domain and antigen protein are derived from the spike (S) protein of SARS-CoV-2. In another example, the antigen is derived from the alpha (B.1.1.7), beta (B.1.351), gamma (P1), epsilon (B.1.429), delta (B.1.617.2) variant, kappa (B.1.617.1), Wuhan (2019-nCoV / USA-WA1 / 2020) strain, or omicron (B.1.1.529) strain of SARS-CoV-2.

[0034] In another example, the S protein is a mutant S protein.

[0035] In one example, the mutant S protein contains mutations in the receptor-binding domain. For example, such mutations include S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, and T478I. The group is selected from P479L, P479L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F490L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S, and Y505H, Y508H. In one example, the mutant S protein contains a mutation in the receptor-binding domain selected from the group consisting of N439K, N439L, L452R, S477N, T478I, V483A, and E484D.

[0036] In one example, the mutant S protein contains mutations in the receptor-binding domain. For example, such mutations include R346K, K417N, K417T, S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F, K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, and S477R. Selected from the group consisting of T478I, T478K, P479L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F490L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S, N501Y, Y505H, and Y508H. In one example, the mutant S protein contains mutations in the receptor-binding domain selected from the group consisting of R346K, K417N, K417T, N439K, N439L, L452R, S477N, T478I, V483A, E484D, E484K, and N501Y.

[0037] In one example, the mutant S proteins are P337S, F338L, F338C, G339D, E340K, V341I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V395I, F400C, R403K, R403S, D This includes mutations selected from the group consisting of 405V, R408I, Q414E, Q414K, Q414P, Q414R, T415S, K417R, K417N, I418V, Y421S, Y423C, Y423F, Y423S, D427Y, R509K, V510L, V511E, V512L, L518I, H519O, A520S, A520V, P521R, P521S, A522P, A522S, and D614G.

[0038] In one example, the mutant S proteins are L18F, D80A, T95I, Y144S, Y145N, D215G, P337S, F338L, F338C, G339D, E340K, V341I, A344S, T345S, R346K, A348S, A348T, W353R, N354D, N354K, N354S, S359N, D364Y, V367F, S373L, V382L, P384L, P384S, T385A, T393P, V 395I, F400C, R403K, R403S, D405V, R408I, Q414E, Q414K, Q414P, Q414R, T415S, K417N, K417T, K417R, I418V, Y421S, Y42 3C, Y423F, Y423S, D427Y, S438F, N439K, N440K, L441I, K444R, V445A, V445I, G446V, G446S, N450K, L452R, L452P, L455F , K458N, N460T, D467V, I468F, I468T, I468V, E471O, I472V, A475V, G476S, S477G, S477I, S477N, S477R, T478I, T478K, P 479L, P479S, N481D, N481H, V483F, V483A, E484D, E484K, E484K, E484O, G485S, Y489H, Y489D, Y489F, Y489C, Y489N, F49 This includes mutations selected from the group consisting of 0L, F490S, P491R, Q493L, S494P, Y495N, T500N, N501S, N501Y, Y505H, Y508H, R509K, V510L, V511E, V512L, L518I, H519O, A520S, A520V, P521R, P521S, A522P, A522S, A570D, D614G, P680H, P681H, A701V, T716I, and D950N.

[0039] In some cases, the S protein contains a D-to-G mutation (i.e., a D614G mutation) at the residue corresponding to position 614 of sequence number 38.

[0040] In one example, the S protein lacks a furin cleavage site at the S1 / S2 boundary and / or the S2' region. Therefore, the amino acid sequence of the furin cleavage site can be modified / substituted to inhibit furin cleavage.

[0041] In some cases, the S protein contains a mutation from RRAR to QQAA at residues corresponding to positions 682-685 of SEQ ID NO: 38.

[0042] In some cases, the S protein contains a mutation from RRAR to GSAS at residues corresponding to positions 682-685 of SEQ ID NO: 38.

[0043] In some cases, the S protein contains the insertion of two proline residues between the residue corresponding to position 986 and position 987 of SEQ ID NO: 38.

[0044] In some cases, the S protein (i) lacks a furin cleavage site at the S1 / S2 boundary and / or (ii) contains a mutation from RRAR to QQAA or from RRAR to GSAS at the residue corresponding to positions 682-685 of SEQ ID NO: 38 and / or (iii) lacks a furin cleavage site at the S2' site and / or (iv) contains a mutation from D to G at the residue corresponding to position 614 of SEQ ID NO: 38 and / or (v) contains the insertion of two proline residues between the residues corresponding to positions 986 and 987 of SEQ ID NO: 38.

[0045] In some examples, the capsid fusion protein includes an S protein containing a TM protein domain and an antigen protein, the S protein containing an amino acid sequence selected from any one of SEQ ID NOs. 29, 31, or 38, or an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs. 29, 31, or 38.

[0046] In some cases, the capsid fusion protein includes an S protein containing a TM protein domain and an antigen protein, and the S protein contains the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 29.

[0047] In some cases, the capsid fusion protein includes an S protein containing a TM protein domain and an antigen protein, and the S protein contains the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 31.

[0048] In some cases, the capsid fusion protein includes an S protein containing a TM protein domain and an antigen protein, and the S protein contains the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 38.

[0049] In some cases, the capsid fusion protein contains an S protein containing the antigen protein, and this S protein contains the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 30. SEQ ID NO: 30 is the amino acid sequence of the SARS-CoV-2 spike protein cleaved at the C-terminus to remove the TM protein domain.

[0050] In one example of this disclosure, recombinant virus-like particles (VLPs) comprising a capsid fusion protein and a lipid bilayer are provided, and the capsid fusion protein is (a) Capsid proteins derived from non-enveloped viruses such as AMV, bacteriophage AP205, or bacteriophage MS2, (b) Transmembrane (TM) protein domains derived from the pre-F protein or F protein of respiratory syncytial virus (RSV), (c) comprising the pre-F protein or antigen protein derived from the F protein of RSV, The capsid protein is encapsulated within a lipid bilayer.

[0051] Therefore, in one example, the TM protein domain and antigen protein are derived from respiratory syncytial virus (RSV). For example, the TM protein domain and antigen protein are selected from RSV surface glycoproteins including fusion (F) protein, glycoprotein (G), small hydrophobic protein (SH), matrix proteins M and M2, nucleocapsid proteins N, P, and L, and non-structural proteins NS1 and NS2.

[0052] In one example, the TM protein domain and antigen protein are derived from the Pre-F protein of RSV.

[0053] In another example, the TM protein domain and the antigen protein originate from different viruses.

[0054] For example, the TM protein domain is derived from the S protein (i.e., its TM protein domain) of SARS-CoV-2, and the antigen protein is derived from the HA protein or NA protein of influenza A virus. In another example, the TM protein domain is the HA protein or NA protein (i.e., its TM protein domain) of influenza A virus, and the antigen protein is the S protein derived from SARS-CoV-2. In one embodiment, the S protein, HA protein, or NA protein is selected from those described herein or those known in the art.

[0055] In another example, the TM protein domain is the S protein (i.e., its TM protein domain) derived from SARS-CoV-2, and the antigen protein is the F protein or Pre-F protein of RSV. In yet another example, the TM protein domain is the F or Pre-F protein (i.e., its TM protein domain) of RSV, and the antigen protein is the S protein (i.e., derived from SARS-CoV-2). In one embodiment, the S protein, HA protein, or NA protein is selected from those described herein or those known in the art.

[0056] In another example, the TM protein domain is the F or Pre-F of RSV (i.e., its TM protein domain), and the antigen protein is the HA or NA protein of influenza A virus. In yet another example, the TM protein domain is the TM protein domain of the HA or NA of influenza A virus (i.e., its TM protein domain), and the antigen protein is the F or Pre-F protein of RSV. In one embodiment, the S protein, HA protein, or NA protein is selected from those described herein or those known in the art.

[0057] In some cases, the capsid fusion protein contains one of the amino acid sequences from SEQ ID NOs: 1-20, or an amino acid sequence that is at least 90% identical to one of SEQ ID NOs: 1-20. The polyhistidine tag GSSHHHHHH (SEQ ID NO: 28) may or may not be present in the sequence of the capsid fusion protein.

[0058] In some cases, the capsid fusion protein contains the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 (optionally lacking the polyhistidine tag GSSHHHHHH).

[0059] In some cases, the capsid fusion protein contains the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 (optionally lacking the polyhistidine tag GSSHHHHHH).

[0060] In some cases, the capsid fusion protein contains the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 15 (optionally lacking the polyhistidine tag GSSHHHHHH).

[0061] In some cases, the capsid fusion protein contains the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 17 (optionally lacking the polyhistidine tag GSSHHHHHH).

[0062] In some cases, the capsid fusion protein contains the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 (optionally lacking the polyhistidine tag GSSHHHHHH).

[0063] In some cases, the capsid fusion protein contains the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 20 (optionally lacking the polyhistidine tag GSSHHHHHH).

[0064] In one example, the VLP includes a second capsid fusion protein. In one embodiment, the second capsid fusion protein includes an antigen protein different from the first antigen protein. In this embodiment, the TM protein domain of the second capsid fusion protein may be the same as the TM protein domain of the first capsid fusion protein. In another embodiment, the second capsid fusion protein includes a TM protein domain different from the first TM protein domain of the first capsid fusion protein.

[0065] In one example, if a second capsid fusion protein is assumed, each antigen may be formulated in a separate VLP or in the same VLP. For example, a capsid fusion protein containing the S protein and a capsid fusion protein containing the F protein or Pre-F protein may be formulated in the same VLP. In another example, a capsid fusion protein containing the S protein and a capsid fusion protein containing the F protein or Pre-F protein may be formulated in different VLPs. In yet another example, a capsid fusion protein containing the HA protein or NA protein and a capsid fusion protein containing the F protein or Pre-F protein may be formulated in the same VLP. In yet another example, a capsid fusion protein containing the HA protein or NA protein and a capsid fusion protein containing the F protein or Pre-F protein may be formulated in different VLPs. In yet another example, a capsid fusion protein containing the HA protein or NA protein and a capsid fusion protein containing the S protein may be formulated in the same VLP. In another example, a capsid fusion protein containing an HA protein or an NA protein and a capsid fusion protein containing an S protein may be formulated within different VLPs.

[0066] In one example, the VLP contains no viral RNA whatsoever. In other words, the VLP lacks RNA capable of infecting a host and replicating within that host. Therefore, the VLPs of this disclosure are considered non-infectious. In another example, the VLP is substantially free of viral RNA or contains about 10% by weight, 5% by weight, 1% by weight, or less than 0.1% by weight, preferably less than about 5% by weight, more preferably less than 1% by weight of viral RNA.

[0067] In one example, the VLP has a diameter of approximately 70nm-160nm, 70nm-150nm, 70nm-140nm, 70nm-130nm, 70nm-120nm, 70nm-110nm, 70nm-100nm, or 70nm-90nm. In another example, the VLP has a diameter of approximately 80nm.

[0068] In one example, the VLP has a diameter of approximately 30nm-120nm, 40nm-110nm, 50nm-100nm, 60nm-90nm, or 70nm-80nm.

[0069] In one example, VLPs are formulated within lipid nanoparticles (LNPs). For example, VLPs are encapsulated within LNPs. In another example, VLPs are bound to LNPs. In yet another example, VLPs are adsorbed onto LNPs.

[0070] In one example, LNP further comprises PEG lipids, structural lipids, and / or neutral lipids. For example, LNP further comprises PEG lipids. In another example, LNP further comprises structural lipids. In yet another example, LNP further comprises neutral lipids.

[0071] In one example, LNPs contain ionizable lipids. For instance, the ionizable lipids are cationic lipids. In another example, the ionizable lipids are zwitterionic lipids.

[0072] In one example, LNPs do not contain ionizable lipids.

[0073] In one example, if multiple VLPs are envisioned, each VLP may be formulated together within an LNP. For example, a capsid fusion protein containing an S protein and a capsid fusion protein containing an F protein or Pre-F protein may be formulated in separate VLPs and then formulated together within an LNP. In another example, a capsid fusion protein containing an HA protein or NA protein and a capsid fusion protein containing an F protein or Pre-F protein may be formulated in separate VLPs and then formulated together within an LNP. In yet another example, a capsid fusion protein containing an S protein and a capsid fusion protein containing an HA protein or NA protein may be formulated in separate VLPs and then formulated together within an LNP.

[0074] In one example, each VLP is formulated separately within an LNP. For example, a capsid fusion protein containing an S protein and a capsid fusion protein containing an F protein or Pre-F protein may be formulated in separate VLPs and then separately formulated within an LNP. In another example, a capsid fusion protein containing an HA protein or NA protein and a capsid fusion protein containing an F protein or Pre-F protein may be formulated in separate VLPs and then separately formulated within an LNP. In yet another example, a capsid fusion protein containing an S protein and a capsid fusion protein containing an HA protein or NA protein may be formulated in separate VLPs and then separately formulated within an LNP.

[0075] This disclosure also states, (a) Capsid proteins derived from non-enveloped viruses, (b) Transmembrane (TM) protein domain and (c) Capsid fusion proteins containing antigen proteins are also provided.

[0076] This disclosure also states, (a) Capsid proteins derived from non-enveloped viruses, (b) We also provide a capsid fusion protein containing an antigen protein.

[0077] In this specification, examples of the characteristics of capsid fusion proteins within VLPs of this disclosure are applicable to capsid fusion proteins of this disclosure as well, with necessary modifications.

[0078] In one example, the Disclosure further provides isolated, recombinant, or synthetic nucleotide sequences encoding the capsid fusion proteins disclosed herein. In another example, the Disclosure further provides isolated, recombinant, or synthetic nucleotide sequences encoding the VLPs disclosed herein.

[0079] In one example, an isolated, recombinant, or synthesized nucleotide sequence encodes a capsid fusion protein, and the nucleotide sequence is arranged in 5' to 3' order. (a) Polynucleotides encoding a capsid protein derived from a non-enveloped virus, such as AMV, bacteriophage AP205, or bacteriophage MS2, (b) A polynucleotide encoding a transmembrane (TM) protein domain derived from the SARS-CoV-2 spike (S) protein, (c) A polynucleotide encoding an antigen protein derived from the SARS-CoV-2 spike (S) protein, The polynucleotide is operably linked to the regulatory element.

[0080] In one example, the regulatory element is a promoter. In another example, the promoter is a synthetic genome promoter.

[0081] In one example, an isolated, recombinant, or synthesized nucleotide sequence further encodes a signal peptide located at the 5' end of the polynucleotide encoding the antigen. In another example, the polynucleotides encoding the TM protein and the antigen protein are operably linked by a polynucleotide encoding a linker.

[0082] In one example, the Disclosure further provides an expression vector comprising a nucleotide sequence encoding a capsid fusion protein disclosed herein. In another example, the Disclosure further provides an expression vector comprising a nucleotide sequence encoding a VLP disclosed herein.

[0083] In one example, this disclosure further provides a pharmaceutical composition comprising a VLP and a pharmaceutically acceptable carrier disclosed herein. In one example, the pharmaceutical composition is an immunogenic composition.

[0084] In another example, the Disclosure further provides a pharmaceutical composition comprising a VLP disclosed herein for use as a vaccine. In another example, the Disclosure further provides a vaccine comprising a pharmaceutical composition or an immunogenic composition.

[0085] In one example, the composition further comprises an adjuvant. In one example, the adjuvant is selected from the group consisting of Freund's adjuvant, incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, GMCSP, BCG, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, monophosphoryl lipid A (MPL), RIBI, MPL, trehalose dimicholate (TDM), Novasomes®, QS21, Quil A (and its derivatives and components), calcium phosphate, calcium hydroxide, zinc hydroxide, MHC antigen, PolyI:C, MF59, glycolipid analogs, octodecyl esters of amino acids, muramyl dipeptide, polyphosphazene, lipoprotein, ISCOM matrix, DC-Chol, ODA, cytokines, and other adjuvants and their derivatives. In one example, the adjuvant is MF59.

