Virus-like particle, preparation method thereof, and use thereof in preparing vaccine

Abstract

A virus-like particle (VLP) includes: an extracellular domain of a hemagglutinin having an amino acid sequence, or the extracellular domain of a spike protein; a matrix protein 1 having an amino acid sequence; and a transmembrane-cytoplasmic domain of the hemagglutinin. A method for preparing the VLP includes: constructing a recombinant plasmid; generating a recombinant bacmid by transformating the recombinant plasmid into a competent cell; generating a recombinant baculovirus by transfecting the recombinant bacmid into a first cell; and harvesting the VLP by transfecting the recombinant baculovirus into a second cell. Also provided is the use of the VLP in preparing a vaccine.

Description

TITLEVIRUS-LIKE PARTICLE, PREPARATION METHOD THEREOF, AND USE THEREOF IN PREPARING VACCINECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 642,864, filed on May 5th, 2024. The content of the application is incorporated herein by reference.BACKGROUND OF THE INVENTION1. FIELD OF THE INVENTION

[0002] The present disclosure relates to a platform for producing virus-like particles (VLPs), particularly for producing VLPs used for preparing vaccines.Sequence Listing

[0003] The present application hereby incorporates by reference the entire contents of the text file named “NHR-P0034-PUS-Sequencing Listing.xml” in XML format. The text file containing the Sequencing Listing of the present application was created on 28 April, 2025 and is 84,666 bytes in size.2. DESCRIPTION OF THE PRIOR ART

[0004] Influenza infection, more commonly known as flu, is an acute respiratory infectious disease caused by the seasonal and animal influenza viruses. According to the World Health Organization (WHO), influenza annually affects nearly 3 to 5 million people worldwide. Historically, flu viruses have been associated with a large number of deaths and hospitalization, especially in children and elderly populations. In the 2021 to 2022 flu season, the USA alone reported 470,000 to 790,000 cases, with nearly half of them requiring medical attention and resulting in a significant number of deaths.

[0005] Current classical quadrivalent virus vaccines mainly rely on embryonic chicken eggs (ECE) to produce either inactivated or live attenuated influenza vaccine. This formulation technique has raised several questions, the possibility of allergic reactions to egg albumin, the time required for vaccine production, and the continuous supply of ECE during an avian influenza outbreak. Moreover, this conventional technology is sometimes associated with unintentional mutations of the virus during propagation in ECE, which may reduce vaccine efficacy. Recently, advanced egg-free technologies have become attractive for the developmentof influenza vaccines. Another problem associated with the egg-based vaccines is the production time. In general, the ECE-based influenza vaccines take nearly six months from preparing the CVV to delivering the ultimate vaccine to the market.

[0006] However, there exists an unmet need in the art to develop an effective approach that ensures rapid and cost-effective in producing vaccines.SUMMARY OF THE INVENTION

[0007] In view of the foregoing, the present disclosure provides a virus-like particle, including: an extracellular domain of a hemagglutinin, having an amino acid sequence at least 75% identical to one selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 12, or the extracellular domain of a spike protein, having an amino acid sequence at least 90% identical to SEQ ID NO: 30; a matrix protein 1 (Ml protein), having an amino acid sequence at least 90% identical to SEQ ID NO: 24; and a transmembrane-cytoplasmic domain (TM-CTD) of the hemagglutinin between the extracellular domain of the hemagglutinin and the matrix protein 1, having an amino acid sequence at least 90% identical to one selected from the group consisting of SEQ ID NOs: 8, 14, and 20.

[0008] The present disclosure further provides a method for preparing a virus-like particle, including: constructing a recombinant plasmid; generating a recombinant bacmid by transformating the recombinant plasmid into a competent cell; generating a recombinant baculovirus by transfecting the recombinant bacmid into a first cell; and harvesting the virus-like particle by transfecting the recombinant baculovirus into a second cell. The recombinant plasmid may include: a first nucleic acid sequence encoding an extracellular domain of a hemagglutinin, wherein the first nucleic acid sequence is at least 85% identical to one selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 11, or a second nucleic acid sequence encoding the extracellular domain of a spike protein, wherein the second nucleic acid sequence is at least 85% identical to SEQ ID NO: 29; a third nucleic acid sequence encoding a matrix protein 1, wherein the third nucleic acid sequence is at least 85% identical to SEQ ID NO: 23; and a fourth nucleic acid sequence encoding a transmembrane-cytoplasmic domain (TM-CTD) of the hemagglutinin, wherein the fourth nucleic acid sequence is at least 85% identical to one selected from the group consisting of SEQ ID NOs: 7, 13, and 19.

[0009] The present disclosure also provides a use of the virus-like particle in preparing an influenza vaccine.

[0010] These and other objectives of the present disclosure will no doubt become obvious to those of ordinary skill in the art after reading the following detailed description of the preferred embodiment that is illustrated in the various figures and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] FIG. 1 is a schematic diagram of recombinant baculoviruses (rBVs) according to at least one embodiment of the present disclosure (the modified pFastBac Dual vector containing three promoters is used for the expression of chimeric HA (hemagglutinin), NA and Ml genes; and NA is under the control of plO promoter and HA and Ml gene under the control of polyhedrin (PH) promoter).

[0013] FIG. 2 is a schematic diagram of timeline for immunization, blood collection and virus challenge according to at least one embodiment of the present disclosure (“i.m ” stands for intramuscular injection; “i.n ” stands for intranasal inoculation; and “dpi” stands for days post-infection).

[0014] The upper panel of FIG. 3 is a line graph illustrating activity of chimeric and homologous A / Hl VLPs in High Five cells at different time points according to at least one embodiment of the present disclosure (HA titer is assessed by HA assay; “A” indicates type A influenza virus; and “Hl” indicates H1N1 subtype).

[0015] The middle panel of FIG. 3 is a line graph illustrating activity of chimeric and homologous A / H3 VLPs in High Five cells at different time points according to at least one embodiment of the present disclosure (HA titer is assessed by HA assay; “A” indicates type A influenza virus; and “H3” indicates H3N2 subtype).

[0016] The lower panel of FIG. 3 is a line graph illustrating activity of chimeric and homologous B / Yamagata VLPs in High Five cells at different time points according to at least one embodiment of the present disclosure (HA titer is assessed by HA assay; “B” indicates typeB influenza virus; and “Yamagata” indicates Yamagata lineage).

[0017] FIG. 4A is an image illustrating HA, NA, and Ml protein expressions of chimeric A / H1-VLP detected using western blot according to at least one embodiment of the present disclosure (arrow indicates HA, NA, and Ml protein; “A” indicates type A influenza virus; “Hl” indicates H1N1 subtype; “Vic” stands for Victoria lineage; and “M” indicates protein marker (kDa)).

[0018] FIG. 4B is an image illustrating HA, NA, and Ml protein expressions of chimeric A / H3-VLP detected using western blot according to at least one embodiment of the present disclosure (arrow indicates HA, NA, and Ml protein; “A” indicates type A influenza virus; “H3” indicates H3N2 subtype; “Vic” stands for Victoria lineage; and “M” indicates protein marker (kDa)).

[0019] FIG. 4C is an image illustrating HA, NA, and Ml protein expressions of chimeric B / Yamagata-VLP detected using western blot according to at least one embodiment of the present disclosure (arrow indicates HA, NA, and Ml protein; “B” indicates type B influenza virus; “Yamagata” indicates Yamagata lineage; “Vic” stands for Victoria lineage; and “M” indicates protein marker (kDa)).

[0020] FIG. 4D is an image illustrating HA, NA, and Ml protein expressions of chimeric Victoria- VLP detected using western blot according to at least one embodiment of the present disclosure (arrow indicates HA, NA, and Ml protein; “B” indicates type B influenza virus; “Victoria” and “Vic” indicates Victoria lineage; and “M” indicates protein marker (kDa)).

[0021] The left panel of FIG. 5A is a graph illustrating serum HAI antibody titers against homologous A / H1N1 (A / Hawaii / 70 / 2019) strain in ferrets immunized with quadrivalent chimeric VLPs and Flublok vaccine antigens according to at least one embodiment of the present disclosure (statistical analysis is analyzed by two-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “**” indicates P < 0.01; “***” indicates P < 0.001; “A” indicates type A influenza virus; “H1N1” indicates H1N1 subtype; and “GSD” stands for geometric standard deviation).

