Recombinant nipah / hendra virus antigen, preparation method therefor and use thereof

Abstract

Provided are a recombinant Nipah / Hendra virus antigen, a nucleic acid encoding same, a vaccine or immunogenic composition based on the recombinant antigen or the nucleic acid encoding same, and the use of each of the products in the preparation of a vaccine for preventing and / or treating Nipah virus and / or Hendra virus infections. The recombinant Nipah / Hendra virus antigen can effectively elicit a strong immune response against multiple strains of HeV and / or NiV, and has great value and broad development prospects in clinical applications.

Description

A Nipah / Hendra virus recombinant antigen, its preparation method and application

[0001] Cross-referencing

[0002] This application claims priority to Chinese Patent Application No. 202510199430.9, filed on February 21, 2025, entitled "A Nipah / Hendra Virus Recombinant Antigen, Its Preparation Method and Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedicine, specifically to a Nipah / Hendra virus recombinant antigen, its preparation method, and its application in the preparation of vaccines for the prevention and / or treatment of Nipah virus and / or Hendra virus infection. Background Technology

[0004] Hendra virus (HeV) and Nipah virus (NiV) are zoonotic pathogens that can cause severe respiratory and neurological diseases in humans, often fatally. Hendra virus was first isolated from horses suffering from severe respiratory and neurological diseases in Brisbane, Australia in 1994. Nipah virus was first discovered between 1998 and 1999 in cases of respiratory and neurological diseases in pigs in Malaysia and Singapore, and subsequently in cases of encephalitis among pig farmers. Studies have shown that the primary natural host of NiV is the fruit bat, and human infection with NiV is related to consuming food contaminated with the saliva or feces of infected bats. NiV can be transmitted from intermediate hosts (such as pigs) to humans, and it has limited transmissibility between humans. The primary natural host of HeV is the flying fox, and human infection with HeV results from the virus spreading from bats to horses, and then through contact with infected horses or their tissues.

[0005] To date, cases of NiV have been reported in Malaysia, Singapore, Bangladesh, India, and the Philippines, while cases of HeV have been reported in Australia. Given the widespread distribution of NiV in bats, the risk of spillover to humans is extremely high. The estimated case fatality rate for NiV in humans is approximately 40-75%. Since the first recorded case of HeV in 1994, although only 7 cases of human infection have been confirmed, there have been 4 deaths, resulting in a case fatality rate of approximately 57%.

[0006] Both HeV and NiV belong to the Paramyxoviridae family and are enveloped, single-stranded, negative-sense RNA viruses. Their broad host and cell tropism primarily depends on glycoproteins (G and F) on the viral envelope. These proteins are responsible for viral adhesion and infection of host cells and are key targets for vaccine and antibody research. Currently, there are no approved antiviral therapies or vaccines for HeV or NiV for human use. Given the potential for environmental transmission, high pathogenicity, and infectivity of HeV and NiV in a variety of mammalian hosts, including humans, the development of safe, broad-spectrum, and highly effective vaccines against these two viruses is particularly urgent. Summary of the Invention

[0007] Purpose of the invention

[0008] In response to the needs of the prior art, the present invention provides a Nipah / Hendra virus recombinant antigen, a polynucleotide encoding the same, a nucleic acid product associated with the polynucleotide, a vaccine or immunogenic composition based on the aforementioned recombinant antigen or polynucleotide, and the use of the aforementioned products in the preparation of a vaccine for the prevention and / or treatment of Nipah / Hendra virus.

[0009] Recombinant protein vaccines or various nucleic acid vaccines based on the Nipah / Hendra virus recombinant antigen or its encoded nucleic acid can effectively elicit a strong immune response against multiple strains of HeV and / or NiV, exhibiting broad-spectrum and strong immunogenicity.

[0010] Specifically, the present invention provides the following technical solutions:

[0011] In a first aspect, the present invention provides a Nipah / Hendra virus recombinant antigen having an amino acid sequence arranged according to one of the structures shown in formulas (I) to (IV):

[0012] (AB)-C-(A-B') (I)

[0013] (A-B')-C-(AB) (II)

[0014] (AB)1-C-(AB)2 (III)

[0015] (A-B')1-C-(A-B')2 (IV)

[0016] In equations (I) to (II):

[0017] (AB) represents an amino acid sequence of the head domain or a portion thereof of the Nipah virus G protein, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to it and has the same or substantially the same immunogenicity as it.