[0086] In one example, an adjuvant such as MF59 is administered simultaneously with the administration of the composition of the disclosure. In another example, an adjuvant such as MF59 is administered sequentially, prior to, or following the administration of the composition of the disclosure.

[0087] In one example, the Disclosure provides a method for treating, preventing, or slowing the progression of a disease, disorder, or condition to a subject in need thereof, the method comprising administering to the subject a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein.

[0088] In one example, this disclosure provides the use of a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the manufacture of a pharmaceutical product for treating, preventing, or delaying the progression of a disease, disorder, or condition in a subject.

[0089] In one example, the Disclosure provides a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in the treatment, prevention, or delay of the progression of a disease, disorder, or condition in a subject.

[0090] In one example, the disease, disorder, or condition is a viral infection.

[0091] In one example, the viral infection could be COVID-19, influenza, or RSV.

[0092] In one example, a subject with a viral infection has at least one symptom of COVID-19, influenza, or RSV. In one example, such symptoms include runny nose, cough, sore throat, fever, headache, muscle aches, or fatigue.

[0093] In one example, the present disclosure provides a method for inducing an immune response in a subject, the method comprising administering a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to a subject in need of such induction of an immune response.

[0094] In one example, this disclosure provides the use of a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the manufacture of a pharmaceutical product for inducing an immune response in a subject that requires such induction of an immune response.

[0095] In one example, the present disclosure provides a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in inducing an immune response in a subject requiring such induction of an immune response.

[0096] In one example, the composition induces a humoral immune response in a subject. For example, the humoral immune response is an antibody-mediated immune response. For example, the production of neutralizing antibodies. In another example, the composition induces a cell-mediated immune response. For example, the cell-mediated immune response includes the activation of antigen-specific cytotoxic T cells. For example, the T cells are CD4 T cells and / or CD8 T cells. In one example, the T cells are CD4 T cells. In another example, the T cells are CD8 T cells. In yet another example, the T cells are CD4 T cells and CD8 T cells.

[0097] In one example, administration of the VLP, pharmaceutical composition, immunogenic composition, or vaccine of this disclosure induces a CD4 T cell-mediated immune response.

[0098] In one example, administration of the VLP, pharmaceutical composition, immunogenic composition, or vaccine of this disclosure induces a CD8 T cell-mediated immune response.

[0099] In one example, administration of the VLPs, pharmaceutical compositions, immunogenic compositions, or vaccines of this disclosure induces CD4 T-cell-mediated and CD8 T-cell-mediated immune responses.

[0100] In one example, the CD4 T cell-mediated immune response is a Th0 response, a Th1 response, and / or a Th2 response. For example, the CD4 T cell-mediated immune response is a Th0 response. In another example, the CD4 T cell-mediated immune response is a Th1 response. In yet another example, the CD4 T cell-mediated immune response is a Th2 response. In one example, the CD4 T cell-mediated immune response is a Th0 response and a Th1 response. In another example, the CD4 T cell-mediated immune response is a Th0 response and a Th2 response. In yet another example, the CD4 T cell-mediated immune response is a Th1 response and a Th2 response. In yet another example, the CD4 T cell-mediated immune response is a Th0 response, a Th1 response, and a Th2 response.

[0101] In one example, the Th0 response cytokine expresses interleukin-2 (IL2+) and / or tumor necrosis factor alpha (TNFα+), and / or is negative for interferon-gamma (IFNg-), IL5-, and / or IL13-. For example, the cytokine is IL2+. In another example, the cytokine is TNFα+. In one example, the cytokine is IFNg-. In yet another example, the cytokine is IL5-. In yet another example, the cytokine is IL13-.

[0102] In one example, the Th1 response cytokine expresses interferon-gamma (IFNg+) and / or is negative for IL5- and / or IL13-. For example, the cytokine is IFNg+. In another example, the cytokine is IL5-. In yet another example, the cytokine is IL13-.

[0103] In one example, the Th2-response cytokine expresses IL5+ and / or IL13+ and / or is negative for IFN g. For example, the cytokine is IL5+. In a further example, the cytokine is IL13+. For example, the cytokine is IFN g-.

[0104] In one example, the immune response is elevated in response to at least one antigen derived from SARS-CoV-2, influenza, or RSV. For example, the immune response is elevated in response to the SARS-CoV-2-derived S protein antigen as described herein. In another example, the immune response is elevated in response to the influenza antigen as described herein, such as the HA protein antigen or NA protein antigen. In yet another example, the immune response is elevated in response to the RSV antigen as described herein, such as the F protein or Pre-F protein.

[0105] In another example, the immune response is sufficient to treat, prevent, or delay the progression of at least one symptom of a viral infection caused by SARS-CoV-2, influenza, or RSV.

[0106] In one example, the Disclosure provides a method for reducing the viral load in a subject, which includes administering a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to a subject requiring a reduction in viral load.

[0107] In one example, this disclosure provides the use of a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the preparation of a pharmaceutical product for reducing the viral load in a subject.

[0108] In one example, this disclosure provides a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in reducing the viral load in a subject.

[0109] In one example, the Disclosure provides a method for treating, preventing, or delaying the progression of pneumonia, which includes administering a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to a subject in need of treatment, prevention, or delaying the progression of pneumonia.

[0110] In one example, the Disclosure provides the use of a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the preparation of a pharmaceutical product for treating, preventing, or delaying the progression of pneumonia in a subject.

[0111] In one example, the Disclosure provides a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in treating, preventing, or delaying the progression of pneumonia in a subject.

[0112] In one example, the Disclosure provides a method for treating, preventing, or delaying the progression of acute respiratory distress syndrome in a subject, which includes administering a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein to a subject in need of treatment, prevention, or delaying the progression of acute respiratory distress syndrome.

[0113] In one example, this disclosure provides the use of a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein in the preparation of a pharmaceutical product for treating, preventing, or delaying the progression of acute respiratory distress syndrome in a subject.

[0114] In one example, the present disclosure provides a VLP, a pharmaceutical composition, an immunogenic composition, or a vaccine disclosed herein for use in treating, preventing, or delaying the progression of acute respiratory distress syndrome in a subject.

[0115] In one example, the subjects are people aged 18 or older. In another example, the subjects are people of any age, for example, from about 1 month to 100 years old, for example, from about 2 months to about 80 years old, from about 6 months to about 3 years old, from about 3 years to about 18 years old, from about 12 years to about 18 years old, from about 18 years to about 55 years old, from about 50 years to about 75 years old, or from about 40 years to about 65 years old. In another example, the subjects are people aged 2 years or older. In another example, the subjects are people aged 18 or older, people aged 30 or older, people aged 40 or older, people aged 50 or older, people aged 60 or older, people aged 70 or older, people aged 80 or older, or people aged about 90 or older. In another example, the subjects are people under 2 years old, under 18 months old, under 12 months old, under 6 months old, or under 3 months old.

[0116] In one example, the composition or vaccine described herein is administered in a single-dose regimen. In another example, the composition is administered in a two-, three-, or four-dose regimen. In this example, these doses may be administered at intervals of approximately one, two, or three months.

[0117] In one example of this disclosure, eukaryotic cells for expressing the VLPs described herein are provided. In one example, the eukaryotic cells are CHO cells, baby hamster kidney 21 (BHK-21) cells, human embryonic kidney 293 (HEK293) cells, CAP-T cell lines derived from human amniotic cells, Vero9 cells, or East Lansing Line-0 (ELL-0) cells.

[0118] In one example, eukaryotic cells contain polynucleotides that encode a capsid fusion protein, and the capsid fusion protein is (a) Capsid proteins derived from non-enveloped viruses, (b) Transmembrane (TM) protein domain and (c) Contains antigen protein and

[0119] In one example of this disclosure, a method for producing VLPs is provided. (a) To provide one or more expression vectors comprising polynucleotides for expressing virus-like particles (VLPs) as described herein, (b) providing host cells, (c) Transfecting a host cell with one or more expression vectors to produce virus-like particles (VLPs) containing one or more antigens, The polynucleotide in question is expressed under conditions sufficient for VLP formation.

[0120] In one example of this disclosure, a method for producing VLPs is provided. (a) To provide an expression vector comprising a polynucleotide encoding a capsid fusion protein, wherein the capsid fusion protein is (i) Capsid proteins derived from non-enveloped viruses, (ii) Transmembrane (TM) protein domain, (iii) To provide an antigen protein and, (b) providing host cells, (c) Transfecting the host cell with the vector to produce virus-like particles (VLPs) containing one or more antigens, The polynucleotide is expressed under conditions sufficient for VLP formation, and once formed, the VLP contains a lipid bilayer. The capsid protein is encapsulated within the lipid bilayer.

[0121] In one example, the method further includes purifying the VLP.

[0122] In one example, the Disclosure also provides a kit comprising at least one composition or vaccine of the Disclosure.

[0123] In one example, the kit comprises the composition or vaccine of the Disclosure, optionally contained in a delivery system and / or a pharmaceutically acceptable carrier or diluent, and is packaged with instructions for use in treating, preventing, or delaying the progression of a viral infection in a subject requiring treatment, prevention, or delaying the progression of such infection. In one example, the composition or vaccine comprises an adjuvant such as MF59. In another example, the kit further comprises a delivery system and / or a pharmaceutically acceptable carrier or diluent, and is packaged with instructions for use in administering the VLP to a subject with or at risk of developing a viral infection.

[0124] Therefore, in one example, the kit is, (a) A VLP disclosed herein, a pharmaceutical composition disclosed herein, an immunogenic composition disclosed herein, or a vaccine disclosed herein, (b) Instructions for use for their use, and optionally, (c) comprising a pharmaceutically acceptable carrier, excipient, or diluent.

[0125] In one example, the composition, immunogenic composition, or pharmaceutical composition of the Disclosure is supplied in a vial. In another example, the immunogenic composition or pharmaceutical composition of the Disclosure is supplied in a syringe.

[0126] Nothing relating to the documents, acts, materials, devices, or articles contained herein should be construed as acknowledging that all or part of such matters constituted part of the prior art foundation or common general knowledge in the art relating to this disclosure, as existing prior to the respective priority dates of the attached claims. [Brief explanation of the drawing]

[0127] [Figure 1]This is a schematic diagram of an exemplary capsid fusion protein, including the capsid protein, linker, transmembrane domain, and antigen protein. [Figure 2] Example 1 describes exemplary VLP structures of the present disclosure. A is an exemplary VLP containing the AMV capsid protein. B is an exemplary VLP containing the bacteriophage capsid protein AP205. C is an exemplary VLP containing the bacteriophage capsid protein MS2. [Figure 3] An exemplary VLP structure of this disclosure, containing an AMV capsid protein. [Figure 4] An exemplary VLP structure of this disclosure, containing the bacteriophage AP205 protein. [Figure 5] This is an exemplary VLP structure of the present disclosure, containing the bacteriophage MS2 protein. [Figure 6] This is an electron microscope image of a VLP containing the MS006 structure. Well-formed VLPs are indicated by a gray frame. [Modes for carrying out the invention]

[0128] general Throughout this specification, unless otherwise specifically stated or the context requires a different interpretation, references to a single step, composition, group of steps, or group of compositions should be understood as encompassing one or more (i.e., one or more) of those steps, compositions, group of steps, or group of compositions.

[0129] Those skilled in the art will understand that this disclosure is subject to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds mentioned or indicated herein, individually or collectively, as well as any combination of such steps or features, or any two or more of them.

[0130] This disclosure is not limited in scope by the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0131] Any example in this disclosure should be considered to be provided in any other example in this disclosure with the necessary modifications, unless otherwise specifically stated. In other words, any specific example in this disclosure can be combined with any other specific example in this disclosure (unless they are mutually exclusive).

[0132] If any example in this disclosure discloses a specific feature or set of features, or a method or steps of a method, it shall be deemed to provide explicit support for disclaiming such specific feature or set of features, or a method or steps of a method.

[0133] Unless otherwise specifically defined, all technical and scientific terms used herein should be understood to have the same meaning as those commonly understood by those skilled in the art (for example, in the fields of cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0134] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard methods well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. This is described and explained throughout the literature, including sources such as al. (ed.) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0135] The term "and / or," for example "X and / or Y," should be understood as meaning either "X and Y" or "X or Y," and should be taken as providing explicit support for both meanings, or for either one of them.

[0136] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” should be understood as implying the inclusion of the elements, components, or steps, or groups of elements, components, or steps described, but not as implying the exclusion of any other elements, integers, or steps, or groups of elements, integers, or steps.

[0137] As used herein, the term "derived from" should be understood to indicate that a specified component can be obtained from a specific source, but not necessarily directly from that source. Similarly, the term "based on" should be understood to indicate that a specified component can be developed from or used from a specific source, but not necessarily directly from that source.

[0138] Selected definition As used herein, the term “fragment” refers to a portion of a nucleotide sequence or polypeptide (protein) of a reference nucleotide sequence or polypeptide disclosed herein that maintains the defined activity of the full-length nucleotide sequence or polypeptide. For example, the defined activity is the induction of an immune response in a subject administered with a composition of this disclosure.

[0139] As used herein, the term “variant” means a nucleotide sequence or polypeptide (e.g., an antigenic polypeptide) that has one or more nucleotide sequences or amino acid sequences that differ from a reference nucleotide sequence or polypeptide disclosed herein, which maintains the defined activity of the nucleotide sequence or polypeptide. The one or more nucleotide sequences or amino acid sequences that differ may result from one or more modifications made to the nucleotide sequence or polypeptide of the Disclosure. For example, the modification is a chemical modification of one or more nucleotides in the nucleotide sequence. For example, at least one naturally occurring nucleotide in RNA is replaced with a chemically modified nucleotide (e.g., pseudouridine (ψ) and 1-methylpseudridine (m1ψ)). For example, the modification includes increasing the G / C content of the nucleotide sequence. For example, the modification includes codon optimization of the nucleotide sequence. For example, the defined activity is the induction of an immune response in a subject administered with a composition of the Disclosure.

[0140] In one example, the variant has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the sequence disclosed herein. In one example, the variant has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the sequence disclosed herein. Variants of nucleotide sequences or polypeptides disclosed herein may have one or more nucleotides or amino acids that are deleted or substituted by different nucleotides or amino acids. In one example, the substitutions are conservative substitutions. Those skilled in the art will understand that conservative substitutions of a polypeptide involve the substitution of an amino acid in the polypeptide with a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). In one example, the substitution is a non-conservative substitution.

[0141] As used herein, the terms “encode,” “encodes,” or “encoding” refer to a region of RNA that is capable of being translated into a polypeptide.

[0142] As used herein, the term “antigen” refers to a molecule or structure containing one or more epitopes that induce, trigger, enhance, or strengthen a cellular and / or humoral immune response. Antigens include, for example, proteins and peptides derived from pathogens such as viruses, bacteria, fungi, protozoa, and plants, or from tumors. For example, an antigen may be derived from a gene of interest. In another example, the antigenic protein may be derived from SARS-CoV-2, RSV, or influenza. In yet another example, the antigenic protein may be derived from the S protein of SARS-CoV-2, the F protein or Pre-F protein of RSV, or the HA or NA protein of influenza.

[0143] As used herein, the term “adjuvant” refers to a compound that, when used in combination with a specific immunogen (e.g., a VLP) in a formulation, enhances, alters, or modifies the resulting immune response. Modification of the immune response includes increasing or expanding the specificity of either or both the antibody-mediated immune response and the cellular-mediated immune response. Modification of the immune response may also mean reducing or suppressing a particular antigen-specific immune response.

[0144] As used herein, the term “naked” refers to nucleic acids that are substantially free of other macromolecules such as lipids, polymers, and proteins. Naked nucleic acids are not formulated with other macromolecules to improve cellular uptake. Therefore, naked nucleic acids are not encapsulated in, adsorbed to, or bound to lipid nanoparticles (LNPs), liposomes, or polymer microparticles.