[0022] The right panel of FIG. 5A is a graph illustrating serum HAI antibody titers against homologous A / H3N2 (A / Minnesota / 41 / 2019) strain in ferrets immunized with quadrivalentchimeric VLPs and Flublok vaccine antigens according to at least one embodiment of the present disclosure (statistical analysis is analyzed by two-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “**” indicates P < 0.01; “***” indicates P < 0.001; “A” indicates type A influenza virus; “H3N2” indicates H3N2 subtype; and “GSD” stands for geometric standard deviation).

[0023] The left panel of FIG. 5B is a graph illustrating serum HAI antibody titers against homologous B / Victoria (B / Darwin / 7 / 2019) strain in ferrets immunized with quadrivalent chimeric VLPs and Flublok vaccine antigens according to at least one embodiment of the present disclosure (statistical analysis is analyzed by two-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “**” indicates P < 0.01; “***” indicates P < 0.001; “B” indicates type B influenza virus; “Victoria” indicates Victoria lineage; and “GSD” stands for geometric standard deviation).

[0024] The right panel of FIG. 5B is a graph illustrating serum HAI antibody titers against homologous B / Yamagata (B / Brisbane / 09 / 2014) strain in ferrets immunized with quadrivalent chimeric VLPs and Flublok vaccine antigens according to at least one embodiment of the present disclosure (statistical analysis is analyzed by two-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “**” indicates P < 0.01; “***” indicates P < 0.001; “B” indicates type B influenza virus; “Yamagata” indicates Yamagata lineage; and “GSD” stands for geometric standard deviation).

[0025] The left panel of FIG. 6A is a graph illustrating serum NT antibody titer against A / Hawaii / 70 / 2019 (HlNl)-like virus (A / Delaware / 55 / 2019) in ferrets at two weeks after two doses of immunization according to at least one embodiment of the present disclosure (statistical analysis is analyzed by one-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “A” indicates type A influenza virus; “H1N1” indicates H1N1 subtype; and “GSD” stands for geometric standard deviation).

[0026] The right panel of FIG. 6A is a graph illustrating serum NT antibody titer against A / H3N2 (A / Minnesota / 41 / 2019) viruses in ferrets at two weeks after two doses of immunization according to at least one embodiment of the present disclosure (statistical analysis is analyzed by one-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “A” indicates type A influenza virus; “H3N2” indicates H3N2 subtype; and “GSD” stands for geometric standard deviation).

[0027] The left panel of FIG. 6B is a graph illustrating serum NT antibody titer against B / Victoria (B / Darwin / 7 / 2019) viruses in ferrets at two weeks after two doses of immunization according to at least one embodiment of the present disclosure (statistical analysis is analyzed by one-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “B” indicates type B influenza virus; “Victoria” indicates Victoria lineage; and “GSD” stands for geometric standard deviation).

[0028] The right panel of FIG. 6B is a graph illustrating serum NT antibody titer against B / Yamagata (B / Brisbane / 09 / 2014) viruses in ferrets at two weeks after two doses of immunization according to at least one embodiment of the present disclosure (statistical analysis is analyzed by one-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “B” indicates type B influenza virus; “Yamagata” indicates Yamagata lineage; and “GSD” stands for geometric standard deviation).

[0029] The left panel of FIG. 7 is a graph illustrating viral clearance from the respiratory tracts according to at least one embodiment of the present disclosure (four ferrets per group at day 3 post-infection and two ferrets per group at day 7 post-infection are anesthetized to determine the B / Darwin / 7 / 2019 live virus titer in nasal washes by TCID50 assays; data are presented as the geometric mean with geometric standard deviation (GSD); statistical analysis is analyzed by two-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “NW” stands for nasal washes; and “DPI” stands for days post-infection).

[0030] The right panel of FIG. 7 is a graph illustrating viral clearance from the respiratory tracts according to at least one embodiment of the present disclosure (at day 3 and day 7 post-infection, two ferrets per group are euthanized to determine the B / Darwin / 7 / 2019 live virus titer in lung tissues by TCID50 assays; data are presented as the geometric mean with geometric standard deviation (GSD); statistical analysis is analyzed by two-way ANOVA with the Tukey’s posttest; indicates P < 0.05; “Lung” stands for lung tissues; and “DPI” stands for days post-infection).

[0031] FIG. 8 is a schematic diagram of a VLP according to at least one embodiment of the present disclosure (“ECD” stands for extracellular domain; “TM-CTD” stands for transmembrane-cytoplasmic domain; “Ml” stands for matrix protein 1).DETAILED DESCRIPTION

[0032] The following embodiments are provided to illustrate the present disclosure in detail. A person having ordinary skills in the art can easily understand the advantages and effects of the present disclosure after reading the disclosure of this specification, and also can implement or apply in other different embodiments. Therefore, it is possible to modify and / or alter the following embodiments for carrying out this disclosure without contravening its scope for different aspects and applications, and any element or method within the scope of the present disclosure disclosed herein can combine with any other element or method disclosed in any embodiments of the present disclosure.

[0033] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

[0034] As used herein, the singular forms “a,” “an,” and “the” include plural referents, unless expressly and unequivocally limited to one referent. For example, “an experiment” means one experiment or more than one experiment, e.g., a plurality of experiments. The term “or” is used interchangeably with the term “and / or” unless the context clearly indicates otherwise.

[0035] As used herein, the term “comprising,” “comprises” “include,” “including,” “have,” “having,” “contain,” “containing,” and any other variations thereof are intended to cover a non-exclusive inclusion. For example, when describing an object “comprises” a limitation, unless otherwise specified, it may additionally include other sequences, domains, ingredients, elements, components, structures, regions, parts, steps, or connections, etc., and should not exclude other limitations.

[0036] As used herein, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently, “at least one of A and / or B”) can refer, inone embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements).

[0037] As used herein, the term “sequence identity” or, for example, comprising a “sequence 90% identical to” refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the polypeptide variant maintains at least one biological activity or function of the reference polypeptide.

[0038] The numeral ranges used herein are inclusive and combinable, any numeral value that falls within the numeral scope herein could be taken as a maximum or minimum value to derive the sub-ranges therefrom. For example, the numeral range “75% to 100%” includes any sub-ranges between the minimum value of 75% to the maximum value of 100%, such as the sub-ranges from 75% to 95%, from 80% to 100%, from 90% to 99% and so on. In addition, a plurality of numeral values used herein can be optionally selected as maximum and minimum values to derive numerical ranges. For instance, the numerical ranges of 90% to 95%, 90% to 100%, and 95% to 100% can be derived from the numeral values of 90%, 95%, and 100%.

[0039] As used here, the term “subject” includes human or other animals. In at least one embodiment of the present disclosure, the subject may be a mammal, such as, but not limited to a human, non-human primate, canine, feline, murine, bovine, equine, porcine, sheep, deer, wolf, fox, and rabbit.

[0040] As used herein, the term “pharmaceutically acceptable carrier” maybe any and all solvents, dispersion media, diluents, excipients, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, buffering agents, humectants, or other components that are suitable for the formulation and administration of the pharmaceutical composition described in the present disclosure. The carrier may be compatible with the active ingredients and may not produce an adverse biological reaction when administered. The term “pharmaceutically acceptable” indicates that the carrier is approved by a regulatory authority such as the U.S. Food and Drug Administration, or is listed in the United States Pharmacopeia or other recognized pharmacopeia for use in animals, particularly in humans. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, sterile water, aqueous dextrose, glycerol, ethanol, propylene glycol, and combinations thereof. Solid carriers may include starch, lactose, sucrose, gelatin, or talc. The choice of carrier will depend on the intended mode of administration, such as oral, intramuscular, subcutaneous, or intravenous routes. Suitable examples and formulations may be found in standard references such as Remington ’s Pharmaceutical Sciences.