[0145] As used herein, the terms “nucleotide sequence” or “nucleic acid sequence” should be understood to mean a series of consecutive nucleotides (or bases) covalently bonded to a phosphate diester backbone. By convention, unless otherwise specified, sequences are presented from the 5' end to the 3' end.

[0146] As used herein, the term “operably ligated to ~” means that a translation initiation sequence (e.g., Kozak consensus sequence, internal ribosome entry site (IRES), subgenome (SG) promoter) or a stabilizing element (e.g., 5'UTR) is positioned relative to a nucleic acid such that the expression of the nucleic acid is controlled or regulated by the sequence or element. For example, a translation initiation sequence can be operably ligated to the 5' end of one or more polynucleotide sequences disclosed herein.

[0147] The terms "polypeptide" or "polypeptide chain" should be understood as referring to a sequence of consecutive amino acids linked by peptide bonds. For example, a protein should be considered to consist of a single polypeptide chain, i.e., a sequence of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains (i.e., polypeptide complexes) linked to one another by covalent or non-covalent bonds. A series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0148] The term "recombinant" should be understood to mean products of artificial genetic modification.

[0149] As used herein, the terms “lipid nanoparticles” or “LNPs” should be understood to refer to any lipid composition, including but not limited to liposomes or vesicles in which an aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., monolayer, being single, or multilayer, being multiple); micelle-like lipid nanoparticles having a non-aqueous core; and solid lipid nanoparticles.

[0150] As used herein, the terms “disease,” “disorder,” or “condition” refer to a failure or interference with normal function.

[0151] As used herein, a subject “at risk” of developing an infectious disease (e.g., a viral infection) may or may not have a detectable disease or symptoms of the infectious disease, and may or may not exhibit a detectable disease or symptoms of the infectious disease prior to treatment as disclosed herein. “At risk” means that the subject has one or more risk factors, which are measurable parameters known in the art and / or described herein, that correlate with the development of the infectious disease.

[0152] As used herein, the terms “treatment” or “treat” a subject include applying or administering the compounds or compositions of the Disclosure to a subject (or applying or administering the compounds of the Disclosure to cells or tissues derived from the subject) for the purpose of delaying, slowing, stabilizing, curing, treating, reducing, alleviating, altering, correcting, reducing, improving, enhancing, or influencing a disease or condition, the symptoms of such disease or condition, or the risk (or susceptibility) to such disease or condition. The terms “treat” include any signs of success in treating or improving an injury, pathology, or condition, and include any objective or subjective parameters such as reduction, remission, slower rate of progression, reduced severity of the disease, stabilization, reduced symptoms, or making the injury, pathology, or condition more tolerable to the subject, slower rate of progression of degeneration or functional decline, or making the final stages of degeneration less fatal.

[0153] As used herein, “prevent” or “prevention” is intended to mean at least reducing the likelihood of acquiring the risk of acquiring a disease or disability (or susceptibility to acquiring a disease or disability) (i.e., preventing at least one clinical symptom of the disease from developing in patients who may be exposed to the disease or may be predisposed to the disease but have not yet experienced or shown any symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known to physicians.

[0154] As used herein, the phrase “to slow the progression of” includes reducing or slowing the progression of a disease or condition, and / or at least one symptom of the disease or condition, in an individual.

[0155] “Effective dose” means the minimum amount effective to achieve the desired outcome in the required dose and duration. For example, the desired outcome may be a therapeutic or prophylactic outcome. The effective dose may be provided in one or more doses. In some examples of this disclosure, the term “effective dose” means the amount required to produce the therapeutic effect of the disease or condition as described herein. In some examples of this disclosure, the term “effective dose” means the amount required to produce the change associated with the disease or condition as described herein. The effective dose may vary depending on the disease or condition to be treated or the factors to be modified, depending on body weight, age, racial background, sex, health status, and / or physical condition, as well as other factors related to the mammal being treated. Typically, the effective dose falls within a relatively broad range (e.g., “dosage” range) that can be determined through routine trial and error by healthcare professionals. Therefore, the term should not be construed as limiting this disclosure to a specific amount. The effective dose may be a single dose, or a single dose may be administered once or multiple times during the treatment period.

[0156] "Therapeutic dose" refers to the minimum concentration required to produce a measurable improvement in a particular disease or condition. The therapeutic dose as defined herein may vary depending on factors such as the disease stage, the patient's age, sex, and weight, and the ability of the VLPs disclosed herein to induce the desired response in the individual. The therapeutic dose is also the amount at which any toxic or adverse effects of the VLP are outweighed by the therapeutically beneficial effects.

[0157] As used herein, the term “preventive effective dose” should be understood to mean a VLP of the present disclosure that is sufficient to prevent, inhibit, or delay the onset of one or more detectable symptoms of a disease or disorder.

[0158] "Subject" can be an animal susceptible to an infectious disease within the scope of this disclosure. The subject of this disclosure can be a mammal, and in certain embodiments, a human, who may be an infant, child, adult, or elderly. "Subject at risk of infection" is any subject that may be exposed to, or has been exposed to, an infectious disease such as SARS-CoV-2, influenza, or RSV. A subject may be a primary contact of an individual diagnosed with the relevant infectious disease. "Subject" includes any human or non-human animal. Therefore, in addition to their usefulness in the treatment of humans, the compounds of this disclosure may also be useful in the veterinary treatment of mammals, including, but not limited to, companion animals and livestock such as dogs, cats, horses, cattle, sheep, and pigs.

[0159] As used herein, the terms “virus-like particle,” “VLP,” “virus-like particle (plural),” or “VLPs (plural)” should be understood to mean a multi-subunit protein- and lipid-based structure that resembles the morphology and / or size of a virus particle but does not contain the genetic material of the virus. VLPs (plural) exhibit an antigen that presents a conformational epitope that elicits an immune response from T cells and / or B cells, but is incapable of replicating host cells and / or infecting host cells. For example, the VLPs of this disclosure include one or more antigens described herein that are suitable for use as a vaccine.

[0160] Virus-like particles Virus-like particles (VLPs) are particles that resemble viruses but do not contain viral nucleic acids and are therefore non-infectious. They generally contain one or more viral capsid proteins or envelope proteins that are capable of self-assembling to form a VLP. VLPs have been produced from components of a wide variety of virological families (Noad and Roy (2003), Trends in Microbiology, 11:438-444; Grugacic et al., (2006), Methods, 40:60-65). Several VLPs have been approved as therapeutic vaccines, such as Engerix-B (for hepatitis B), Cervarix, and Gardasil (for human papillomavirus).

[0161] Where different types of capsid fusion proteins are envisioned, such capsid fusion proteins may be contained within a single VLP or a number of VLPs. Those skilled in the art will understand that VLPs can be synthesized through the individual expression of viral structural proteins, which can then self-assemble into a virus-like structure. Recombinant VLPs can be prepared using combinations of structural capsid proteins derived from different viruses. In addition, antigens or their immunogenic fragments can be fused to the surface of VLPs. By means of non-limiting examples, the antigens or their immunogenic fragments of the disclosure may be conjugated to VLPs using a SpyCatcher-SpyTag system (as described by Brune, Biswas, and Howarth).

[0162] Capsid fusion proteins may further contain signal sequences for targeting the fusion protein to a specific site within the host cell (e.g., ER, chloroplasts) or for directing the extracellular secretion of the fusion protein. If the signal peptide is absent, this results in translation of the protein in the cytoplasm. Any signal sequence suitable for the host cell may be used, or it may be omitted. Signal sequences have been found to be conserved across phyla and kingdoms, and generally, almost any signal sequence can be used. See Bennett and Scheller, PNAS 90:2559-2563, 1993; Luirink and Sinning, Biochim. Biophys. Acta 1694:17-35, 2005; Doudna and Batey, Ann. Rev. Biochem. 73:539-557, 2004; Stern, et al., Trends in Cell and Mol. Biol. 2:1-17, 2007.

[0163] In one example, the fusion protein of the Disclosure comprises the antigen peptide and TM peptide domain of the Disclosure, as well as one or more of the following: hepatitis B surface antigen (HBSAg), human papillomavirus (HPV) 18 L1 protein, HPV 16 L1 protein, and / or hepatitis E P239, preferably the hepatitis B surface antigen. In one example, the one or more fusion proteins may take the form of a VLP. This is because, although not theoretically bound, HPSAg, HPV 18 L1 protein, HPB 16 L1 protein, and hepatitis E P239 protein are known to spontaneously form VLPs during recombinant expression, and this structure is maintained when HPSAg, HPV 18 L1 protein, HPB 16 L1 protein, and / or hepatitis P239 protein exist in the form of a fusion protein combined with the antigen and TM domain of the Disclosure.

[0164] Capsid fusion protein This disclosure provides recombinant virus-like particles (VLPs) comprising a capsid fusion protein and a lipid bilayer, wherein the capsid fusion protein is (a) Capsid proteins derived from non-enveloped viruses, (b) Transmembrane (TM) protein domain and (c) Antigen protein, and, The capsid protein is encapsulated within the lipid bilayer.

[0165] Those skilled in the art will understand that the capsid fusion proteins for use in this disclosure are capable of self-assembling into recombinant VLPs during expression in suitable cells such as CHO cells or HEK-293 cells. During self-assembly, the lipid bilayer encapsulating the capsid protein is formed from the host cell. In this embodiment of the disclosure, the VLP provides a more controlled immunogenic response by causing the immune response to be induced against the antigen rather than the capsid protein of the capsid fusion protein.

[0166] In one embodiment, the capsid fusion protein includes a capsid protein derived from alfalfa mosaic virus (AMV), a capsid protein derived from bacteriophage AP205, or a capsid protein derived from bacteriophage MS2, with a transmembrane protein domain and antigen linked by a linker, as shown in the schematic diagram provided in Figure 1. In one example, the antigen and TM protein domains are derived from the same virus. For example, the antigen and TM protein domains are derived from the S protein of SARS-CoV-2. In another example, the antigen and TM protein domains are derived from the F protein or Pre-F protein of RSV. In yet another example, the antigen and TM protein domains are derived from the HA or NA protein of influenza. In one example, the antigen and TM protein domains are derived from different viruses. In this embodiment, it is assumed that an immunogenic response is induced as a result of antigen expression. In one example, the antigen is derived from the S protein of SARS-CoV-2, and the TM protein domain is derived from the F protein or Pre-F protein of RSV. In another example, the antigen is derived from the HA or NA protein of influenza, and the TM protein domain is derived from RSV. In yet another example, the antigen is derived from the S protein of SARS-CoV-2, and the TM protein domain is derived from the HA or NA protein of influenza.

[0167] In another example, a capsid fusion protein that is particularly useful, but not limited to, for use in this disclosure is illustrated in Figure 2. 1. AMV004, which includes a capsid protein derived from alfalfa mosaic virus (AMV), a spike (S) protein derived from SARS-CoV-2 containing the TM protein domain and antigen, and a signal peptide, 2. AP003, which includes a capsid protein derived from bacteriophage AP205, a SARS-CoV-2-derived spike (S) protein containing the TM protein domain and antigen, and a signal peptide, 3. AP007, which includes a capsid protein dimer derived from bacteriophage AP205, a SARS-CoV-2 spike (S) protein containing the TM protein domain and antigen, and a signal peptide, 4. MS003, which includes a capsid protein derived from bacteriophage MS2, a SARS-CoV-2-derived spike (S) protein containing the TM protein domain and antigen, and a signal peptide, 5. MS007 comprises a capsid protein dimer derived from bacteriophage MS2, a SARS-CoV-2 spike (S) protein containing the TM protein domain and antigen, and a signal peptide.

[0168] In any of the capsid fusion proteins listed above, the TM protein domain and antigen can be linked to the capsid protein by a linker amino acid sequence.

[0169] Capsid protein The capsid protein for use in the capsid fusion protein of this disclosure may be any suitable capsid protein derived from a non-enveloped virus, as understood by those skilled in the art. Non-enveloped viruses include norovirus, enterovirus, adenovirus, and rhinovirus. While non-enveloped viruses are understood in the art to be non-contained lipid membranes, a lipid bilayer membrane is formed from the host cell expressing the VLP during the expression of the VLP in a suitable cell according to this disclosure. Suitable capsid proteins derived from non-enveloped viruses include alfalfa mosaic virus (AMV), bacteriophage AP205, or bacteriophage MS2.

[0170] AMV capsid proteins are examples of non-enveloped viruses suitable for use in this disclosure and are porous particles that provide efficient binding of antigens to the N-terminus.

[0171] The capsid protein of bacteriophage AP205 is another non-enveloped virus suitable for use in this disclosure, and includes exposed, closely spaced N-terminus and C-terminus. This structural configuration provides easy antigen addition.

[0172] The bacteriophage MS2 capsid protein is another non-enveloped virus suitable for use in this disclosure, and includes exposed, closely spaced N-terminus and C-terminus. Its triple symmetry is ideal for the addition of trimer antigens.

[0173] Transmembrane (TM) protein domain In one embodiment, the capsid fusion protein of this disclosure includes a transmembrane domain. The terms “TM domain,” “transmembrane domain,” and “transmembrane protein domain” are used interchangeably and refer to a protein sequence that spans the lipid bilayer of the VLP. The TM protein domain may be derived from any protein containing any of those described herein or known in the art. Methods for identifying the TM of a protein are known in the art (Elofsson et al. (2007) Annu. Rev. Biochem. 76:125-140, Bemsel et al. (2005) Protein Science 14:1723-1728).

[0174] The transmembrane domain may originate from either a natural or recombinant source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein.

[0175] In one example, the TM protein domain is the TM protein domain of the S protein derived from SARS-CoV-2, and the antigen protein is the HA or NA protein of influenza A virus. In another example, the TM protein domain is the TM domain protein of the HA or TM domain protein of the NA of influenza A virus, and the antigen protein is the S protein derived from SARS-CoV-2. In one embodiment, the S protein, HA protein, or NA protein is selected from those described herein or those known in the art.

[0176] In another example, the TM protein domain is the TM protein domain of the S protein derived from SARS-CoV-2, and the antigen protein is the F protein or Pre-F protein of RSV. In yet another example, the TM protein domain is the TM protein domain of the F or Pre-F protein of RSV, and the antigen protein is the S protein derived from SARS-CoV-2. In one embodiment, the S protein, F protein, or Pre-F protein is selected from those described herein or those known in the art.

[0177] In another example, the TM protein domain is the TM protein domain of RSV's F or Pre-F protein, and the antigen protein is the HA protein or NA protein of influenza A virus. In yet another example, the TM protein domain is the TM protein domain of the HA protein or NA protein of influenza A virus, and the antigen protein is the F or Pre-F protein of RSV. In one embodiment, the F or Pre-F protein or the F or Pre-F protein is selected from those described herein or those known in the art.

[0178] The TM domain may include one or more additional amino acids adjacent to the transmembrane domain, for example, one or more amino acids associated with the extracellular domain of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids from the extracellular domain) and / or one or more additional amino acids associated with the intracellular domain of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids from the intracellular domain).

[0179] In some cases, transmembrane protein domains can attach to the extracellular region of a chimeric polypeptide via hinges, such as human protein-derived hinges. For example, in one embodiment, the hinge may be a human Ig (immunoglobulin) hinge, such as an IgG4 hinge or a CD8α hinge.

[0180] Those skilled in the art will understand that the hinge region is a flexible amino acid extension in the central part of the heavy chain of IgG and IgA immunoglobulin classes, distinct from the spacer region and linker region, and that it links these two chains by a disulfide bond. In particular, the hinge region forms a flexible linker between the Fab arm and the Fc portion of a given antibody. It will also be understood that the length and flexibility of the hinge region can vary widely among IgG subclasses, and that those skilled in the art can determine a hinge suitable for use in the recombinant VLP described herein.

[0181] The capsid fusion proteins of this disclosure may include a linker (hereinafter interchangeably referred to as a linker peptide, spacer, or spacer peptide). The linker may be used to link two or more functional domains of the fusion protein of this disclosure. For example, the linker may link the capsid protein to a TM protein domain. In another example, particularly when the TM protein domain and antigen are derived from different viruses (e.g., the S protein of SARS-CoV-2 and the F or Pre-F protein of RSV), the TM protein domain may be linked to the antigen by the linker. The use of linkers in fusion proteins is commonplace in the art, and any conventional linker protein may be used in the fusion proteins of this disclosure, provided that the resulting fusion protein retains the desired functional properties of the antigen.

[0182] The linker may be a short peptide with a length of up to about 30 amino acids, for example, about 5-30 amino acids, about 5-25 amino acids, about 5-20 amino acids, about 10-20 amino acids, about 5-15 amino acids, or about 10-15 amino acids. In some embodiments, the linker is about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, or about 20 amino acids.

[0183] Rigid linker In one example, a rigid linker may be used in the fusion protein of this disclosure. Rigid linkers are conventionally used when it is necessary to maintain a certain distance between different domains / parts of a fusion protein and preserve their independent functions. Rigid linkers may also be used when spatial separation of fusion protein domains is important for maintaining the stability or biological activity of the fusion protein. An empirical rigid linker having the sequence A(EAAAK)nA(n=2~5) exhibits an α-helix conformation, the α-helix structure being stabilized by a Glu-Lys+ salt bridge. A non-limiting example of a rigid linker is EAAAKEAAAKEAAAK (also referred to as EAAAKh). Rigid linkers may be used for the expression of the fusion protein of this disclosure in mammalian cells such as HEK293 cells.

[0184] In some embodiments, flexible linkers may be used in the fusion proteins of this disclosure. Flexible linkers are used conventionally when the domains to be linked require a certain degree of mobility or interaction. Flexible linkers typically contain or consist of small amino acid residues such as glycine, threonine, arginine, serine, asparagine, glutamine, alanine, aspartic acid, proline, glutamic acid, lysine, leucine, and / or valine, particularly glycine, serine, alanine, leucine, and / or valine. Flexible linkers containing or consisting of glycine, serine, and / or alanine are preferred, with glycine and serine being particularly preferred. Thus, the most commonly used flexible linkers have sequences consisting mainly of elongations of Gly and Ser residues ("GS" linkers), and such flexible linkers contain the sequence (Gly-Gly-Gly-Gly-Ser)n. Non-limiting examples of GS linkers include GS5, GS 10 GS 15 GS 20 , and GS 25 It includes.

[0185] antigen Suitable antigens for use in the VLPs and compositions described herein will be apparent to those skilled in the art. For example, the antigen may be the spike (S) protein from SARS-CoV-2, the F or Pre-F protein from RSV, or the HA or NA protein from influenza.

[0186] SARS-CoV-2 antigen In one example, the present disclosure provides a VLP comprising a capsid fusion protein containing an antigen derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as the spike (S) protein. Thus, in the context of the present disclosure, the antigen is a pathogenic antigen. For example, the antigen of interest is an antigenic protein, immunogenic fragment, and / or variant thereof that can induce an immune response in a subject.

[0187] The SARS-CoV-2 genome encodes at least four major structural proteins: the spike (S) protein, the membrane (M) protein, the envelope (E) protein, the nucleocapsid (N) protein, and other accessory proteins that assist the replication process and facilitate entry into cells. The M protein is the most abundant component of the viral envelope and directs the assembly of the coronavirus through its interaction with all other structural proteins. The E protein is a small membrane protein or biloporin that is thought to facilitate the budding of the viral particle by plucking the cell membrane surface. The S protein is a class I fusion protein that mediates the attachment of SARS-CoV-2 to human angiotensin-converting enzyme 2 (ACE2), the primary cell surface receptor. Due to its exposed conformation on the surface of the virus, the S protein is highly immunogenic and is the main focus of current vaccine development. The N protein packages the RNA genome to form a nucleocapsid and, while not necessarily required for envelope formation, appears to play a crucial role in complete virion assembly and stability, as well as enhancing VLP yield.

[0188] The S protein contains three domains: (i) a large ectodomain, (ii) a transmembrane protein domain (which crosses the viral envelope once), and (iii) a short intracellular tail. The ectodomain consists of a trimer stalk made up of three receptor-binding subunits (3×S1) and three membrane-fusion subunits (3×S2). Thus, the SARS-CoV-2 S protein is homotrimer. During viral entry, S1 binds to receptors on the host cell surface to allow the virus to attach, and S2 fuses the host membrane and the viral membrane, enabling the viral genome to enter the host cell. Receptor binding and membrane fusion are crucial early steps in the coronavirus infection cycle. There is remarkable diversity in the receptors targeted by different CoVs.

[0189] The structure of the SARS-CoV-2 S protein is described, for example, in Cai et al. (Science (2020) 369:1586-1592), which is incorporated herein by reference in its entirety. Each S1 subunit of the SARS-CoV-2 S protein contains an N-terminal domain (NTD), a receptor-binding domain (RBD), and two C-terminal domains (CTD). Prior to fusion with the host cell membrane, the S1 subunit of the SARS-CoV-2 S protein protects the S2 subunit. Upon binding to ACE2, the SARS-CoV-2 S protein refolds in a "jackknife" manner, forming a long, coiled coil at its center, ultimately leading to membrane fusion and viral entry into the host cell.

[0190] Given the tendency of RNA viruses such as SARS-CoV-2 to mutate, the present inventors provide a VLP containing an S protein that may contain mutations found in different strains of SARS-CoV-2, thereby enabling the vaccine composition to find specific utility in the treatment of target strains of SARS-CoV-2, including the Omicron strain of SARS-CoV-2.

[0191] The VLPs, compositions, and vaccines of this disclosure may also be useful in the treatment of variants of SARS-CoV-2, including B.1.1.7 SARS-CoV-2 strain (also known as 201 / 501Y.V1, which was first detected in the UK and is now known as the alpha variant), B.1.351 SARS-CoV-2 strain (also known as 20H / 501.V2, which was first detected in South Africa and is now known as the beta variant), P.1 SARS-CoV-2 strain (also known as 20J / 501Y.V3, which was first detected in Japan and Brazil and is now known as the gamma variant), B.1.427 and B.1.429 SARS-CoV-2 strains (first detected in California and is now known as the epsilon variant), and / or B.1.617.2 SARS-CoV-2 strain (first detected in India and is now known as the delta variant). The VLPs, compositions, and vaccines of this disclosure may also be useful in the treatment of the Wuhan (original) strain of SARS-CoV-2.

[0192] According to the CDC (SARS-CoV-2 Variant Classifications and Definitions (cdc.govl)), the alpha variant includes the following mutations in the S protein: deletion of residues 69, 70, and 144, (E484K*), (S494P*), N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, and (K1191N*). The major mutations found are deletions of residues 69 / 70 and 144Y, as well as substitutions of N501Y, A570D, D614G, and P681H. The beta variant includes the following mutations: D80A, D215G, 241 deletion, 242 deletion, 243 deletion, K417N, E484K, N501Y, D614G, and A701V, with the major mutations being the substitutions of K417N, E484K, N501Y, and D614G. The gamma variant includes the following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T10271, with the major mutations being E484K, K417N / T, N501Y, and D614G. The delta variant includes the following mutations: T19R, (G142D*), 156 deletion, 157 deletion, R158G, L452R, T478K, D614G, P681R, and D950N, with the major mutations being L452R, E484Q, and T478K. The epsilon variant includes the following mutations: S131, W152C, 30 L452R, and D614G, with the major mutation being L452R. Therefore, this disclosure encompasses VLPs containing S protein-derived antigens that include one or more or all of the above mutations.

[0193] In one example, the VLP contains antigens derived from the S, M, and E proteins of the SARS-CoV-2 Omicron strain. In another example, the VLP contains antigens derived from the S and M proteins of the SARS-CoV-2 Omicron strain. In yet another example, the VLP contains antigens derived from the S and E proteins of the SARS-CoV-2 Omicron strain. In yet another example, the VLP contains antigens derived from the E and M proteins of the SARS-CoV-2 Omicron strain. In one example, the Omicron variant may be BA.1 or BA.2.

[0194] influenza Influenza, also known as "flu," is an infectious disease caused by the influenza virus. It will be apparent to those skilled in the art that there are currently four types of influenza viruses: A, B, C, and D. Influenza A is the most common influenza virus, infecting humans, animals, and birds, while influenza B infections primarily occur in humans. Influenza C infections do not cause any serious symptoms in humans or mammals, and influenza D has so far infected only pigs and cattle.

[0195] In one example of a VLP in this disclosure, the antigen is derived from an influenza A virus strain. For example, the antigen is the influenza A virus hemagglutinin (HA) protein, neuraminidase (NA) protein, matrix (M) protein, nucleoprotein (NP), non-structural (NS) protein, or an immunogenic fragment or variant thereof. In one example, the antigen is a subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 of the hemagglutinin (HA) of influenza A, and / or a subtype N1, N2, N3, N4, N5, N6, N7, N8, or N9 of the neuraminidase (NA) of influenza A, and / or a subtype M1 or M2 of the matrix (M) protein of influenza A, and / or a subtype NS1 or NS2 of the non-structural (NS) protein of influenza A.

[0196] Those skilled in the art will recognize that pandemic strains of influenza viruses are generally influenza A virus strains of the H1, H2, H3, H5, H6, H7, or H9 subtypes. For example, H1N1, H2N2, H3N2, H5N1, H5N3, H6N1, H7N2, H7N3, H7N7, H7N9, and H9N2 strains. In one example, the antigen is the H1N1 antigen derived from the A / Delaware / 55 / 2019 virus strain. In another example, the antigen is the H1 antigen derived from the A / Delaware / 55 / 2019 virus strain.

[0197] In one example, the antigen is an influenza A virus strain of subtype H1, H2, H3, H5, H6, H7, or H9. For example, the antigen is H1 hemagglutinin, or H2 hemagglutinin, or H3 hemagglutinin, or H5 hemagglutinin, or H6 hemagglutinin, or H7 hemagglutinin, or H9 hemagglutinin. For example, the antigen is an influenza A virus strain of subtype H5 (i.e., H5 hemagglutinin). In one example, H5 hemagglutinin is the A / turkey / Turkey / 1 / 2005 virus strain. In one example, H3 hemagglutinin is the A / Delaware / 39 / 2019 virus strain.

[0198] In one example, the antigen is an influenza A virus strain of subtype N1, N2, N3, N7, or N9. For example, the antigen is N1 neuraminidase, or N2 neuraminidase, or N3 neuraminidase, or N7 neuraminidase, or N9 neuraminidase. For example, the antigen is an influenza A virus strain of subtype N1 neuraminidase. In one example, N1 neuraminidase is the A / turkey / Turkey / 1 / 2005 strain. In one example, N2 neuraminidase is the A / Delaware / 39 / 2019 virus strain.

[0199] In one example, the antigen is a strain of influenza B virus. Those skilled in the art will recognize that influenza B viruses are not classified into subtypes, but rather into two lineages, namely B / Yamagata and B / Victoria.

[0200] In one example, the antigen is a B / Yamagata influenza B virus strain. For example, the influenza B virus strain is the B / Singapore / INFTT 16 0610 / 16(By) virus strain. In another example, the antigen is derived from a B / Victoria influenza B virus strain.

[0201] Respiratory syncytial virus (RSV) RSV is an enveloped, unsegmented, negative-strand RNA virus belonging to the genus Pneumonia Virus in the family Paramyxoviridae. Like other enveloped viruses such as influenza viruses, paramyxoviruses like RSV require fusion of the viral membrane with the host cell membrane to infect host cells. In the case of RSV, a conserved fusion protein (RSV-F glycoprotein) fuses the viral membrane with the cell membrane by linking irreversible protein refolding with membrane juxtaposition. Based on paramyxovirus research, the RSV-F protein initially folds into a metastable pre-fusion conformation. Upon cell entry, the pre-fusion conformation undergoes refolding and conformational changes to transition to its stable post-fusion conformation.

[0202] In one example, the antigen is derived from RSV. For instance, the antigen is an RSV surface glycoprotein selected from the fusion (F) protein, glycoprotein (G), small hydrophobic protein (SH), matrix proteins M and M2, nucleocapsid proteins N, P, and L, and non-structural proteins NS1 and NS2. In a particular example, the antigen is the RSV-F antigen. In another example, the antigen is derived from the pre-fusion (Pre-F) protein of RSV. The F protein presents two distinct conformations: the lollipop-shaped Pre-F, which exists on the viral surface before the interaction between the virus and the cell, and the crutch-shaped post-fusion (Post-F) state, which is acquired by an unknown mechanism that spontaneously initiates rearrangement from the highly metastable pre-F to the energetically favorable post-F conformation after the fusion of the viral membrane with the cell membrane or from the highly metastable pre-F to the post-F conformation. These two forms are antigenically distinct and both are considered potential vaccine candidates.

[0203] The RSV F glycoprotein is a type I single-pass endogenous membrane protein with four common domains: an N-terminal ER transition signal sequence (SS), an ectodomain (ED), a transmembrane protein domain (TM), and a cytoplasmic tail (CT). The CT contains a single palmitoylated cysteine ​​residue. The F protein sequence is highly conserved among RSV isolates but evolves over time. Unlike most paramyxoviruses, the F protein in RSV can mediate entry and syncytial formation independently of other viral proteins (other paramyxoviruses typically require HN in addition to F).

[0204] The RSV-F glycoprotein is translated from mRNA into a protein of approximately 574 amino acids called F0. Post-translational processing of F0 includes removal of the N-terminal signal peptide by signal peptidases in the endoplasmic reticulum. F0 is also cleaved in the trans-Golgi apparatus at two sites (approximately 109 / 110 and approximately 136 / 137) by cellular proteases (particularly furin). This cleavage results in the removal of a short intervening sequence, generating two subunits called F1 (approximately 50 kDa, C-terminus, approximately residues 137-574) and F2 (approximately 20 kDa, N-terminus, approximately residues 1-109), which remain related to each other. F1 contains a hydrophobic fusion peptide at its N-terminus and also contains two amphiphilic heptarepeat regions (HRA and HRB). HRA is located near the fusion peptide, and HRB is located near the transmembrane domain. The three F1-F2 heterodimers assemble within the virion as an F1-F2 homotrimer.

[0205] Suitable RSV-F antigens for inclusion in the VLPs or compositions described herein include RSV-F glycoproteins and RSV-F glycoprotein variants. Suitable RSV-F glycoprotein variants include, for example, cleaved variants such as full-length F protein and soluble ectodomain, each optionally containing one or more mutations, such as furin cleavage mutations, trypsin cleavage mutations, fusion peptide mutations (e.g., whole or partial deletion), mutations that stabilize the HRB trimer, and mutations that destabilize the HRA trimer.

[0206] Full-length RSV-F glycoproteins and cleaved RSV-F glycoproteins, including those having one or more such mutations in various combinations, are well known in the art, for example, disclosed in WO2011 / 008974, which is incorporated herein by reference in its entirety.

[0207] In one example, the compositions described herein include an additional antigen. In one example, the additional antigen is a virus, bacteria, fungus, or protozoan.

[0208] Viral antigens In one example, the VLP of this disclosure further comprises an additional capsid fusion protein and a lipid bilayer, wherein the capsid fusion protein is (a) Capsid proteins derived from non-enveloped viruses, (b) Transmembrane (TM) protein domain and (c) Antigen protein, and, The capsid protein is encapsulated within the lipid bilayer.

[0209] In one example, the additional capsid fusion protein differs from the first capsid fusion protein described herein and contains a different TM protein domain and / or antigen protein. In one example, the additional antigen protein is a viral antigen protein.