[0041] As used herein, the term “effective amount” refers to the amount of an active agent or a pharmaceutical composition that is sufficient to bring about an effect on treating, preventing, or ameliorating a disorder, disease, or condition of a subject in need thereof, such as but not limited to, inducing an immune response in a subject in need thereof. The effective amount may vary by a person ordinarily skilled in the art, depending on excipient usage, routes of administration, the possibility of co-usage with other therapeutic treatment, or the condition to be treated, but the present disclosure is not limited thereto.

[0042] As used herein, the term “administer,” “administering” or “administration” refer to the placement of an active ingredient into a subject by a method or route which results in at least partial localization of the active ingredient at a desired site to produce the desired effect. For example, the active ingredient of the present disclosure may be administered to a subject by oral administration, injection, subcutaneous administration, intramuscular administration, topical administration, or nasal administration, but the present disclosure is not limited thereto.

[0043] In some embodiments of the present disclosure, human epidemic events are mainly caused by two types of influenza viruses: Type A and Type B. Based on the surface glycoproteins, hemagglutinin (HA), and neuraminidase (NA) of the virus, Type A viruses are further divided into many subtypes, with H1N1 and H3N2 being involved in human seasonal outbreaks andpandemics (World Health, 2023). Although type B viruses are not responsible for pandemics, they are potentially associated with a significant number of severe acute respiratory diseases. To confer protection against deadly influenza viruses, the most effective way is through annual vaccination. Before the onset of the new flu season, WHO recommends the Candidate Vaccine Virus (CVV) strains for the southern (September) and northern (February) hemispheres based on the global influenza surveillance data. Before the 2013 to 2014 flu season, only trivalent vaccines containing two circulating type A viruses and one type B virus, either Yamagata or Victoria were recommended. However, many surveillance data have shown that human infections were associated with the co-infection of Yamagata and Victoria lineage viruses. Since the 2014 to 2015 flu season, WHO has released recommendations for quadrivalent CVV strains which include both circulating B lineage viruses.

[0044] In some embodiments of the present disclosure, VLP may be a great choice since they may significantly reduce the manufacturing time to four months compared to that of the egg-based vaccines, which may have a great impact for pandemic mitigations. Apart from the influenza virus, many other VLP vaccines have gained popularity in the market, especially against human papilloma virus, hepatitis B virus, hepatitis E virus. Despite the favorable advantages of baculovirus expression vector system (BEVS) for VLP production, no insect cell-based quadrivalent influenza VLP vaccine has been licensed for human use to date. The present disclosure has developed quadrivalent seasonal influenza VLPs for influenza A / H1N1, A / H3N2, B / Victoria, and V / Yamagata and found that the B / Victoria has the best productivity of influenza HA antigen yield. The present disclosure improves the productivity of insect cell-based influenza VLPs using the B / Victoria (e.g., B / Darwin / 7 / 2019 and B / Brisbane / 63 / 2014) as templates, ensuring the expression of functional chimeric HA proteins. Additionally, a process that enables the rapid production of cost-effective, and immunogenic influenza VLPs vaccine candidates is also developed.

[0045] In some embodiments of the present disclosure, a VLP may include HA protein having an extracellular domain (ECD) and a transmembrane-cytoplasmic domain (TM-CTD) and a ML The ECD of the HA may be heterologous from the TM-CTD of the HA, or may be replaced by a membrane protein of the other viruses (e.g., spike protein of SARS-CoV-2) (FIG. 8).

[0046] In at least one embodiment of the present disclosure, the competent cell may be an Escherichia coli. and the first cell and the second cell may be sf-21 cell and / or High Five cell.

[0047] In at least one embodiment of the present disclosure, the extracellular domain may be derived from a type A influenza virus of a H1N1 subtype of a A / Hawaii / 70 / 2019 virus, and the transmembrane-cytoplasmic domain may be derived from a type B influenza virus of a Victoria lineage of a B / Brisbane / 63 / 2014 virus. Since the amino acid sequences of the extracellular domain derived from the type A influenza virus of the H1N1 subtype and / or the amino acid sequences of the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Victoria lineage may slightly mutate annually, it is predictable that variations of these amino acid sequences still fall within the scope of the present disclosure and should not be constrained by the examples illustrated herein. For instance, in other embodiments, the extracellular domain may be derived from a type A influenza virus of a H1N1 subtype of other strains such as A_Texas_WRAIR_1254P_2009 virus or A / Singapore / GP 1908 / 2015 virus, whose amino acid sequence is slightly different from that of the A / Hawaii / 70 / 2019 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). In some other embodiments, the amino acid sequences of the transmembrane-cytoplasmic domain may be derived from the type B influenza virus of the Victoria lineage of other strains such as B / Victoria / 504 / 2000 virus, whose amino acid sequence is slightly different from that of the B / Brisbane / 63 / 2014 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). Similarly, in the process of preparing a VLP, the variations of nucleic acid sequences encoding the extracellular domain derived from the type A influenza virus of the H1N1 subtype and / or the variations of nucleic acid sequences encoding the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Victoria lineage are also predictable in the construction of the recombinant plasmid due to annual variations of the corresponding amino acid sequences in the aforementioned strains.

[0048] In at least one embodiment of the present disclosure, the extracellular domain may be derived from a type A influenza virus of a H3N2 subtype of a A / Minnesota / 41 / 2019 virus, and the transmembrane-cytoplasmic domain may be derived from a type B influenza virus of a Victoria lineage of a B / Brisbane / 63 / 2014 virus. Since the amino acid sequences of the extracellular domain derived from the type A influenza virus of a H3N2 subtype and / or the amino acid sequences of the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Victoria lineage may slightly mutate annually, it is predictable that variations of these sequences still fall within the scope of the present disclosure and should not be constrained by the examples illustrated herein. For instance, in other embodiments, the extracellular domain may be derived from a type A influenza virus of a H3N2 subtype of other strains such as A / Singapore / GP2050 / 2015 virus or A / Fujian / 140 / 2000 virus, whose amino acidsequence is slightly different from that of the A / Minnesota / 41 / 2019 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). In some other embodiments, the amino acid sequences of the transmembrane-cytoplasmic domain may be derived from the type B influenza virus of the Victoria lineage of other strains such as B / Victoria / 504 / 2000 virus, whose amino acid sequence is slightly different from that of the B / Brisbane / 63 / 2014 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). Similarly, in the process of preparing a VLP, the variations of nucleic acid sequences encoding the extracellular domain derived from the type A influenza virus of the H3N2 subtype and / or variations of nucleic acid sequences encoding the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Victoria lineage are also predictable in the construction of the recombinant plasmid due to the annual variations of the corresponding amino acid sequences in the aforementioned strains.

[0049] In at least one embodiment of the present disclosure, the extracellular domain and the transmembrane-cytoplasmic domain may be derived from a type B influenza virus of a Yamagata lineage of a B / Brisbane / 09 / 2014 virus. Since the amino acid sequences of the extracellular domain and / or the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Yamagata lineage may slightly mutate annually, it is predictable that variations of these sequences still fall within the scope of the present disclosure and should not be constrained by the examples illustrated herein. For instance, in other embodiments, the extracellular domain may be derived from a type B influenza virus of the Yamagata lineage of other strains such as B / hanfang / 383 / 1999 (B / Yamagata) virus or B / Yamagata / 16 / 88 (B / Yamagata) virus, whose amino acid sequence is slightly different from that of the B / Brisbane / 09 / 2014 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). In some other embodiments, the transmembrane-cytoplasmic domain may be derived from the type B influenza virus of the Yamagata lineage of other strains such as B / Panama / 45 / 90 (B / Yamagata) virus or B / Yamagata / 16 / 88 (B / Yamagata) virus, whose amino acid sequence is slightly different from that of the B / Brisbane / 09 / 2014 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). Similarly, in the process of preparing a VLP, the variations of nucleic acid sequences encoding the extracellular domain and / or the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Yamagata lineage are also predictable in the construction of the recombinant plasmid due to annual variations of the corresponding amino acid sequence in the aforementioned strains.