[0210] Additional viral antigens are obvious to those skilled in the art and include, for example, orthomyxoviruses (e.g., influenza A, B, and C), paramyxoviridae viruses (pneumonia virus genus (e.g., respiratory syncytial virus (RSV), bovine respiratory syncytial virus, mouse pneumonia virus, and turkey rhinotracheitis virus), paramyxovirus types 1-4 (PIV), mumps, Sendai virus, monkey virus 5), bovine parainfluenza virus, nipah virus, henipavirus, and Newcastle disease virus), poxviridae (e.g., Smallpox virus (including large and small varicella), metapneumovirus (e.g., human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), etc.), morbillivirus (e.g., measles), picornaviridae (e.g., enterovirus, rhinovirus, heparnavirus, parechovirus, cardiovirus and aftovirus), enterovirus (e.g., poliovirus type 1, 2 or 3, coxsackie A virus types 1-22 and 24) Coxsackie B viruses types 1-6, echoviruses (ECHO) types 1-9, 11-27, and 29-34, and enteroviruses 68-71), Bunyaviridae (e.g., California encephalitis virus), Phleboviruses (e.g., Rift Valley fever virus), Nairoviruses (e.g., Crimean-Congo hemorrhagic fever virus), Heparnaviridae (e.g., Hepatitis A virus (HAV)), Togaviridae (e.g., rubella virus, Alphavirus, or Alterivirus), Flaviviridae (e.g., tick-borne encephalitis virus (TBE)). )viruses, dengue (type 1, 2, 3 or 4) virus, yellow fever virus, Japanese encephalitis virus, Kyasanur forest disease virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Poissant encephalitis virus), Pestivirus genus (e.g., bovine viral diarrhea (BVDV), classical swine fever (CSFV) or border disease (BDV)), Hepadnaviridae family (e.g., hepatitis B virus and hepatitis C virus), Rhabdoviridae family (e.g., lyssavirus genus (rabies virus) and becyclovirus genus (VSV)),Caliciviridae (e.g., Norwalk virus and Norwalk-like viruses (e.g., Hawaii virus and Snow Mountain virus)), Coronavirus genus (e.g., Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome (MERS) Coronavirus (MERS-CoV), Avian Infectious Bronchitis Virus (IBV), Mouse Hepatitis Virus (MHV), and Porcine Infectious Gastroenteritis Virus (TGEV)), Retroviridae (e.g., Oncovirus, Lentivirus, or Spumavirus genera), Reoviridae (e.g., Orthoreovirus, Rotau This includes proteins and peptides derived from the genera Illus, Orbivirus, or Cortivirus, Parvoviridae (e.g., Parvovirus B19), Hepatitis Delta virus (HDV), Hepatitis E virus (HEV), Human herpesviruses (e.g., Herpes simplex virus (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human herpesvirus 6 (HHV6), Human herpesvirus 7 (HHV7), and Human herpesvirus 8 (HHV8)), Papovaviridae (e.g., Papillomavirus and Polyomavirus), Adenoviridae, and Arenaviridae.

[0211] In one example, the additional viral antigen is derived from the parainfluenza virus.

[0212] In one example, the additional viral antigens originated from the metapneumonia virus.

[0213] In one example, the additional viral antigen is derived from rhinovirus.

[0214] In one example, the additional viral antigen is derived from a coronavirus.

[0215] In one example, the additional viral antigen is derived from adenovirus.

[0216] In one example, the additional viral antigen is derived from the bocavirus.

[0217] In one example, the additional antigen may be derived from a single strain (i.e., monovalent) of the influenza virus, or from multiple strains (i.e., polyvalent).

[0218] In one example, the additional antigens are influenza A, B, and / or C virus strains.

[0219] In one example, the additional antigen is an influenza A virus strain. For instance, the antigen may be the hemagglutinin (HA) protein, neuraminidase (NA) protein, matrix (M) protein, nucleoprotein (NP), non-structural (NS) protein of the influenza A virus, or an immunogenic fragment or variant thereof. In one example, the additional antigens are subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 of influenza A hemagglutinin (HA), and / or subtypes N1, N2, N3, N4, N5, N6, N7, N8, or N9 of influenza A neuraminidase (NA), and / or subtypes M1 or M2 of the matrix (M) protein of influenza A, and / or subtypes NS1 or NS2 of the non-structural (NS) protein of influenza A.

[0220] Those skilled in the art will recognize that pandemic strains of influenza viruses are generally influenza A virus strains of the H1, H2, H3, H5, H6, H7, or H9 subtypes. For example, H1N1, H2N2, H3N2, H5N1, H5N3, H6N1, H7N2, H7N3, H7N7, H7N9, and H9N2 strains.

[0221] In one example, the additional antigen is derived from an influenza A virus strain of subtype H1, H2, H3, H5, H6, H7, or H9. For example, the additional antigen may be H1 hemagglutinin, or H2 hemagglutinin, or H3 hemagglutinin, or H5 hemagglutinin, or H6 hemagglutinin, or H7 hemagglutinin, or H9 hemagglutinin. For example, the additional antigen may be derived from an influenza A virus strain of subtype H5 (i.e., H5 hemagglutinin). In one example, H5 hemagglutinin is from the A / turkey / Turkey / 1 / 2005 virus strain. In another example, H3 hemagglutinin is from the A / Delaware / 39 / 2019 virus strain.

[0222] In one example, the additional antigen is an influenza A virus strain of subtype N1, N2, N3, N7, or N9. For example, the additional antigen is N1 neuraminidase, or N2 neuraminidase, or N3 neuraminidase, or N7 neuraminidase, or N9 neuraminidase. For example, the additional antigen is an influenza A virus strain of subtype N1 neuraminidase. In one example, N1 neuraminidase is the A / turkey / Turkey / 1 / 2005 strain. In one example, N2 neuraminidase is the A / Delaware / 39 / 2019 virus strain.

[0223] Infections such as influenza and coronavirus infections are major causes of ARDS. Therefore, in one example of this disclosure, ARDS is associated with influenza, RSV, or SARS-CoV-2 infection. In one example, ARDS is associated with SARS-CoV-2 infection. Therefore, those skilled in the art will understand that antigens targeting SARS-CoV-2 infection or influenza, including those listed above, may be suitable antigens for the treatment of ARDS.

[0224] bacterial antigen In one example, the additional antigens of this disclosure are bacterial antigens.

[0225] Bacterial antigens are obvious to those skilled in the art, and these include, for example, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Neisseria catalaris, Bordetella pertussis, Burkholderia species (e.g., Bacillus melioides, Bacillus pseudomeroides and Bacillus cepacia), Staphylococcus aureus, Haemophilus influenzae, Neisseria tetanus (tetanus), Clostridium perfringens, Clostridium botulinum, Neisseria diphtheriae (diphtheria), Pseudomonas aeruginosa, Legionella pneumophila, Coxiella bruneti, Brucella species (e.g., B. avoltus, B. canis, B. melitensis, B. neotomae, B. obis, B. suiss, and B. pinipedier), Francisella species (e.g. This includes proteins and peptides derived from *F. nobicida*, *F. phyllomirazia*, and *F. tularemia*, Group B Streptococcus, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum (syphilis), Haemophilus ducray, Streptococcus fecal, Enterococcus faecium, Helicobacter pylori, Staphylococcus saprophyticus, Yersinia enterocolitica, Escherichia coli, Bacillus anthracis (anthrax), Plague bacillus (plague), Mycobacterium tuberculosis, Rickettsia species, Listeria species, Chlamydia pneumoniae, Vibrio cholerae, Salmonella typhi (typhoid fever), Borrelia borrelia (Lyme disease), Porphyromonas species, and Klebsiella species.

[0226] fungal antigen In one example, the additional antigens of this disclosure are fungal antigens.

[0227] Fungal antigens that can be encoded by RNA or provided in polypeptide form pursuant to this disclosure will be apparent to those skilled in the art, and these include, for example, dermatophytes (Epidermophyton floccosum, Microsporum oozani, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypseum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton Trichophyton galine, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton cinceanum, Trichophyton rubrum, Trichophyton schoenreini, Trichophyton tonsurans, Trichophyton belcosum, T. belcosum album varietal, T. discoides varietal, T. ocraceim varietal, Trichophyton violaceum, and / or Trichophyton fabiforme), Aspergillus fumigatus, A Spergillus flavus, Aspergillus niger, Aspergillus nidurans, Aspergillus teleus, Aspergillus sidwi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida crusei, Candida parapsis, Candida stellatoidea, Candida kusei, Candida paraquesei, Candida This includes proteins and peptides derived from *Dida lucitani*, *Candida pseudotropicalis*, *Candida gilliermondii*, *Cladosporum carionii*, *Coccidioides imitis*, *Blastomyces dermatichidis*, *Cryptococcus neoformans*, *Geotrichum clavatum*, *Histoplasma capsulatum*, *Klebsiella pneumoniae*, microsporids, *Encephalitozoon*, *Septata intestinalis*, and *Enterocitozoon vienusi*.

[0228] Protozoan antigens In one example, the additional antigens of this disclosure are protozoan antigens.

[0229] Protozoan antigens are obvious to those skilled in the art and include, for example, proteins and peptides derived from Entamoeba histolytica, Giardia lamblia, Cryptosporidium nigricans, Cyclospora caietanensis, and Toxoplasma.

[0230] Polynucleotides This disclosure also provides polynucleotides suitable for encoding the VLPs disclosed herein and forming compositions or vaccines for treating infectious diseases such as viral infections, when expressed under sufficient conditions. The term polynucleotide encompasses both DNA sequences and RNA sequences. Hereinafter, the terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably. Thus, antigens derived from SARS-CoV-2, influenza, or RSV, as well as capsid fusion proteins, may be encoded or expressed by DNA or RNA contained within one or more expression cassettes or expression vectors.

[0231] As a non-limiting example, if a VLP contains different capsid fusion proteins containing different protein antigens (e.g., those derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and those derived from RSV), one or more of these antigens may be expressed by monocistronic polynucleotides, or each of these antigens may be expressed by polycistronic polynucleotides.

[0232] One or more polynucleotides (e.g., DNA or RNA) encoding a capsid fusion protein may be optimized for expression within a cell. As used herein, the term “optimized” includes, with respect to the optimization of the expression of a capsid fusion protein, both codon optimization (in terms of both nucleic acid sequence and other modifications) and / or other modifications to the polynucleotide, which increase the expression level and / or duration of the capsid fusion protein derived from said polynucleotide within the cell.

[0233] One or more polynucleotides (e.g., DNA or RNA) according to this disclosure may be included in an expression vector, thereby promoting the expression of a capsid fusion protein. Typically, in such expression structure, the one or more polynucleotides are operably ligated to a suitable promoter(s). One or more polynucleotides may be ligated to a suitable terminator(s) sequence(s). One or more polynucleotides may also be ligated to both a promoter(s) and a terminator(s). Suitable promoter and terminator sequences are well known in the art.

[0234] One or more polynucleotides (e.g., DNA or RNA) encoding a capsid fusion protein may additionally include a leader sequence(s). Any suitable leader sequence may be used, including conventional leader sequences known in the art. Suitable leader sequences include human tissue plasminogen activator leader sequences (tPAs), which are routinely used in viral and DNA-based vaccines, as well as for protein vaccines, to aid in secretion from mammalian cells.

[0235] One or more viral vectors, expression vectors, or DNA vectors (or DNA plasmids) may comprise one or more polynucleotides encoding the capsid fusion proteins described herein. Preferably, the one or more viral vectors or DNA vectors (or DNA plasmids) encode at least one antigen described herein. Multiple capsid fusion proteins may be expressed by a single viral vector or DNA vector (or DNA plasmid), by multiple viral vectors or DNA vectors (or DNA plasmids), or by a combination thereof. In a non-limiting example, if different capsid fusion proteins are envisioned, these capsid fusion proteins may be expressed by a single viral vector or DNA vector (or DNA plasmid), or each capsid fusion protein may be expressed by a separate viral vector or DNA vector (or DNA plasmid).

[0236] One or more vectors may be DNA vectors, such as DNA plasmids. One or more vectors may be RNA vectors, such as mRNA vectors or self-amplifying RNA vectors. One or more DNA vectors and / or RNA vectors of this disclosure are typically expressible in eukaryotic cells, in particular in any host cell type described herein.

[0237] Typically, DNA vectors and / or RNA vectors can be expressed in human cells, E. coli cells, or yeast cells. One or more vectors may be phage vectors, such as the AAV / phage hybrid vector described in Hajitou et al., Cell 2006;125(2)pp.385-398, which is incorporated herein by reference.

[0238] The nucleic acid molecules and vectors of this disclosure can be prepared using any suitable process known in the art. Therefore, nucleic acid molecules can be produced using chemical synthesis techniques. Alternatively, the nucleic acid molecules and vectors of this disclosure may be prepared using molecular biological techniques.

[0239] Method of production Preferred methods for producing the VLPs of this disclosure are obvious to those skilled in the art and / or are described herein.

[0240] Typically, plasmid DNA is produced by inserting a polynucleotide sequence encoding at least one antigen into a DNA vector. Suitable DNA vectors for use are obvious to those skilled in the art, and the polynucleotide sequences of this disclosure can be purchased from any commercial supplier. Insertion of a nucleotide sequence(s) into a DNA vector can be carried out using methods standard in the art.

[0241] In one example, the protein antigens described herein are produced using plasmid DNA. Those skilled in the art will understand that plasmid DNA is relatively stable. Briefly, viable bacterial cells (e.g., E. coli) are transformed with a DNA plasmid encoding the protein antigen described herein. Individual bacterial colonies are isolated, and the resulting plasmid DNA is amplified in an E. coli culture.

[0242] In one example, plasmid DNA is isolated after fermentation. For example, plasmid DNA is isolated using a commercially available kit (e.g., Maxiprep DNA kit) or other common methods known to those skilled in the art. After isolation, plasmid DNA is linearized by restriction digestion (i.e., using restriction enzymes). The restriction enzymes are removed using methods known in the art, including, for example, phenol extraction / chloroform extraction and ethanol precipitation.

[0243] composition This disclosure provides immunogenic compositions, including the VLPs of this disclosure. In one example, the immunogenic composition is a vaccine. This disclosure also provides pharmaceutical compositions, including the immunogenic compositions of this disclosure and pharmaceutically acceptable carriers.

[0244] In one example, the VLP described herein may be administered in a composition comprising an adjuvant for enhancing immunogenicity. In one example, the adjuvant is selected from the group consisting of Freund's adjuvant, incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, GMCSP, BCG, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, monophosphoryl lipid A (MPL), RIBI, MPL, trehalose dimicholate (TDM), Novasomes®, QS21, Quil A (and its derivatives and components), calcium phosphate, calcium hydroxide, zinc hydroxide, MHC antigens, PolyI:C, MF59, glycolipid analogs, octodecyl esters of amino acids, muramyl dipeptides, polyphosphazenes, lipoproteins, ISCOM matrix, DC-Chol, ODA, cytokines, and other adjuvants and their derivatives. In one example, the adjuvant is MF59. In one example, MF59 is administered concurrently with the administration of the VLP, composition, or vaccine of the Disclosure. In another example, MF59 is administered sequentially, prior to, or following the administration of the VLP, composition, or vaccine of the Disclosure.

[0245] It will be apparent to those skilled in the art, and / or as described herein, that the VLPs of this disclosure may exist as VLPs or in combination with lipids, polymers, or other delivery systems that facilitate entry into cells.

[0246] Delivery system In one example, the pharmaceutical composition of the present disclosure further comprises lipid nanoparticles (LNPs) and / or polymer microparticles. For example, VLPs are encapsulated within the LNPs and / or polymer microparticles, bound to the LNPs and / or polymer microparticles, or adsorbed onto the LNPs and / or polymer microparticles.

[0247] Lipid nanoparticles In one example, the pharmaceutical composition of this disclosure further comprises LNP.

[0248] It should be understood that the terms “lipid nanoparticles” or “LNPs” include, but are not limited to, any lipid composition, including liposomes or vesicles in which aqueous volume is encapsulated by an amphiphilic lipid bilayer (e.g., monolayer, being single, or multilayer, being multiple); micelle-like lipid nanoparticles having a non-aqueous core; and solid lipid nanoparticles. Methods for preparing LNPs are known to those skilled in the art and / or are described herein. In one example, LNPs are prepared using a staggered herringbone mixer, as described, for example, in U.S. Patent Application No. 20120276209. In another example, liposomes are prepared using a microfluidic device, as described, for example, in WO2018220553.

[0249] Lipid nanoparticles suitable for use in this disclosure will be obvious to those skilled in the art and / or are described herein. For example, LNPs include ionizable lipids.

[0250] As used herein, the terms "ionizable lipid" or "ionizable lipid(s)" refer to lipids having at least one protonable or deprotonable group. For example, lipids are positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH (e.g., above physiological pH). For example, lipids are cationic lipids.

[0251] Suitable ionizable lipids may have anionic, cationic, or zwitterionic hydrophilic head groups. Exemplary phospholipids (anionic or zwitterionic) for use in this disclosure include, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. In one example, the lipid is a cationic lipid. Examples of cationic lipids include, but are not limited to, dioleoyltrimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and 2,5-bis((9Z,12Z)-octadeca-9,12-diene-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750). In one example, the phospholipid is 2,5-bis((9Z,12Z)-octadeca-9,12-diene-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and dodecylphosphocholine. Lipids can be saturated or unsaturated.

[0252] Suitable lipid moieties for use in LNPs are obvious to those skilled in the art, and these include, for example, fatty acids, isoprenoids, and combinations thereof. In one example, the lipid moiety is selected from the group consisting of isoprenoids, triglycerides, phospholipids, cholesteryl esters, and combinations thereof.

[0253] In one example, the lipid nanoparticles further include PEG lipids, sterol-structured lipids, and / or neutral lipids. In another example, the lipid nanoparticles do not contain cationic lipids.

[0254] PEG-lipids In one example, this disclosure provides LNPs containing PEGylated lipids.

[0255] It will be apparent to those skilled in the art that PEGylated lipids refer to lipids modified with polyethylene glycol. Exemplary PEGylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, PEG lipids include PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and combinations thereof.

[0256] neutral lipid In one example, this disclosure provides LNPs containing neutral lipids.

[0257] Suitable neutral or zwitterionic lipids for use in this disclosure will be apparent to those skilled in the art, and include, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 dietherPC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difytanol-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero This includes 3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. The lipids can be saturated or unsaturated.

[0258] structural lipids In one example, this disclosure provides LNPs containing structural lipids.

[0259] Exemplary structural lipids include, but are not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatin, ursolic acid, and alpha-tocopherol.

[0260] In one example, the structural lipid is a sterol. For example, the structural lipid is cholesterol. In another example, the structural lipid is campesterol.

[0261] Polymer microparticles In one example, the pharmaceutical composition of the present disclosure further includes polymer microparticles.

[0262] Those skilled in the art will recognize that various polymers can form microparticles and thereby encapsulate or adsorb the protein antigen or VLP of the present disclosure. The use of substantially non-toxic polymers means that the particles are safe, and the use of biodegradable polymers means that long-term persistence can be avoided by the particles being metabolized after delivery. Useful polymers are also sterilizable, thereby assisting in the preparation of pharmaceutical-grade formulations.

[0263] Exemplary non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acid), polyhydroxybutyric acid, polylactones (including polycaprolactone), polydioxanone, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl-pyrrolidone or polyester-amides, and combinations thereof.

[0264] Pharmaceutically acceptable carrier Preferably, in a composition or method for administering the VLP, vaccine, or composition of the present disclosure to a subject, the VLP, vaccine, or composition is combined with a pharmaceutically acceptable carrier, as understood in the art. Thus, one example of the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising the VLP of the present disclosure (and any delivery system, e.g., LNP) combined with a pharmaceutically acceptable carrier.

[0265] Generally, a "carrier" means any solid or liquid filler, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating agent, or lubricant that can be safely administered to any subject, e.g., a human. Depending on the particular route of administration, various acceptable carriers known in the art can be used, as described, for example, in Remington’s Pharmaceutical Sciences (Mack Publishing Co., N.J., USA, 1991).

[0266] The VLP, composition, or vaccine of the present disclosure is useful for parenteral, topical, oral, or local, intramuscular, aerosol, or transdermal administration for prophylactic or therapeutic treatment. In one example, the VLP, composition, or vaccine is administered parenterally, such as intramuscularly, subcutaneously, or intravenously. For example, RNA is administered intramuscularly.

[0267] The formulation of the VLPs, compositions, or vaccines of this disclosure to be administered will vary depending on the selected route of administration and formulation form (e.g., solution, emulsion, capsule). A suitable pharmaceutical composition comprising the VLPs, compositions, or vaccines to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include aqueous / alcoholic aqueous solutions, emulsions, or suspensions, for example, physiological saline and a buffer medium. Parenteral vehicles may include sodium chloride solution, dextrose-added Ringer's solution, dextrose, and sodium chloride, lactated Ringer's solution, or a fixative oil. A variety of suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered physiological saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles may contain additives, preservatives, or fluids, nutrients, or electrolyte replacements (see, for general, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The composition may optionally contain pH adjusters and buffers, as well as pharmaceutically acceptable adjuvants necessary to approximate physiological conditions, such as toxicity modifiers, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. VLPs, compositions, or vaccines may be stored in liquid form or lyophilized for storage and reconstituted in suitable carriers before use according to lyophilization and reconstitution techniques known in the art.

[0268] The optimal concentration of the active ingredient(s) in the selected medium can be determined empirically according to a procedure known to those skilled in the art and will depend on the desired final pharmaceutical formulation.

[0269] When formulated, the compositions of the present disclosure will be administered in a manner compatible with the administered formulation and in a therapeutically / prophylactically effective amount. The dosage range for administering the molecules of the present disclosure is large enough to produce the desired effect. For example, a composition comprises an effective amount of the VLP, composition, or vaccine of the present disclosure. In one example, the composition comprises a therapeutically effective amount of the VLP, composition, or vaccine of the present disclosure. In another example, the composition comprises a prophylactically effective amount of the VLP, composition, or vaccine of the present disclosure.

[0270] The dosage should not be so high as to cause adverse side effects. Generally, the dosage varies depending on the patient's age, condition, sex, and the severity of the disease, and can be determined by those skilled in the art. The dosage may be adjusted by the individual physician if any complications arise.

[0271] The dosage can vary over a day or several days, administered at least once a day, from approximately 0.1 mg / kg to approximately 300 mg / kg, for example, from approximately 0.2 mg / kg to approximately 200 mg / kg, for example, from approximately 0.5 mg / kg to approximately 20 mg / kg.

[0272] In some cases, the VLPs, compositions, or vaccines of this disclosure are administered at an initial (or loading) dose higher than the subsequent (maintenance) dose. For example, the VLPs, compositions, or vaccines of this disclosure are administered at an initial dose of about 10 mg / kg to about 30 mg / kg. The VLP is then administered at a maintenance dose of about 0.0001 mg / kg to about 10 mg / kg. The maintenance dose may be administered every 7 to 35 days, for example, every 7, 14, or 28 days.

[0273] In some cases, a dose-escalation regime is used, in which the VLP, composition, or vaccine of this disclosure is administered initially at a lower dose than that used in subsequent administrations. This dose regime is useful when subjects exhibit adverse events early on.

[0274] In patients not responding adequately to treatment, multiple doses per week may be administered. Alternatively, or in addition, the dose may be increased.

[0275] Subjects may be re-treated with the VLPs, compositions, or vaccines of this disclosure by being given two or more exposure or dose sets, such as at least about two exposures to mRNA, for example, about 2 to 60 exposures, and more specifically about 2 to 40 exposures, and most specifically about 2 to 20 exposures.

[0276] In one instance, optional retreatment may be performed when signs or symptoms of the disease recur.

[0277] In one instance, optional retreatment may be performed when there has been no recurrence of signs or symptoms of the disease.

[0278] In another example, any retreatment may be performed at predetermined intervals. For example, subsequent exposures may be administered at various intervals, such as approximately 3-4 weeks, 4-12 weeks, 24-28 weeks, or 48-56 weeks or longer. For example, such exposures may be administered at intervals of approximately 3-4 weeks, 4-8 weeks, 4-12 weeks, 24-26 weeks, approximately 38-42 weeks, or approximately 50-54 weeks.

[0279] In another example, for subjects experiencing adverse reactions, the initial (or loading) dose may be divided over several days within a week, or over a number of consecutive days.

[0280] The administration of the VLPs, compositions, or vaccines of this disclosure by the method of this disclosure may be continuous or intermittent, depending, for example, the physiological state of the administerer, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the subject and those skilled in the art. The administration of VLPs may be essentially continuous over a pre-selected period, or it may be a series of intervals, for example, during or after the onset of a condition.

[0281] Screening assay Virus-like particle (VLP) antigen In one example, a composition comprising VLPs is evaluated for antigen expression. For example, antigen expression is detected using an antibody against the S protein of SARS-CoV-2, the Pre F or F protein of RSV, or the HA or NA protein of influenza virus. In one example, the number of cells positive for antigen expression is measured, for example, by fluorescence-activated cell sorting (FACS). In another example, the mean fluorescence intensity (MFI) is determined, for example, using FACS.

[0282] Quantification of virus-like particle (VLP) release In one example, a composition comprising VLPs is evaluated for the formation and release of cell-derived VLPs. For example, the release of VLPs from cells is analyzed using an antibody against the VLP antigen. In a further example, the association between VLP antigens is determined, for example, by Western blot analysis, using antibody-mediated co-immunoprecipitation and / or detection of VLP antigens in co-immunoprecipitation samples.

[0283] Micro-neutralization assay In one example, a composition comprising VLPs is evaluated for the antibody response. For example, a composition comprising VLPs is evaluated using a micro-neutralization assay. The method of performing a micro-neutralization assay will be apparent to those skilled in the art. In one example, the micro-neutralization assay is a short-duration assay. For one example, a virus fluorescence focus-based micro-neutralization assay is performed. In another example, the micro-neutralization assay is a long-duration assay.

[0284] Hemagglutination inhibition (HAI) assay In one example, VLPs are evaluated for their antibody response. For instance, VLPs are evaluated using a hemagglutination inhibition (HAI) assay. The method for performing a HAI assay is obvious to those skilled in the art and / or is described, for example, in the WHO (2011) Manual for the laboratory diagnosis and virological surveillance of influenza: WHO Press, World Health Organization.

[0285] Antigen-specific T cell response In one example, a composition containing (naked and / or formulated) VLPs is evaluated for its ability to induce an antigen-specific T cell response. Methods for evaluating the induction of an antigen-specific T cell response are obvious to those skilled in the art and / or are described herein.

[0286] For example, antigen-specific T cell detection is performed on spleen cultures. In short, the spleen cell culture is established in T cell medium, and the cell culture is either stimulated with the antigen peptide or not. In one example, the antigen-specific T cell response is determined using flow cytometry.

[0287] Methods of treatment or prevention This disclosure provides a method for using the immunogenic composition or pharmaceutical composition of this disclosure as a vaccine.

[0288] This disclosure also provides a method for treating or preventing a disease or condition in a subject, which includes administering an immunogenic composition or pharmaceutical composition of this disclosure. For example, the disease or condition is a respiratory viral infection such as influenza, SARS-CoV-2 infection, COVID-19, or respiratory syncytial virus (RSV). In another example, the disease or condition is acute respiratory distress syndrome (ARDS).

[0289] influenza Influenza, also known as "flu," is an infectious disease caused by the influenza virus. Symptoms can range from mild to severe, and the most common symptoms include high fever, runny nose, sore throat, muscle and joint pain, headache, cough, and fatigue. Symptoms typically appear two days after exposure to the virus and most resolve within a week. Complications of influenza may include viral pneumonia, secondary bacterial pneumonia, sinus infections, and exacerbation of pre-existing health problems such as asthma or heart failure. Viral pneumonia can also lead to acute respiratory distress syndrome (ARDS).

[0290] It is evident to those skilled in the art that there are currently four types of influenza viruses: A, B, C, and D. Influenza A is the most common influenza virus, infecting humans, animals, and birds, while influenza B infection occurs primarily in humans. Infection with influenza C does not cause any serious symptoms in humans or mammals, and influenza D has so far infected only pigs and cattle.

[0291] Therefore, in some examples of the present disclosure, the subject has an influenza virus infection. In one example, the subject has influenza. In particular, influenza is associated with ARDS. In one example, the method of the present disclosure can be used to treat or prevent ARDS in a subject suffering from an influenza virus infection. In one example, the method of the present disclosure can be used to treat or prevent ARDS in a subject suffering from influenza.

[0292] In one example, the methods described herein include identifying a subject who has or is suspected of having influenza. In this example, the subject may have one or more of the above-described symptoms and may be classified as having mild or severe influenza.

[0293] Coronavirus disease 2019 (COVID-19) This disclosure provides, for example, a method for treating or preventing COVID-19. This disclosure also provides, for example, a method for treating or preventing SARS-CoV-2 infection. In some examples of this disclosure, the subjects have SARS-CoV-2 infection but do not have clinically diagnosed COVID-19.

[0294] COVID-19 is an infectious disease caused by SARS-CoV-2. It was first identified in Wuhan, Hubei Province, China in December 2019, and has resulted in an ongoing pandemic. Common symptoms include fever, cough, fatigue, shortness of breath, and loss of smell and taste. The majority of cases result in mild symptoms, while some progress to ARDS (Acute Respiratory Disorders of Severity). The time from exposure to the onset of symptoms is typically about 5 days, but can range from 2 to 14 days.

[0295] Therefore, in some cases, the subjects have SARS-CoV-2 infection. In one case, the subjects have COVID-19, for example, severe COVID-19. In particular, severe COVID-19 often results in ARDS. By using the method of this disclosure, ARDS can be treated or prevented in subjects suffering from severe COVID-19.

[0296] In one example, the methods described herein include identifying a subject who has or is suspected of having SARS-CoV-2. In this example, the subject may have one or more of the above-described symptoms and may be classified as having mild or severe SARS-CoV-2.

[0297] In one example, the methods or uses described herein further include identifying subjects who have or are suspected of having mild COVID-19 based on SARS-CoV-2 positive RT-PCR or molecular test results and one or more of the following symptoms: - Fever, - Sore throat, -headache, - Muscle pain (Mialgia) -Gastrointestinal symptoms, -cough, - Chest congestion, -snot, -wheezing, -Skin rash, - Eye irritation or discharge, -cold, - Novel or altered olfactory or gustatory disorders, - Feet or toes that appear reddish or bruised, - Chills or shivers, - Fatigue (loss of appetite, general malaise, fatigue, decreased physical strength)

[0298] In another example, the methods or uses described herein are used in conjunction with SARS-CoV-2-positive RT-PCR or molecular test results, and the following novel or worsened signs or symptoms, namely, - Respiratory rate of 20 breaths / minute or more - Abnormal oxygen saturation levels, however, above 93% when breathing indoor air at sea level. - Clinical or radiological evidence of pneumonia, -Radiological evidence of DVT, -Shortness of breath or difficulty breathing (either one of these) Or any new or worsening signs or symptoms, namely, - Fever, - Heart rate of 90 beats / minute or more - Chills or shivers, - Novel or altered olfactory or gustatory disorders, - Sore throat, - Fatigue, -headache, -cough, - Muscle pain (Mialgia) -Gastrointestinal symptoms, -Reddish, The further step includes identifying subjects who have moderate or suspected COVID-19 based on either two bruise-like features on their feet or toes.