[0050] In at least one embodiment of the present disclosure, the extracellular domain and thetransmembrane-cytoplasmic domain may be derived from a type B influenza virus of a Victoria lineage of a B / Darwin / 7 / 2019 virus. Since the amino acid sequences of the extracellular domain and / or the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Victoria lineage may slightly mutate annually, it is predictable that variations of these sequence still fall within the scope of the present disclosure and should not be constrained by the examples illustrated herein. For instance, in other embodiments, the extracellular domain may be derived from a type B influenza virus of a Victoria lineage of other strains such as B / Sichuan / 207 / 1999 virus or B / Victoria / 504 / 2000 virus, whose amino acid sequence is slightly different from the B / Darwin / 7 / 2019 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). In some other embodiments, the transmembrane-cytoplasmic domain may be derived from the type B influenza virus of the Victoria lineage of other strains such as B / Victoria / 504 / 2000 virus, whose amino acid sequence is slightly different from that of the B / Darwin / 7 / 2019 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). Similarly, in the process of preparing a VLP, the variations of nucleic acid sequences encoding the extracellular domain and / or the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Victoria lineage are also predictable in the construction of the recombinant plasmid due to the annual variations of the corresponding amino acid sequences in the aforementioned strains.

[0051] In at least one embodiment of the present disclosure, the extracellular domain may be derived from a spike protein of a SARS-CoV-2 virus, and the transmembrane-cytoplasmic domain may be derived from a type B influenza virus of a Victoria lineage of a B / Brisbane / 63 / 2014 virus. Since the amino acid sequences of the extracellular domain derived from the spike protein of a SARS-CoV-2 virus and / or the amino acid sequences of the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Victoria lineage may slightly mutate annually, it is predictable that variations of these sequences still fall within the scope of the present disclosure and should not be constrained by the examples illustrated herein. For instance, in other embodiments, the extracellular domain may be derived from a spike protein of a SARS-CoV-2 virus of other strains such as hCoV-19 / USA / VA-CAV_VAS3N_00022271_01 / 2024|EPI_ISL_l 97488531 virus or hCoV-19 / USA / WI-MCRI-015538 / 2025|EPI_ISL_19855081 virus, whose amino acid sequence is slightly different from the SARS-CoV-2 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). In some other embodiments, the amino acid sequences of the transmembrane-cytoplasmic domain may be derived from the type B influenza virus of the Victoria lineage of other strains such as B / Victoria / 504 / 2000 virus,whose amino acid sequence is slightly different from that of the B / Brisbane / 63 / 2014 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). Similarly, in the process of preparing a VLP, the variations of nucleic acid sequences encoding the extracellular domain derived from the spike protein of the SARS-CoV-2 virus and / or the variations of nucleic acid sequences encoding the transmembrane-cytoplasmic domain derived from the type B influenza virus of the Victoria lineage are also predictable in the construction of the recombinant plasmid due to the annual variations of the corresponding amino acid sequences in the aforementioned strains.

[0052] In at least one embodiment of the present disclosure, the matrix protein 1 may be derived from a type B influenza virus of a Victoria lineage of a B / Brisbane / 63 / 2014 virus. Since the amino acid sequence of the matrix protein 1 derived from a type B influenza virus of a Victoria lineage may slightly mutate annually, it is predictable that variations of these sequences still fall within the scope of the present disclosure and should not be constrained by the examples illustrated herein. For instance, in other embodiments, the matrix protein 1 may be derived from a type B influenza virus of a Victoria lineage of other strains such as or B / Norway / 1 / 84 virus, whose amino acid sequence is slightly different from the B / Brisbane / 63 / 2014 virus but maintains at least one biological activity or function thereof (e.g., stimulating immune response of the subject). Similarly, in the process of preparing a VLP, the variations of nucleic acid sequences encoding the extracellular domain derived from a type B influenza virus of a Victoria lineage are also predictable in the construction of the recombinant plasmid due to the difference of the aforementioned strains.

[0053] In at least one embodiment of the present disclosure, the VLP may include an extracellular domain of a hemagglutinin having an amino acid sequence at least 75% identical to one selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 12. In some embodiments, the amino acid sequence may have 75% to 100%, 85% to 100%, or 95% to 100 sequence identity to one selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 12. For example, the amino acid sequence may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 12, or the amino acid sequence may be 100% identical to one selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 12. However, the present disclosure is not limited thereto.

[0054] In at least one embodiment of the present disclosure, the VLP may include theextracellular domain of a spike protein having an amino acid sequence at least 90% identical to SEQ ID NO: 30. In some embodiments, the amino acid sequence may have 90% to 100%, 92% to 100%, or 94% to 100 sequence identity to SEQ ID NO: 30. For example, the amino acid sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30, or the amino acid sequence may be 100% identical to SEQ ID NO: 30. However, the present disclosure is not limited thereto.

[0055] In at least one embodiment of the present disclosure, the VLP may include a matrix protein 1 having an amino acid sequence at least 90% identical to SEQ ID NO: 24. In some embodiments, the amino acid sequence may have 90% to 100%, 95% to 100%, or 99% to 100 sequence identity to SEQ ID NO: 24. For example, the amino acid sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24, or the amino acid sequence may be 100% identical to SEQ ID NO: 24. However, the present disclosure is not limited thereto.

[0056] In at least one embodiment of the present disclosure, the VLP may include a transmembrane-cytoplasmic domain of the hemagglutinin having an amino acid sequence at least 90% identical to one selected from the group consisting of SEQ ID NOs: 8, 14, and 20. In some embodiments, the amino acid sequence may have 90% to 100%, 95% to 100%, or 97% to 100 sequence identity to one selected from the group consisting of SEQ ID NOs: 8, 14, and 20. For example, the amino acid sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one selected from the group consisting of SEQ ID NOs: 8, 14, and 20, or the amino acid sequence may be 100% identical to one selected from the group consisting of SEQ ID NOs: 8, 14, and 20. However, the present disclosure is not limited thereto.

[0057] In at least one embodiment of the present disclosure, the recombinant plasmid may include a first nucleic acid sequence encoding an extracellular domain of a hemagglutinin having the first nucleic acid sequence at least 85% identical to one selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 11. In some embodiments, the first nucleic acid sequence may have 85% to 100%, 90% to 100%, or 95% to 100 sequence identity to one selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 11. For example, the first nucleic acid sequence may be at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 11, or the first nucleic acid sequence may be 100% identical to one selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 11. However, the present disclosure is not limited thereto.

[0058] In at least one embodiment of the present disclosure, the recombinant plasmid may include a second nucleic acid sequence encoding the extracellular domain of a spike protein having the second nucleic acid sequence at least 85% identical to SEQ ID NO: 29. In some embodiments, the second nucleic acid sequence may have 85% to 100%, 90% to 100%, or 95% to 100 sequence identity to SEQ ID NO: 29. For example, the second nucleic acid sequence may be at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, or the second nucleic acid sequence may be 100% identical to SEQ ID NO: 29. However, the present disclosure is not limited thereto.

[0059] In at least one embodiment of the present disclosure, the recombinant plasmid may include a third nucleic acid sequence encoding a matrix protein 1 having the third nucleic acid sequence at least 85% identical to SEQ ID NO: 23. In some embodiments, the third nucleic acid sequence may have 85% to 100%, 90% to 100%, or 95% to 100 sequence identity to SEQ ID NO: 23. For example, the third nucleic acid sequence may be at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23, or the third nucleic acid sequence may be 100% identical to SEQ ID NO: 23. However, the present disclosure is not limited thereto.

[0060] In at least one embodiment of the present disclosure, the recombinant plasmid may include a fourth nucleic acid sequence encoding a transmembrane-cytoplasmic domain of the hemagglutinin having the fourth nucleic acid sequence at least 85% identical to one selected from the group consisting of SEQ ID NOs: 7, 13, and 19. In some embodiments, the fourth nucleic acid sequence may have 85% to 100%, 90% to 100%, or 95% to 100 sequence identity to one selected from the group consisting of SEQ ID NOs: 7, 13, and 19. For example, the fourth nucleic acid sequence may be at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one selected from the group consisting of SEQ ID NOs: 7, 13, and 19, or the fourth nucleic acid sequence may be 100% identical to one selected from the group consisting of SEQ ID NOs: 7, 13, and 19. However, the present disclosure is not limited thereto.