[0299] In another example, the methods or uses described herein further include the step of identifying a subject who has or is suspected of having severe COVID-19 based on SARS-CoV-2 positive RT-PCR or molecular test results and any one or more of the following: - Resting clinical signs suggestive of a serious systemic disease (respiratory rate of 30 breaths / min or more, heart rate of 125 beats / min or more, SpO2 of 93% or less when breathing indoor air at sea level, or PaO2 / FiO2 of 300 mmHg or less), - Respiratory failure (defined as requiring high-flow oxygen therapy, non-invasive ventilation, mechanical ventilation, or extracorporeal membrane oxygenation), - Findings of shock (defined as systolic blood pressure of 90 mmHg or less, diastolic blood pressure of 60 mmHg or less, or requiring vascular pressurizers), - Marked acute renal dysfunction, hepatic dysfunction, or neurological dysfunction, - Admission to the intensive care unit, -death

[0300] Acute respiratory distress syndrome (ARDS) This disclosure provides, for example, a method for treating or preventing ARDS in a subject.

[0301] ARDS is a life-threatening condition characterized by bilateral pulmonary infiltration, severe hypoxemia, and alveolar capillary membrane damage (i.e., pulmonary vascular leakage), leading to non-cardiogenic pulmonary edema. Currently, there is no effective pharmacological treatment.

[0302] Infectious pathogenic factors, including influenza and coronavirus infections, are major causes of ARDS. Therefore, in one example of this disclosure, ARDS is associated with influenza or coronavirus infection. For example, ARDS is associated with influenza. In another example, ARDS is associated with coronavirus infection, such as SARS-CoV infection. In one example, ARDS is associated with SARS-CoV-2 infection.

[0303] ARDS, classified according to the Berlin definition, include the following: (1) Presentation within one week of clinical stimulation or onset of respiratory symptoms, (2) Acute hypoxic respiratory failure, as determined by a PaO2 / FiO2 ratio of 300 mmHg or less with continuous positive airway pressure (CPAP) or positive end-expiratory pressure (PEEP) at a minimum of 5 cm (PaO2 is the partial pressure of oxygen in arterial blood, and FiO2 is the fraction of inspired oxygen), (3) Bilateral radiopaque images of the lungs that are not fully explained by pleural effusion, lung parenchymal infiltration, or atelectasis. (4) Edema / respiratory failure not fully explained by heart failure or fluid overload.

[0304] In one example, the subject has or is suffering from ARDS (i.e., the subject meets the Berlin definition of ARDS). For example, the subject requires treatment (i.e., needs it).

[0305] In one example, a subject has or is suffering from symptoms associated with ARDS. Methods for identifying ARDS-related symptoms and subjects at risk of developing ARDS are apparent to those skilled in the art and / or are described herein. For example, a subject has one or more or all of the following symptoms: a) A respiratory rate exceeding 30 breaths per minute, b) Indoor air breathing with an oxygen saturation (SpO2) of 93% or less, c) Ratio of arterial oxygen partial pressure to the fraction of inhaled oxygen below 300 mmHg (PaO2 / FiO2), d) SpO2 / FiO2 ratio less than 218, and e) Pulmonary infiltrates exceeding 50% observed on radiographic images Currently, ARDS is classified into mild, moderate, or severe based on the associated increased mortality rate. The severity of ARDS can be classified according to the Berlin definition as follows: (i) Mild ARDS: PaO2 / FiO2 of 200-300 mmHg with CPAP or PEEP at least 5 cm, (ii) Moderate ARDS: PaO2 / FiO2 of 100-200 mmHg at least 5 cm PEEP, (iii) Severe ARDS: PaO2 / FiO2 less than 100 mmHg at least 5 cm PEEP

[0306] In one example, ARDS is mild ARDS. In another example, ARDS is moderate ARDS. In yet another example, ARDS is severe ARDS. In one example, the methods described herein include identifying a subject who has or is suspected of having ARDS. In this example, the subject may have one or more of the above-described symptoms and may be classified as having mild or severe ARDS.

[0307] The method described herein can be used to prevent the onset of ARDS in addition to existing treatments for ARDS. Therefore, in one example, the subject does not have ARDS.

[0308] Respiratory syncytial virus (RSV) This disclosure provides, for example, methods for treating, preventing, or slowing the progression of RSV. RSV is an enveloped, unsegmented, negative-strand RNA virus belonging to the genus Pneumonia Virus of the family Paramyxoviridae. For paramyxoviruses like RSV to infect host cells, they require fusion of the viral membrane with the host cell membrane, similar to other enveloped viruses such as influenza viruses.

[0309] In one example, a subject has or is suffering from symptoms associated with RSV. Methods for identifying RSV-related symptoms and subjects at risk of developing RSV are apparent to those skilled in the art and / or are described herein. For example, a subject may have the following symptoms suggestive of mild RSV, namely: a) Nasal congestion or runny nose, b) Dry cough, c) slight fever, d) Sore throat, e) sneezing, f) Headache, Or in severe cases, a) Short, shallow, and rapid breathing, b) Difficulty breathing, that is, the chest muscles and skin are pulled inward with each breath. c) Cough; d) Poor eating habits, e) Abnormal fatigue (lethargy), f) Having one or more, or all, of the following:

[0310] Therefore, in one example, RSV is mild RSV. In another example, RSV is severe RSV.

[0311] In one example, the methods described herein include identifying a subject who has or is suspected of having RSV. In this example, the subject may have one or more of the above-described symptoms and may be classified as having mild or severe RSV.

[0312] The method described herein can be used to prevent the onset of RSV in addition to existing treatments for RSV. Therefore, in one example, the subject does not have RSV.

[0313] kit Another example of the present disclosure is the provision of a kit containing a composition or vaccine of the present disclosure that is useful for treating or preventing the diseases or disorders described above (e.g., SARS-CoV-2 infection, COVID-19, RSV, influenza, ARDS).

[0314] In one example, the kit comprises the composition or vaccine of the Disclosure, optionally contained in a delivery system and / or a pharmaceutically acceptable carrier or diluent, and is packaged with instructions for use in treating, preventing, or delaying the progression of an infection in a subject requiring treatment, prevention, or delay of the progression of such infection. In one example, the composition or vaccine comprises a VLP containing a fusion capsid protein, optionally containing an adjuvant such as MF59. In another example, the kit further comprises a delivery system and / or a pharmaceutically acceptable carrier or diluent, and is packaged with instructions for use in administering the composition or vaccine to a subject with or at risk of contracting a viral infection such as SARS-CoV-2 infection, COVID-19, RSV, or influenza.

[0315] Therefore, in one example, the kit is, (a) A VLP disclosed herein, a pharmaceutical composition disclosed herein, or a vaccine disclosed herein, (b) Instructions for use for their use, and optionally, (c) comprising a pharmaceutically acceptable carrier, excipient, or diluent.

[0316] In this example of the Disclosure, the accompanying information is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold or contain the composition effective for the disease or disorder of the Disclosure and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper puncturable by a subcutaneous needle). At least one activator in the composition is a VLP. The label or accompanying information indicates that the composition is to be used to treat therapeutically eligible subjects, e.g., subjects with SARS-CoV-2 infection, RSV, influenza, and / or COVID-19, who have or are predisposed to developing ARDS or pneumonia, and this includes specific guidance on dosage and administration intervals, as well as any other medicinal products administered. The kit may further include additional containers containing pharmaceutically acceptable diluent buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0317] This disclosure includes the following non-limiting embodiments: [Examples]

[0318] Example 1: Generation of VLP The VLPs for use in this disclosure described herein can be synthesized through the individual expression of viral structural proteins, which can then self-assemble into a virus-like structure. The plasmid DNA encoding the VLP of this disclosure can be produced by inserting a polynucleotide sequence encoding a fusion capsid protein into a DNA vector. Suitable DNA vectors for use will be apparent to those skilled in the art. Insertion of nucleotide sequences into a DNA vector can be carried out using methods standard in the art.

[0319] In short, viable bacterial cells (e.g., Escherichia coli) can be transformed with a DNA plasmid encoding the fusion capsid protein described herein. Individual bacterial colonies are isolated, and the resulting plasmid DNA is amplified in an Escherichia coli culture. Plasmid DNA can be isolated after fermentation. For example, plasmid DNA can be isolated using a commercially available kit (e.g., Maxiprep DNA kit) or other common methods known to those skilled in the art. After isolation, plasmid DNA can be linearized by restriction digestion (i.e., using restriction enzymes). Restriction enzymes are removed using methods known in the art, including, for example, phenol extraction / chloroform extraction and ethanol precipitation.

[0320] The production of VLPs for use as vaccines in accordance with this disclosure can be achieved by cell-based expression of the individual components constituting the VLPs. For example, the expression of viral structural proteins can subsequently self-assemble into virus-like structures, which can be achieved in HEK-293T cells or CHO cells. In this scenario, the VLPs lack an infectious mechanism, express the relevant TM protein domains and antigen proteins, and optionally bind to a capsid protein by a suitable linker.

[0321] The VLPs generated in accordance with this disclosure are schematically shown in Figures 2A-C and include the following: 1. AMV004 comprises a capsid protein derived from alfalfa mosaic virus (AMV), a spike (S) protein derived from SARS-CoV-2 containing the TM protein domain and antigen, and a signal peptide. Various regulatory VLP structures such as AMV001, AMV002, and AMV003 can also be generated in accordance with this disclosure. 2. AP003, which contains a capsid protein derived from bacteriophage AP205, a SARS-CoV-2 spike (S) protein including the TM protein domain and antigen, and a signal peptide. 3. AP007, comprising a capsid protein dimer derived from bacteriophage AP205, a SARS-CoV-2 spike (S) protein containing the TM protein domain and antigen, and a signal peptide. Various regulatory VLP structures such as AP001 and AP001 can also be generated in accordance with this disclosure. 4. MS003 contains a capsid protein derived from bacteriophage MS2, a SARS-CoV-2 spike (S) protein including the TM protein domain and antigen, and a signal peptide. 5. MS007 comprises a capsid protein dimer derived from bacteriophage MS2, a SARS-CoV-2 spike (S) protein containing the TM protein domain and antigen, and a signal peptide. Various regulatory VLP structures such as MS001 and MS006 can also be generated in accordance with this disclosure.

[0322] In any of the capsid fusion proteins listed above, the TM protein domain and antigen may be linked to the capsid protein by a linker amino acid sequence and may further include a detection tag such as a His6 tag.

[0323] Example 2: Effect of VLP on immunogenicity in vitro The effects of VLP outlined in Example 1 can be tested in vitro for immunogenicity by evaluating outcome indicators such as LV microneutralization tests, PV microneutralization tests, and ACE2 binding inhibition. The effects of VLP can also be tested in the presence of adjuvants such as MF-59.

[0324] More specifically, VLPs can be evaluated for antibody response using microneutralization assays, such as short-acting assays (e.g., viral fluorescence focus-based microneutralization assays). In another example, a microneutralization assay is a long-acting assay. VLPs can also be evaluated for antibody response. For example, VLPs can be evaluated using hemagglutination inhibition (HAI) assays. Methods for performing HAI assays are obvious to those skilled in the art and / or are described, for example, in the WHO (2011) Manual for the laboratory diagnosis and virological surveillance of influenza: WHO Press, World Health Organization.

[0325] VLPs can also be evaluated for their ability to induce antigen-specific T cell responses. Methods for evaluating the induction of antigen-specific T cell responses are obvious to those skilled in the art and / or are described herein. For example, antigen-specific T cell detection can be performed on spleen cultures. Briefly, the spleen cell culture is established in T cell medium, and the cell culture can be either stimulated with an antigen peptide or not. In one example, the antigen-specific T cell response can be determined using flow cytometry.

[0326] Example 3: VLP expression results The VLP was produced using the method described in Example 1. The structures described in this example are schematically shown in Figures 3 to 5 and include the following: A. AMV001 contains capsid protein and signal peptide derived from alfalfa mosaic virus (AMV). AMV002 contains a capsid protein derived from B.AMV and a polyhistidine tag with 6 residues. AMV003 contains a capsid protein, signal peptide, and a 6-residue polyhistidine tag derived from C.AMV. AMV004 contains a capsid protein derived from D.AMV, a SARS-CoV-2-derived spike (S) protein including the TM protein domain and antigen, and a signal peptide. AMV005 contains a capsid protein derived from E.AMV, an S protein derived from SARS-CoV-2 that lacks a TM protein domain, and a signal peptide. AMV006 contains a capsid protein derived from F.AMV, as well as an S protein derived from SARS-CoV-2, including the TM protein domain and antigen. AMV007 contains a capsid protein derived from G.AMV and an S protein derived from SARS-CoV-2 that lacks the TM protein domain. AMV008 contains a capsid protein derived from H.AMV, a TM protein domain with a mutation at amino acid position 614 (D614G), an antigen, a SARS-CoV-2 derived S protein, a signal peptide, and a 6-residue polyhistidine tag. I. AP001, containing a capsid protein derived from bacteriophage AP205 and a 6-residue polyhistidine tag. AP003 contains a capsid protein derived from J. bacteriophage AP205, a SARS-CoV-2 derived S protein including the TM protein domain and antigen, and a signal peptide. AP006 contains a capsid protein dimer derived from K. bacteriophage AP205 and a 6-residue polyhistidine tag. AP007 contains a capsid protein dimer derived from L. bacteriophage AP205, a SARS-CoV-2-derived S protein including the TM protein domain and antigen, a signal peptide, and a 6-residue polyhistidine tag. AP008 contains a capsid protein derived from M. bacteriophage AP205, a SARS-CoV-2 derived S protein including the TM protein domain and antigen, and a 6-residue polyhistidine tag. MS001 contains a capsid protein derived from N. bacteriophage MS2 and a 6-residue polyhistidine tag. MS003 contains a capsid protein derived from O. bacteriophage MS2, a SARS-CoV-2-derived S protein including the TM protein domain and antigen, a signal peptide, and a 6-residue polyhistidine tag. MS006 contains a capsid protein dimer derived from P. bacteriophage MS2 and a 6-residue polyhistidine tag. Q. MS007 contains a capsid protein dimer derived from bacteriophage MS2, the TM protein domain and antigen, the SARS-CoV-2-derived S protein, a signal peptide, and a 6-residue polyhistidine tag. A SARS-CoV-2 derived S protein comprising a capsid protein dimer, TM protein domain, and antigen derived from R. bacteriophage MS2, wherein the S protein lacks a furin cleavage site, and includes a signal peptide and a 6-residue polyhistidine tag, MS008 MS009 contains a capsid protein dimer derived from S. bacteriophage MS2, a SARS-CoV-2-derived S protein including the TM protein domain and antigen, a signal peptide, and a 6-residue polyhistidine tag. A SARS-CoV-2 derived S protein comprising a capsid protein dimer, TM protein domain, and antigen derived from T. bacteriophage MS2, wherein the S protein lacks a furin cleavage site, and comprises a SARS-CoV-2 derived S protein, a signal peptide, and a 6-residue polyhistidine tag, MS010

[0327] The amino acid sequences of all the above structures are provided in the table below.

[0328] The expression of several AMV VLP structures was evaluated in 293F and CHO cell lines. The results are summarized in Table 1 below. In short, VLP expression was observed for structures AMV002 and AMV005 in 293F cells, while VLP expression was observed for structures AMV001, AMV002, AMV003, and AMV005 in CHO cells. These results suggest that recombinant expression may successfully produce VLPs, and that CHO cells are more effective for AMV structures. It was also observed that the signal peptide sequence is not essential for VLP secretion. Furthermore, the AMV capsid protein can tolerate additions at its N-terminus. [Table 1]

[0329] The expression of several AP205 VLP structures was evaluated in 293F and CHO cell lines. These test results are summarized in Table 2 below. Briefly, VLP expression was observed for structures AP001 and AP006 in 293F cells, while VLP expression was observed for structures AP001, AP003, and AP007 in CHO cells. These results suggest that dimeric AP205 can enable binding to large antigens. Furthermore, the expression of these structures appears to increase as the amount of antigen present decreases. Importantly, AP205 has been used as a basis for RBD-VLP-based vaccines. [Table 2]

[0330] The expression of MS2 VLP structures was evaluated in 293F and CHO cell lines. The results of these tests are summarized in Table 3 below. Briefly, VLP expression was observed for structures MS001, MS006, MS007, MS008, MS009, and MS010 in 293F cells, while VLP expression was observed for all MS2 structures in CHO cells. Based on expression in these cell types, these results suggest that the MS2 VLP structures are the most promising, demonstrating that both monomeric and dimeric MS2 can express spike proteins. [Table 3] Figure 6 shows an electron micrograph of recombinantly expressed VLPs.

[0331] Exemplary sequences of the present disclosure [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] [Table 4-15] [Table 4-16] [Table 4-17]

[0332] Numbered entries in this disclosure This disclosure provides at least the following numbered information:

[0333] 1. Recombinant virus-like particles (VLPs) comprising a capsid fusion protein and a lipid bilayer, wherein the capsid fusion protein is (a) Capsid proteins derived from non-enveloped viruses, (b) Transmembrane (TM) protein domain and (c) Antigen protein, and, The capsid protein is encapsulated within the lipid bilayer. Recombinant virus-like particles.

[0334] 2. Recombinant VLP as described in item 1, wherein the capsid protein derived from a non-enveloped virus is derived from alfalfa mosaic virus (AMV), bacteriophage MS2, or bacteriophage AP205.

[0335] 3. Recombinant VLP as described in item 2, wherein the capsid protein is a dimer.

[0336] 4. A recombinant VLP as described in any one of items 1 to 3, wherein the capsid protein is fused to the TM protein domain by a peptide linker.

[0337] 5. A recombinant VLP as described in any one of items 1 to 4, wherein the TM protein domain and antigen protein are derived from a virus.

[0338] 6. Recombinant VLPs as described in item 5, wherein the TM protein domain and antigen protein are derived from the same virus.

[0339] 7. Recombinant VLPs as described in item 5, in which the TM protein domain and antigen protein are derived from different viruses.

[0340] 8. Recombinant VLPs as described in any one of items 1 to 6, wherein the antigen protein and TM protein domain are derived from SARS-CoV-2.

[0341] 9. Recombinant VLP as described in any one of items 1-6 or 8, wherein the antigen protein and TM protein domain are derived from the SARS-CoV-2 spike (S) protein.

[0342] 10. Recombinant VLP as described in item 9, wherein the S protein lacks a furin cleavage site at the S1 / S2 boundary and / or S2' site.

[0343] 11. Recombinant VLPs as described in any one of items 1 to 6, wherein the antigen protein and TM protein domain are derived from influenza.

[0344] 12. Recombinant VLP as described in any one of items 1-6 or 11, wherein the antigen protein is hemagglutinin (HA) protein or neuraminidase (NA) protein derived from influenza.

[0345] 13. Recombinant VLP as described in any one of items 1 to 6, wherein the antigen protein and TM protein are derived from respiratory syncytial virus (RSV).

[0346] 14. Recombinant VLP as described in any one of items 1-6 or 13, wherein the antigen protein is the F protein derived from RSV.

[0347] 15. A recombinant VLP described in any one of items 1 to 14, in which the VLP does not contain any viral RNA.

[0348] 16. Recombinant VLPs as described in any one of items 1 to 15, wherein the VLPs have a diameter of approximately 70 nm to 160 nm.

[0349] 17. Recombinant VLP as described in item 16, wherein the VLP has a diameter of approximately 80 nm.

[0350] 18. Recombinant VLP as described in any one of items 1 to 17, wherein the VLP is formulated within lipid nanoparticles (LNPs).

[0351] 19. An isolated, recombinant, or synthetic nucleotide sequence encoding a VLP as described in any one of items 1 to 17.

[0352] 20. An expression vector containing the nucleotide sequence described in item 19.

[0353] 21. An immunogenic composition comprising any one of the VLPs described in item 1 to 18.

[0354] 22. A pharmaceutical composition comprising a VLP described in any one of items 1 to 18 and a pharmaceutically acceptable carrier.

[0355] 23. A pharmaceutical composition as described in item 22, for use as a vaccine.

[0356] 24. A vaccine comprising the pharmaceutical composition described in item 22.

[0357] 25. An immunogenic composition as described in item 21, a pharmaceutical composition as described in item 22, or a vaccine as described in item 24, further comprising an adjuvant.

[0358] 26. An immunogenic composition, pharmaceutical composition, or vaccine as described in item 25, wherein the adjuvant is MF59.

[0359] 27. A method for treating, preventing, or delaying the progression of a disease, disorder, or condition in a subject requiring treatment, prevention, or delay of the progression of said disease, disorder, or condition, the method comprising administering to said subject a VLP as described in any one of items 1 to 18, an immunogenic composition as described in any one of items 21, 25, or 26, a pharmaceutical composition as described in any one of items 22, 23, 25, or 26, or a vaccine as described in any one of items 24 to 26.

[0360] 28. Use of a VLP as described in any one of items 1 to 18, an immunogenic composition as described in any one of items 21, 25, or 26, a pharmaceutical composition as described in any one of items 22, 23, 25, or 26, or a vaccine as described in any one of items 24 to 26, in the manufacture of a medicinal product for treating, preventing, or delaying the progression of a disease, disorder, or condition in a subject.

[0361] 29. A VLP as described in any one of items 1 to 18, an immunogenic composition as described in any one of items 21, 25, or 26, a pharmaceutical composition as described in any one of items 22, 23, 25, or 26, or a vaccine as described in any one of items 24 to 26, for use in the treatment, prevention, or delay of progression of a disease, disorder, or condition.

[0362] 30. A VLP, composition, or vaccine for use as described in item 27, as described in item 28, or as described in item 29, for use in which the disease is a viral infection such as COVID-19, influenza, or RSV.

[0363] 31. A method for inducing an immune response in a subject, the method comprising administering a VLP described in any one of items 1 to 18, an immunogenic composition described in any one of items 21, 25, or 26, a pharmaceutical composition described in any one of items 22, 23, 25, or 26, or a vaccine described in any one of items 24 to 26 to a subject in need thereof.

[0364] 32. Use of a VLP described in any one of items 1 to 18, an immunogenic composition described in any one of items 21, 25, or 26, a pharmaceutical composition described in any one of items 22, 23, 25, or 26, or a vaccine described in any one of items 24 to 26 in the manufacture of a pharmaceutical product for inducing an immune response in a subject that requires such induction of an immune response.

[0365] 33. A VLP as described in any one of items 1 to 18, an immunogenic composition as described in any one of items 21, 25, or 26, a pharmaceutical composition as described in any one of items 22, 23, 25, or 26, or a vaccine as described in any one of items 24 to 26, for use in inducing an immune response in subjects requiring the induction of an immune response.

[0366] 34. A VLP, composition, or vaccine for the method described in item 31, the use described in item 32, or the use described in item 33, wherein the immune response is a humoral immune response and / or a cell-mediated immune response.

[0367] 35. An immune response sufficient to treat, prevent, or delay the progression of at least one symptom of a viral infection caused by SARS-CoV-2, influenza, or RSV, using, or a VLP, composition, or vaccine as described in item 34.

[0368] 36. A method for reducing the viral load in a subject having a viral infection, comprising administering a VLP described in any one of items 1 to 18, an immunogenic composition described in any one of items 21, 25, or 26, a pharmaceutical composition described in any one of items 22, 23, 25, or 26, or a vaccine described in any one of items 24 to 26 to the subject in need of reduction of the viral load.

[0369] 37. In the preparation of pharmaceuticals for reducing the viral load in subjects with viral infections, use of a VLP described in any one of items 1 to 18, an immunogenic composition described in any one of items 21, 25, or 26, a pharmaceutical composition described in any one of items 22, 23, 25, or 26, or a vaccine described in any one of items 24 to 26.

[0370] 38. A VLP as described in any one of items 1 to 18, an immunogenic composition as described in any one of items 21, 25, or 26, a pharmaceutical composition as described in any one of items 22, 23, 25, or 26, or a vaccine as described in any one of items 24 to 26, for use in reducing the viral load in subjects with a viral infection.

[0371] 39. A VLP, vaccine, or composition for use described in any one of the following items, 27, 30, 31, or 34-36, for use described in any one of the following items, 28, 30, 32, 34, 35, or 37, for use described in any one of the following items, 29, 30, 33, 34, 35, or 38, for use in humans aged 18 years or older.

[0372] 40. The method, use, or use of a VLP, vaccine, or composition as described in item 39, wherein the VLP, vaccine, or composition is administered in a single-dose regimen.

[0373] 41. The VLP, vaccine, or composition is administered in a two-, three-, or four-dose regimen, with the doses given at intervals of approximately one, two, or three months, as described in item 39, or for use as described in item 39.

[0374] 42. Eukaryotic cells for expressing VLP as described in any one of items 1 to 17.

[0375] 43. Cells as described in item 42, wherein the cells are CHO cells or HEK-293 cells.

[0376] 44. A method for producing VLPs, (a) To provide one or more expression vectors containing polynucleotides for expressing virus-like particles (VLPs) as described in any one of items 1 to 17, (b) providing host cells, (c) Transfecting host cells with one or more expression vectors to produce virus-like particles (VLPs) containing one or more antigens, Includes, The polynucleotide is expressed under conditions sufficient for VLP formation. method.

[0377] 45. The method described in item 44, further comprising purifying the VLP.

[0378] 46. (a) A VLP as described in any one of items 1 to 18, a pharmaceutical composition as described in any one of items 22, 23, or 25 to 26, an immunogenic composition as described in any one of items 21, 25, or 26, or a vaccine as described in any one of items 24 to 26, (b) Instructions for use for their use, and optionally, (c) With a pharmaceutically acceptable carrier, excipient or diluent, A kit that includes this.

Claims

1. Recombinant virus-like particles (VLPs) comprising a capsid fusion protein and a lipid bilayer, wherein the capsid fusion protein is (a) Capsid proteins derived from non-enveloped viruses, (b) Transmembrane (TM) protein domain, (c) Antigen protein, and The capsid protein is encapsulated within the lipid bilayer. Recombinant virus-like particles (VLPs).

2. The recombinant VLP according to claim 1, wherein the capsid protein derived from a non-enveloped virus is derived from alfalfa mosaic virus (AMV), bacteriophage MS2, or bacteriophage AP205.

3. The recombinant VLP according to claim 2, wherein the capsid protein is a dimer.

4. The recombinant VLP according to claim 1, wherein the capsid protein is fused to the TM protein domain by a peptide linker.

5. The recombinant VLP according to claim 1, wherein the TM protein domain and the antigen protein are derived from a virus.

6. The recombinant VLP according to claim 5, wherein the TM protein domain and the antigen protein are derived from the same virus.

7. The recombinant VLP according to claim 5, wherein the TM protein domain and the antigen protein are derived from different viruses.

8. The recombinant VLP according to claim 1, wherein the antigen protein and the TM protein domain are derived from SARS-CoV-2.

9. The recombinant VLP according to claim 1, wherein the antigen protein and the TM protein domain are derived from the SARS-CoV-2 spike (S) protein.

10. The recombinant VLP according to claim 9, wherein the S protein lacks furin cleavage sites at the S1 / S2 boundary and the S2' region.

11. The recombinant VLP according to claim 1, wherein the antigen protein and the TM protein domain are derived from influenza.

12. The recombinant VLP according to claim 1, wherein the antigen protein is an influenza-derived hemagglutinin (HA) protein or neuraminidase (NA) protein.

13. The recombinant VLP according to claim 1, wherein the antigen protein and the TM protein are derived from respiratory syncytial virus (RSV).

14. The recombinant VLP according to claim 1, wherein the antigen protein is an F protein derived from RSV.

15. The recombinant VLP according to claim 1, wherein the VLP does not contain any viral RNA.

16. The recombinant VLP according to claim 1, wherein the VLP has a diameter of about 70 nm to 160 nm.

17. The recombinant VLP according to claim 16, wherein the VLP has a diameter of about 80 nm.

18. The recombinant VLP according to claim 1, wherein the VLP is formulated within lipid nanoparticles (LNPs).

19. An isolated, recombinant, or synthetic nucleotide sequence encoding the VLP described in claim 1.

20. An expression vector comprising the nucleotide sequence described in claim 19.

21. An immunogenic composition comprising the VLP described in claim 1.

22. A pharmaceutical composition comprising the VLP described in claim 1 and a pharmaceutically acceptable carrier.

23. A pharmaceutical composition according to claim 22 for use as a vaccine.

24. A vaccine comprising the pharmaceutical composition described in claim 22.

25. The immunogenic composition according to claim 21, the pharmaceutical composition according to claim 22, or the vaccine according to claim 24, further comprising an adjuvant.

26. The immunogenic composition, pharmaceutical composition, or vaccine according to claim 25, wherein the adjuvant is MF59.

27. A method for treating, preventing, or delaying the progression of a disease, disorder, or condition in a subject that requires treatment, prevention, or delaying the progression of the said disease, disorder, or condition, comprising administering the VLP described in claim 1 to the subject.

28. Use of the VLP according to claim 1 in the manufacture of a pharmaceutical product for treating, preventing, or delaying the progression of a disease, disorder, or condition in a subject.

29. A VLP according to claim 1 for use in the treatment, prevention, or delay of progression of a disease, disorder, or condition.

30. The method according to claim 27, the use according to claim 28, or the use according to claim 29, wherein the disease is a viral infection such as COVID-19, influenza, or RSV.

31. A method for inducing an immune response in a subject, comprising administering the VLP described in claim 1 to the subject requiring induction of the immune response.

32. The use of the VLP according to claim 1 in the manufacture of a pharmaceutical product for inducing an immune response in a subject requiring induction of an immune response.

33. The VLP according to claim 1 for use in inducing an immune response in subjects requiring induction of an immune response.

34. The method according to claim 31, the use according to claim 32, or a VLP for the use according to claim 33, wherein the immune response is a humoral immune response and / or a cell-mediated immune response.

35. The method, use, or VLP, composition, or vaccine for use according to claim 34, wherein the immune response is sufficient to treat, prevent, or delay the progression of at least one symptom of a viral infection caused by SARS-CoV-2, influenza, or RSV.

36. A method for reducing the viral load in a subject having a viral infection, comprising administering the VLP described in claim 1 to the subject requiring a reduction in viral load.

37. The use of the VLP according to claim 1 in the preparation of a pharmaceutical product for reducing the viral load in a subject having a viral infection.

38. The VLP according to claim 1, for use in reducing the viral load in subjects having a viral infection.

39. A VLP for the method according to claim 27, the use according to claim 28, or the use according to claim 29, wherein the subject is a human being 18 years of age or older.

40. The method, use, or use of the VLP, vaccine, or composition according to claim 39, wherein the VLP, vaccine, or composition is administered in a single-dose regimen.

41. The method, use, or VLP, vaccine, or composition for use according to claim 39, wherein the VLP, vaccine, or composition is administered in a two-, three-, or four-dose regimen, with the doses given at intervals of approximately one, two, or three months.

42. Eukaryotic cells for expressing the VLP described in claim 1.

43. The cell according to claim 42, wherein the cell is a CHO cell or a HEK-293 cell.

44. A method for producing VLP, (a) To provide one or more expression vectors comprising polynucleotides for expressing virus-like particles (VLPs) as described in claim 1, (b) Providing host cells, (c) Transfecting the host cells with one or more expression vectors to produce virus-like particles (VLPs) containing one or more antigens, The aforementioned polynucleotide is expressed under conditions sufficient for VLP formation. method.

45. The method according to claim 44, further comprising purifying the VLP.

46. (a) A VLP according to claim 1, a pharmaceutical composition according to claim 22, an immunogenic composition according to claim 21, or a vaccine according to claim 24, (b) Instructions for use for their use, and optionally, (c) With a pharmaceutically acceptable carrier, excipient, or diluent, A kit that includes this.

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