[0061] In at least one embodiment of the present disclosure, the VLP may include the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 16, 26, 28, and 32. In some embodiments, the amino acid sequence may have 90% to 100%, 95% to 100%, or 98% to 100 sequence identity to one selected from the group consisting of SEQ ID NOs: 10, 16, 26, 28, and 32. For example, the amino acid sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to one selected from the group consisting of SEQ ID NOs: 10, 16, 26, 28, and 32, or the amino acid sequence may be 100% identical to one selected from the group consisting of SEQ ID NOs: 10, 16, 26, 28, and 32. However, the present disclosure is not limited thereto.

[0062] In at least one embodiment of the present disclosure, the VLP may include the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9, 15, 25, 27, and 31. In some embodiments, the nucleic acid sequence may have 90% to 100%, 95% to 100%, or 98% to 100 sequence identity to one selected from the group consisting of SEQ ID NOs: 9, 15, 25, 27, and 31. For example, the nucleic acid sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one selected from the group consisting of SEQ ID NOs: 9, 15, 25, 27, and 31, or the nucleic acid sequence may be 100% identical to one selected from the group consisting of SEQ ID NOs: 9, 15, 25, 27, and 31. However, the present disclosure is not limited thereto.

[0063] In at least one embodiment of the present disclosure, the extracellular domain of the hemagglutinin may have the amino acid sequence of SEQ ID NO: 2, and the transmembrane-cytoplasmic domain of the hemagglutinin may have the amino acid sequence of SEQ ID NO: 20.

[0064] In at least one embodiment of the present disclosure, the extracellular domain of the hemagglutinin may have the amino acid sequence of SEQ ID NO: 4, and the transmembrane-cytoplasmic domain of the hemagglutinin may have the amino acid sequence of SEQ ID NO: 20.

[0065] In at least one embodiment of the present disclosure, the extracellular domain and the transmembrane-cytoplasmic domain of the hemagglutinin may have the amino acid sequence of SEQ ID NO: 10.

[0066] In at least one embodiment of the present disclosure, the extracellular domain and the transmembrane-cytoplasmic domain of the hemagglutinin may have the amino acid sequence of SEQ ID NO: 16.

[0067] In at least one embodiment of the present disclosure, the extracellular domain of the spike (S) protein may have the amino acid sequence of SEQ ID NO: 30, and thetransmembrane-cytoplasmic domain of the hemagglutinin may have the amino acid sequence of SEQ ID NO: 20.

[0068] In at least one embodiment of the present disclosure, the extracellular domain of the hemagglutinin may have the nucleic acid sequence of SEQ ID NO: 1, and the transmembrane-cytoplasmic domain of the hemagglutinin may have the nucleic acid sequence of SEQ ID NO: 19.

[0069] In at least one embodiment of the present disclosure, the extracellular domain of the hemagglutinin may have the nucleic acid sequence of SEQ ID NO: 3, and the transmembrane-cytoplasmic domain of the hemagglutinin may have the nucleic acid sequence of SEQ ID NO: 19.

[0070] In at least one embodiment of the present disclosure, the extracellular domain and the transmembrane-cytoplasmic domain of the hemagglutinin may have the nucleic acid sequence of SEQ ID NO: 9.

[0071] In at least one embodiment of the present disclosure, the extracellular domain and the transmembrane-cytoplasmic domain of the hemagglutinin may have the nucleic acid sequence of SEQ ID NO: 15.

[0072] In at least one embodiment of the present disclosure, the extracellular domain of the spike (S) protein may have the nucleic acid sequence of SEQ ID NO: 29, and the transmembrane-cytoplasmic domain of the hemagglutinin may have the nucleic acid sequence of SEQ ID NO: 19.

[0073] In at least one embodiment of the present disclosure, the virus-like particle of the present disclosure may be used in preparing vaccines, such as influenza vaccines or SARS-CoV-2 vaccines, but the present disclosure is not limited thereto. Some embodiments may provide the use of the virus-like particle in manufacture of a pharmaceutical composition for preventing or treating influenza and / or SARS-CoV-2.

[0074] In at least one embodiment of the present disclosure, a pharmaceutical composition may include an effective amount of the virus-like particle of the present disclosure. In some embodiments, the pharmaceutical composition may further include a pharmaceuticallyacceptable carrier and / or an adjuvant. In some embodiments, the pharmaceutical composition may be a vaccine, such as an influenza vaccine or a SARS-CoV-2 vaccine, but the present disclosure is not limited thereto.

[0075] In at least one embodiment of the present disclosure, a method for inducing an immune response in a subject in need thereof is provided. The method may include administering an effective amount of the vaccine containing an effective amount of the virus-like particle of the present disclosure. In some embodiments, the immune response may be against influenza or SARS-CoV-2, but the present disclosure is not limited thereto.EXAMPLES

[0076] Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed to limit the scope of the present disclosure.Material and methodMethod for producing chimeric virus-like particlesViruses, cell lines and culture media

[0077] Influenza viruses are cultured in Madin-Darby canine kidney (MDCK) cells using serum-free OptiPRO medium (ThermoFisher, US), and Reed-Muench method (Reed and Muench, 1938) is performed to determine the 50% tissue culture infectious dose (TCID50). Experiments related to the live viruses are conducted in the Biosafety Level 2 (BSL-2) facility. For cultivation of Insect Sf-21 cells and High Five cells, Sf-21 cells are grown in Grace’s medium (ThermoFisher, US) supplemented with 10% FBS, while High Five cells are cultured in ESF-921 medium (Expression Systems, US).Construction of plasmid

[0078] Two essential genes, NA and Ml genes of B / Brisbane / 63 / 2014 (B / Victoria), are amplified by PCR from the cDNA of the strain listed in Table 1 and cloned into a modified pFastBac Dual vector (ThermoFisher, US) (Lai et al., 2019). Subsequently, the Chimeric HA by fusing the HA extracellular domain (ECD) of HA genes of H1N1 or H3N2 with the gene of transmembrane-cytoplasmic domain (TM-CTD) of B / Brisbane / 63 / 2014 (B / Victoria); the HAgene of B / Brisbane / 09 / 2014 (B / Yamagata); or the HA gene of B / Darwin / 7 / 2019 (B / Victoria) is introduced separately into the vector system. The recombinant baculoviruses (rBVs) are generated through the Bac-to-Bac baculovirus expression system according to the manufacturer’s protocol (ThermoFisher, 2018). The scheme for constructing the recombinant baculoviruses is shown in FIG. 1. Moreover, the SEQ ID NO: of each aforementioned gene sequence and corresponding amino acid sequence is listed in Table 2 and Table 3.Table 1 : List of Influenza proteins included in the plasmids that are used in the productions of quadrivalent chimeric VLP vaccines (“*” indicates HA nucleic acid sequences of CVV are chosen according to the WHO recommendations for influenza vaccine formulations for the 2020 to 2021 season).Table 2: List of DNA and amino acid sequences included in the plasmids for chimeric VLP (“nt” stands for nucleotide; “bp” stands for base pair: and “a. a.” stands for amino acid).

[0079] In some embodiments of the present disclosure, the ECD gene of transmembrane protein of other virus (e.g., spike protein of SARS-CoV-2 virus) may fuse with the TM-CTD of B / Victoria of B / Brisbane / 63 / 2014 for encoding a chimeric protein (chimera) (Table 3), and be introduced into the aforementioned vector system for generating chimeric VLPs.Table 3: List of DNA and amino acid sequences included in the plasmids for chimeric VLP.

[0080] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a.1-524) of HA protein of A_Texas_WRAIR_1254P_2009 (SEQ ID NO: 33) has an identity of 77.86% to SEQ ID NO: 2.

[0081] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a.1-525) of HA protein of A / Singapore / GP 1908 / 2015 (SEQ ID NO: 34) has an identity of 97.5% to SEQ ID NO: 2.

[0082] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a.1-526) of HA protein of A / Singapore / GP2050 / 2015 (SEQ ID NO: 35) has an identity of 97.15% to SEQ ID NO: 4.

[0083] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a.1-526) of HA protein of A / Fujian / 140 / 2000 (SEQ ID NO: 36) has an identity of 90.87% to SEQ ID NO: 4.

[0084] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a. 1-543) of HA protein of B / hanfang / 383 / 1999 (B / Yamagata) (SEQ ID NO: 37) has an identity of 97.79% to SEQ ID NO: 6.

[0085] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a. 1-542) of HA protein of B / Yamagata / 16 / 88 (B / Yamagata) (SEQ ID NO: 38) has an identity of 95.95% to SEQ ID NO: 6.

[0086] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a.1-544) of HA protein of B / Sichuan / 207 / 1999 (SEQ ID NO: 39) has an identity of 97.43% to SEQ ID NO: 12.

[0087] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a.1-543) of HA protein of B / Victoria / 504 / 2000 (SEQ ID NO: 40) has an identity of 92.63% to SEQ ID NO: 12.

[0088] In some embodiments of the present disclosure, an amino acid sequence of Ml protein (a.a. 1-249) of BVic Ml of B / Victoria / 504 / 2000 (SEQ ID NO: 41) has an identity of 100% to SEQ ID NO: 24.

[0089] In some embodiments of the present disclosure, an amino acid sequence of Ml protein (a.a. 1-249) of BVic Ml of B / Norway / 1 / 84 (SEQ ID NO: 42) has an identity of 99.19% to SEQ ID NO: 24.

[0090] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a. 1-1208) of S protein of hCoV-19 / USA / VA-CAV_VAS3N_00022271_01 / 2024|EPI_ISL_l 97488531 (SEQ ID NO: 43) has an identity of 94.29% to SEQ ID NO: 30.

[0091] In some embodiments of the present disclosure, an amino acid sequence of ECD domain (a.a. 1-1209) of S protein of hCoV-19 / USA / WI-MCRI-015538 / 2025|EPI_ISL_19855081 (SEQ ID NO: 44) has an identity of 94.21% to SEQ ID NO: 30.

[0092] In some embodiments of the present disclosure, an amino acid sequence of TM-CTD domain (a.a. 543-584) of HA protein of B / Panama / 45 / 90 (B / Yamagata) (SEQ ID NO: 45) has an identity of 97.62% to SEQ ID NO: 8.

[0093] In some embodiments of the present disclosure, an amino acid sequence of TM-CTD domain (a.a.543-584) of HA protein of B / Yamagata / 16 / 88 (B / Yamagata) (SEQ ID NO: 46) has an identity of 97.62% to SEQ ID NO: 8.

[0094] In some embodiments of the present disclosure, an amino acid sequence of TM-CTD domain (a.a. 542-583) of HA protein of B / Washington / 02 / 2019 (SEQ ID NO: 47) has an identity of 100% to SEQ ID NO: 14.

[0095] In some embodiments of the present disclosure, an amino acid sequence of TM-CTD domain of HA protein (a.a. 542-583) of B / Victoria / 504 / 2000 (SEQ ID NO: 48) has an identity of 95.12% to SEQ ID NO: 14.

[0096] In some embodiments of the present disclosure, an amino acid sequence of TM-CTD domain (a.a. 545-586) of HA protein of B / Netherlands / 2 / 2006 (SEQ ID NO: 49) has an identityof 100% to SEQ ID NO: 20.

[0097] In some embodiments of the present disclosure, an amino acid sequence of TM-CTD domain of HA protein (a.a. 542-583) of B / Victoria / 504 / 2000 (SEQ ID NO: 50) has an identity of 95.12% to SEQ ID NO: 20.Production of recombinant baculovirus (rBV) and virus-like particles (VLPs)

[0098] Sf-21 cells (Invitrogen, US) cultured in Grace’s insect basal medium (Invitrogen, US) supplemented with 10% FBS (Gibco, USA) are transfected with bacmid DNAto generate the rBV. The virus titer of the rBV is determined by plaque assay in Sf21 cells. To produce the VLPs, High Five cells are suspension-cultured at 27°C in serum-free ESF-921 medium (Expression System, US) at a density of 2xlOA6 cells / mL, followed by rBV infection with a Multiplicity of Infection (MOI) ranging from 1 to 2.5. VLPs are harvested at different time points to monitor the cell viability and HA protein production by measuring HA activity in the supernatant and pellet. After harvesting, the cell debris is removed by low-speed centrifugation, and the supernatant is collected and stored at 4° C until purification.Purification of the virus-like particles (VLPs)

[0099] The influenza VLP in supernatant is pelleted by ultracentrifugation (135,000xg for 4 hours at 4°C) in the sucrose cushion method. The sedimented VLPs are re-suspended in the phosphate buffered saline (PBS) with or without trehalose (final cone, is 20%).Hemagglutination assay

[0100] The hemagglutination (HA) assay is carried out by following the guideline of World Health Organization (WHO) (World Health, 2002). In brief, VLPs are subjected to a 2-fold serial dilution in PBS within a 96-well plate. These diluted VLPs are mixed with equal volume of 0.5% suspension of Turkey Red Blood Cell (TRBC) then incubated to room temperature (RT) for 40 minutes. The HA titer is determined as the maximum dilution factor of the VLPs that entirely agglutinated the TRBC.Western blot and SDS-PAGE analysis

[0101] The presence of the desired three proteins (HA, NA, and Ml) in the purified chimeric A / Hl, A / H3, B / Yamagata, and B / Victoria VLPs is confirmed through western blot analysis. To detect the HA proteins in the chimeric VLPs, primary antibodies, including anti-HA antibodies against A / Guangdong-Maonan / SWL1536 / 2019 (NIBSC NO: 19 / 314, UK) for H1N1, A / Hong Kong / 2671 / 2019 (NIBSC NO: 19 / 316, UK) for H3N2, B / Phuket / 3073 / 2013 (NIBSC NO: 14 / 248, UK) for B / Yamagata and B / Washington / 02 / 2019 (NIBSC NO: 19 / 218, UK) for B / Victoria are used. Anti-NA antibodies (GTX128540, Taiwan) and anti-Ml antibodies (GTX128537, Taiwan) for influenza B are used to detect the NA and Ml protein in all VLPs. All primary antibodies are diluted (1 :4000, v / v) and incubated at room temperature (RT) for 1 hour. Subsequently, the membrane is washed with PBST (phosphate-buffered saline with Tween 20 detergent) and incubated at RT for 1 hour with horseradish peroxidase (HRP) conjugated secondary antibodies (1 : 10000, v / v). Anti-sheep / goat IgG-HRP (Merck, US) are used for HA proteins, while anti-rabbit IgG-HRP (GTX213110-01, Taiwan) are employed for NA and Ml proteins. The HA, NA, and Ml proteins on the membrane are visualized using an imaging system (Amersham™ Imager 600, UK).Single radial immunodiffusion assay (SRID)

[0102] HA protein content of the VLPs samples and comparator vaccine antigens are determined by the standard Single Radial Immunodiffusion (SRID) assay protocol described previously (Wood et al., 1977). In brief, the procedure involved the initial incubation of VLP samples, commercial vaccines, and HA standards with a 1% Zwittergent (ZW) solution for 30 minutes. Subsequently, the diffusion takes place in a 1% agarose gel that contains the appropriate standard anti-HA serum. It is crucial to note that the specific HA standard antigens and serum for A / H1N1, A / H3N2, B / Yamagata, and B / Victoria strains, are provided by the National Institute for Biological Standards and Control (NIBSC).Total protein and HA protein quantification

[0103] Total protein concentration in the purified VLPs and commercial vaccine group used herein are quantified by modified Lowry assay (ThermoFisher, USA) according to the manufacturer’s protocols.Ferret Immunization and influenza virus challenge

[0104] In some embodiments of the present disclosure, ferret is the most relevant non-primate animal model for evaluating influenza pathology and vaccine immunity (Basu Thakur et al., 2024; Belser et al., 2020). Four- to eight-month-old female ferrets are obtained through a laboratory breeding program at Institute of Preventive Medicine, National Defense Medical Center and are confirmed seronegative for seasonal influenza viruses (H1N1, H3N2, B / Vitoria, and B / Yamagata) using the hemagglutination inhibition assay.

[0105] A microchip capable of monitoring temperature is implanted beneath the skin between the shoulder blades. Each ferret is numbered and housed individually in animal biosafety level 2 laboratory with controlled conditions (22 to 24°C; and 55 to 60% relative humidity) throughout the experiment. Ferrets (4 ferrets per group) are immunized intramuscularly with two doses of vaccine antigen or PBS (mock group) at a 2-week interval. Four treatment groups, consisting of quadrivalent chimeric VLP vaccines or recombinant HA vaccine (Flublok) with two HA dosages (1.5 pg and 3 pg), are evaluated. Sera are collected at Day 0 (pre-vaccination), Day 14 (post-dose 1), and Day 28 (2 weeks post-dose 2) for measuring hemagglutination inhibition (HAI) and neutralizing antibody titers (NT). On Day 28, ferrets are intranasally inoculated with influenza B / Darwin / 07 / 2019 (Victoria lineage) at a dose of 3.56 x 107TCID50 in 500 pl per ferret. Clinical signs, including body weight and temperature, are monitored daily for 7 days post-infection (dpi). On Days 31 (3 dpi) and 35 (7 dpi), two ferrets from each group are euthanized, and blood, nasal washes, and respiratory tract samples are collected for viral titer measurement using TCID50 assay (FIG. 2).Hemagglutination inhibition (HAI) titers

[0106] At different time points, blood samples are collected from the ferrets and placed into serum separation tube (BD Microtrainer, BD, US), as previously described. The blood samples undergo centrifugation at 3,500 rpm for 10 minutes to collect the serum from supernatant. Following heat inactivation at 56°C for 30 minutes, the serum samples are stored at -20°C for subsequent analysis. To determine hemagglutination inhibition (HAI) titers, serum samples undergo processing in accordance with the guidelines set forth by the WHO, as outlined in a previous study (Chia et al., 2015). In a nutshell, the serum samples are treated with Receptor Destroying Enzyme (RDE) (Denka Seiken, Japan) at a 1 :4 ratio, allowing for an overnight incubation at 37°C. The serum dilution process commences with an initial 1 : 10 dilution. After that, the diluted serum is mixed with 8 HAU of the corresponding inactivated viruses and incubated at RT for 30 min. Following this, the mixtures are incubated with a 0.5% suspension of turkey RBCat RT for 40 minutes. The HAI titer is determined as the equal of the maximum dilution that exhibits inhibition of HA activity. If no inhibition is detected at the starting serum dilution (1 : 10), the HAI is recorded as <10, and a HAI titer of 5 is employed for geometric mean titers (GMT) calculation.Virus neutralization (NT) assay

[0107] Virus neutralization (NT) assay is done using a modified protocol based on the manual on Animal Influenza Diagnosis and Surveillance published by WHO (World Health, 2002), as previously described by Chia et al. (Chia et al., 2015). Briefly, serum samples are subjected to serial 2-fold dilution, starting from an initial dilution of 1 : 10. These diluted sera are then incubated with 2000 TCID50 / ml live virus at 35°C for 2 hours. The serum-virus mixture is subsequently added to the 96-well plate including a monolayer of MDCK cells. Following 4 to 5 days of incubation, Cytopathic Effects (CPE) are examined to determine serum neutralizing antibody titers. Titer is stated as equal of the maximum serum dilution that shown less than 50% of virus induced CPE.Tissue-culture infectious dose (TCID50) assay

[0108] To quantify the infectious viral titer for challenging the ferrets and viral titer presented in both nasal wash and 10% lung homogenate samples 3 dpi and 7 dpi, the 50% tissue culture infectious doses (TCID50) determination assay, as described in prior studies (Lin et al., 2017), is performed. MDCK cell monolayers are cultured in 96-well plates and infected with 10-fold serial dilutions of the clarified nasal wash and lung samples. Following 4 to 5 days of incubation, virus titers are determined by identifying the maximum dilution at which 50% of the cells exhibited virus-induced cytopathic effects (CPE). Subsequently, TCID50 results are calculated using the Reed-Muench formula.Statistical analysis

[0109] GraphPad Prism 9.5.1 software (GraphPad Co., San Diego, CA) is used for statistical analysis. The statistical significance in the GMT among different vaccine groups is determined by one-way ANOVA or two-way ANOVA with Tukey’s posttest. Statistical significance is expressed as / ?-values less that 0.05 (P<0.05).Example 1. Expression and characterization of chimeric VLPs

[0110] An insect cell-based baculovirus expression system to generate rBVs bearing chimeric influenza HA genes or parental HA gene from 4 seasonal influenza vaccine strains is successfully utilized. Then, influenza VLPs by infecting High Five cells with relevant rBVs are produced and the HA activities are determined at different time points. For chimeric A / Hl and A / H3 VLPs, the HA activity peaks at 1024 HAU / 50 pl, and 2048 HAU / 50 pl at 48 hours and 64 hours post-infection, respectively. Meanwhile, both chimeric B / Yamagata (i.e., combination of SEQ ID NO: 9 and SEQ ID NO: 23; or combination of SEQ ID NO: 10 and SEQ ID NO: 24) and chimeric B / Victoria VLP (i.e., combination of SEQ ID NO: 15 and SEQ ID NO: 23; or combination of SEQ ID NO: 16 and SEQ ID NO: 24) demonstrate their highest HA activity, reaching 1024 HAU / 50 pl, at 64 hours and 48 hours post infection, respectively. The HA activity of chimeric A / Hl, A / H3 and B / Yamagata VLPs with previously produced homologous A / H1N1, A / H3N2, B / Yamagata VLPs is compared. The results clearly indicate that the chimeric VLPs reach their peak HA activity earlier than its homologous VLPs (FIG. 3).

[0111] The supernatant is clarified and subsequently purified them using ultra-centrifugation. The presence of the desired proteins is confirmed by Western blot. As expected, HA, NA, and Ml proteins are observed at around 70 kDa, 53 kDa, and 22 kDa, respectively (FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D).

[0112] The HA productivity of the chimeric VLPs is then determined by assessing HA titers by HA assay, HA protein content by SRID assay, and total protein concentration by modified Lowry assay (Table 4). After purification, SRID data reveals varying levels of HA productivity among four different influenza VLPs, including 8.5 mg / L for chimeric A / Hl, 14.7 mg / L for chimeric A / H3, 5.9 mg / L for chimeric B / Yamagata and 10.6 mg / L for B / Victoria. After comparing with total protein and HA protein concentration, HA protein content of the chimeric VLP antigens exceeds over 18% (Table 4), which all pass the requirement (16.6%) of influenza virus vaccine (World Health, 2011). Finally, the productivity of chimeric VLPs with their homologous counterparts is compared. The results indicate that in chimeric VLPs, HA protein production increase for 29% to 150% compared to that of the homologous constructs (Table 5).Table 4: HA yield of chimeric seasonal influenza VLPs produced in insect cells.Estimated HA yield (mg / L) is based on the HA content (pg / ml) and volume (1.2ml) after purification using 40 ml working volume and is calculated by converting working volume from 40 ml to one liter.Table 5: Comparative HA yield after purification.Example 2. HAI and NT antibody responses of chimeric VLPs in ferrets

[0113] The immunogenicity and protective effects of a quadrivalent chimeric seasonal flu VLP vaccine (Hl, H3, B / Yam, and B / Vic) and the commercial rHA flu vaccine (Flublok) at two dosages (1.5 pg / strain and 3 pg / strain) in ferrets are assessed. No significant side effects or reactogenicity are observed within 7 days post-vaccination after both the first and second doses. Each group consists of four ferrets (n = 4), and their hemagglutination inhibition (HAI) and neutralization (NT) titers are evaluated on day 0 (pre-vaccination), day 14 (post-dose 1), and day 28 (post-dose 2). After the first immunization, ferrets receiving the chimeric VLP vaccine show mild HAI titers against the homologous A / H3N2 (A / Minnesota / 41 / 2019) antigen on day 14.Notably, two weeks following the booster dose, the high-dose chimeric VLP group demonstrate significantly higher HAI antibody responses against homologous vaccine antigens at day 28 compared to that of the low-dose chimeric VLP and Flublok groups. Among the four vaccine strains, A / H3N2, B / Victoria (B / Darwin / 7 / 2019), and B / Yamagata (B / Brisbane / 09 / 2014) elicit notably higher antibody responses, with HAI geometric mean titers (GMT) of 67.3, 28.3, and 16.8, respectively. In contrast, the Flublok group does not show HAI titers at day 14, and only the high-dose Flublok group exhibits mild HAI titers at day 28 against the A / H3N2 antigen (FIG. 5A and FIG. 5B). Furthermore, neutralization (NT) antibody titers against the vaccine viruses are measured in ferret serum on day 28 post-immunization. Ferrets vaccinated with chimeric VLPs display higher NT titers against the homologous vaccine viruses compared to that of the Flublok group. The high-dose chimeric VLP group show significantly higher NT titers against H3N2, B / Victoria, and B / Yamagata viruses than that of the other groups (FIG. 6A and FIG. 6B). These results suggest that the chimeric VLP vaccine has the potential to induce strong immunity, potentially offering enhanced protection against seasonal influenza viruses.Example 3. Efficacy of chimeric VLP vaccine against influenza challenges in ferretsExample 3-1. Viral clearance from the respiratory tracts

[0114] Following challenge with B / Darwin / 07 / 2019 (B / Victoria candidate vaccine virus), no clinical symptoms are observed in any of the five groups within 7 days post-challenge. However, both the chimeric VLPs and Flublok groups exhibit lower live virus titers in the nasal wash at 3 dpi compared to that of the control group (PBS), with the high dosage (3 pg / strain) chimeric VLP group showing undetectable levels of live virus (the left panel of FIG. 7). Additionally, the chimeric VLP groups (1.5 pg / strain and 3 pg / strain) demonstrate non-detectable viral copies and significantly lower live virus titers in lung homogenates compared to both the control and Flublok groups at 3 dpi (the right panel of FIG. 7). Taken together, these results demonstrate that quadrivalent chimeric seasonal flu VLP vaccines (Hl, H3, B / Yam, and B / Vic) are found to be both immunogenic and protective against the B / Victoria virus in ferrets.

[0115] In some embodiments of the present disclosure, a method for producing chimeric seasonal influenza VLPs vaccine antigens is developed. Through optimization of production techniques using an insect cell-based baculovirus expression system, these chimeric VLPs have shown their efficiency, exhibiting early peak HA activity and significantly increased HA protein production compared to their homologous counterparts. Assessment of productivity data revealsthat the HA content of the chimeric VLPs surpasses the required composition for influenza virus vaccine by World Health Organization. Therefore, these chimeric VLPs are very promising for formulating quadrivalent or trivalent seasonal flu vaccines. One of the major advantages of chimeric VLP production lies in its independence from the adaptation and culture of live viruses in embryonated chicken eggs or mammalian cells. By simply retrieving HA sequences from reliable sources, the generation of chimeric VLPs can be readily achieved in a shorter time. These chimeric VLPs have shown their potential as a quadrivalent vaccine by inducing consistent hemagglutination inhibition antibody and neutralizing antibody responses against homologous vaccine viruses in ferrets than the commercial recombinant HA protein vaccine (Flublok). Moreover, during the intranasal challenge with influenza B / Victoria virus, chimeric VLPs have exhibited better protective efficacy than Flublok. Overall, the potential of chimeric VLPs as a promising candidate for a seasonal flu vaccine is indicated, offering a rapid production, high productivity, and cost effectiveness and paving the way for their integration into vaccination strategies for combating against seasonal and pandemic influenza viruses.

[0116] Those skilled in the art will readily observe that numerous modifications and alterations of the embodiments may be made while retaining the teachings of the disclosure. Accordingly, the above disclosure should be construed as limited only by the metes and bounds of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A virus-like particle, comprising: an extracellular domain of a hemagglutinin, having an amino acid sequence at least 75% identical to one selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 12, or the extracellular domain of a spike protein, having an amino acid sequence at least 90% identical to SEQ ID NO: 30; a matrix protein 1, having an amino acid sequence at least 90% identical to SEQ ID NO: 24; and a transmembrane-cytoplasmic domain of the hemagglutinin between the extracellular domain of the hemagglutinin and the matrix protein 1, having an amino acid sequence at least 90% identical to one selected from the group consisting of SEQ ID NOs: 8, 14, and 20.

2. The virus-like particle of claim 1, wherein the extracellular domain of the hemagglutinin has the amino acid sequence of SEQ ID NO: 2, and the transmembrane-cytoplasmic domain of the hemagglutinin has the amino acid sequence of SEQ ID NO: 20.

3. The virus-like particle of claim 1, wherein the extracellular domain of the hemagglutinin has the amino acid sequence of SEQ ID NO: 4, and the transmembrane-cytoplasmic domain of the hemagglutinin has the amino acid sequence of SEQ ID NO: 20.

4. The virus-like particle of claim 1, wherein the extracellular domain and the transmembrane-cytoplasmic domain of the hemagglutinin has the amino acid sequence of SEQ ID NO: 10.

5. The virus-like particle of claim 1, wherein the extracellular domain and the transmembrane-cytoplasmic domain of the hemagglutinin has the amino acid sequence of SEQ ID NO: 16.

6. The virus-like particle of claim 1, wherein the extracellular domain of the spike protein has the amino acid sequence of SEQ ID NO: 30, and the transmembrane-cytoplasmic domain of the hemagglutinin has the amino acid sequence of SEQ ID NO: 20.

7. The virus-like particle of claim 1, comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 16, 26, 28,and 32.

8. A method for preparing a virus-like particle, comprising: constructing a recombinant plasmid, wherein the recombinant plasmid comprising: a first nucleic acid sequence encoding an extracellular domain of a hemagglutinin, wherein the first nucleic acid sequence is at least 85% identical to one selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 11, or a second nucleic acid sequence encoding the extracellular domain of a spike protein, wherein the second nucleic acid sequence is at least 85% identical to SEQ ID NO: 29; a third nucleic acid sequence encoding a matrix protein 1, wherein the third nucleic acid sequence is at least 85% identical to SEQ ID NO: 23; and a fourth nucleic acid sequence encoding a transmembrane-cytoplasmic domain of the hemagglutinin, wherein the fourth nucleic acid sequence is at least 85% identical to one selected from the group consisting of SEQ ID NOs: 7, 13, and 19; generating a recombinant bacmid by transformating the recombinant plasmid into a competent cell; generating a recombinant baculovirus by transfecting the recombinant bacmid into a first cell; and harvesting the virus-like particle by transfecting the recombinant baculovirus into a second cell.

9. The method of claim 8, wherein the competent cell is an Escherichia coli. and the first cell and the second cell are sf-21 cell and / or High Five cell.

10. The method of claim 8, wherein the extracellular domain of the hemagglutinin has the nucleic acid sequence of SEQ ID NO: 1, and the transmembrane-cytoplasmic domain of the hemagglutinin has the nucleic acid sequence of SEQ ID NO: 19.

11. The method of claim 8, wherein the extracellular domain of the hemagglutinin has the nucleic acid sequence of SEQ ID NO: 3, and the transmembrane-cytoplasmic domain of the hemagglutinin has the nucleic acid sequence of SEQ ID NO: 19.

12. The method of claim 8, wherein the extracellular domain and the transmembrane-cytoplasmic domain of the hemagglutinin has the nucleic acid sequence of SEQ ID NO: 9.

13. The method of claim 8, wherein the extracellular domain and the transmembrane-cytoplasmic domain of the hemagglutinin has the nucleic acid sequence of SEQ ID NO: 15.

14. The method of claim 8, wherein the extracellular domain of the spike protein has the nucleic acid sequence of SEQ ID NO: 29, and the transmembrane-cytoplasmic domain of the hemagglutinin has the nucleic acid sequence of SEQ ID NO: 19.

15. The method of claim 8, comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9, 15, 25, 27, and 31.

16. A use of the virus-like particle of claim 1 in preparing a vaccine.

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