[0018] (A-B') represents the amino acid sequence of the head domain or a portion thereof of the Hendra virus G protein, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and has the same or substantially the same immunogenicity as it.

[0019] C is either empty or a connected subsequence;

[0020] In equations (III) to (IV):

[0021] (AB)1 and (AB)2 each independently represent the amino acid sequence of the head domain or a portion thereof of the Nipah virus G protein, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and has the same or substantially the same immunogenicity as it.

[0022] (A-B')1 and (A-B')2 each independently represent an amino acid sequence of the head domain or a portion thereof of the Hendra virus G protein, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity as it.

[0023] C represents a sequence of no or no connected subsequences.

[0024] In feasible implementation schemes, equations (I) to (II) are as follows:

[0025] (AB) represents the G177-T602 segment or L166-T602 segment of the head domain of the Nipah virus G protein;

[0026] And / or, (A-B') represents the G177-S604 segment or L166-S604 segment of the Hendra virus G protein head domain;

[0027] And / or, C is none or is a connector (GGS) n , where n is an integer between 1 and 10.

[0028] In the preferred embodiment, in formula (I):

[0029] (AB) represents the G177-T602 segment of the head domain of the Nipah virus G protein; preferably, (AB) represents the amino acid sequence shown in SEQ ID NO:1, or the amino acid sequence shown in SEQ ID NO:1 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it.

[0030] And / or, (A-B') represents the L166-S604 segment of the head domain of the Hendra virus G protein; preferably, (A-B') represents the amino acid sequence as shown in SEQ ID NO:18, or the amino acid sequence as shown in SEQ ID NO:18 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity.

[0031] And / or, C is none or is a connector (GGS) n Where n is an integer between 1 and 10; preferably, C is none;

[0032] More preferably, the amino acid sequence of formula (I) is shown in SEQ ID NO:20.

[0033] SEQ ID NO:20-NiV-HeV-G-head dimer sequence (SEQ ID NO:1+SEQ ID NO:18):

[0034] (Among them, the normal font part is SEQ ID NO:1, and the bold font part is SEQ ID NO:18).

[0035] In the preferred embodiment, in formula (II):

[0036] (A-B') represents the G177-S604 segment of the head domain of the Hendra virus G protein; preferably, (A-B') represents the amino acid sequence as shown in SEQ ID NO:17, or the amino acid sequence as shown in SEQ ID NO:17 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity.

[0037] And / or, (AB) represents the L166-T602 segment of the head domain of the Nipah virus G protein; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO:2, or the amino acid sequence as shown in SEQ ID NO:2 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it.

[0038] And / or, C is none or is a connector (GGS) n Where n is an integer between 1 and 10; preferably, C is none;

[0039] More preferably, the amino acid sequence of formula (II) is shown in SEQ ID NO:21.

[0040] SEQ ID NO:21-HeV-NiV-G-head dimer sequence (SEQ ID NO:17+SEQ ID NO:2):

[0041] (Among them, the normal font part is SEQ ID NO:17, and the bold font part is SEQ ID NO:2).

[0042] In the preferred embodiment, in formula (III):

[0043] (AB)1 represents the G177-T602 segment of the head domain of the Nipah virus G protein; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO:1 or 8, or the amino acid sequence as shown in SEQ ID NO:1 or 8 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity.

[0044] And / or, (AB)2 represents the L166-T602 segment of the head domain of the Nipah virus G protein; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:2 or 9, or the amino acid sequence as shown in SEQ ID NO:2 or 9 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it.

[0045] And / or, C is none or is a connector (GGS) n , where n is an integer between 1 and 10; preferably, C is none.

[0046] The above (AB)1 and (AB)2 can be the corresponding segments of the head domain of the Nipah virus G protein of the same or different strains, preferably the corresponding segments of the head domain of the Nipah virus G protein of different strains.

[0047] More preferably, the amino acid sequence of formula (III) is shown in one of SEQ ID NO:7, 10, 11.

[0048] SEQ ID NO:7-NiV-Indian strain-G-head dimer sequence (SEQ ID NO:1+SEQ ID NO:2):

[0049] (Among them, the normal font part is SEQ ID NO:1, and the bold font part is SEQ ID NO:2).

[0050] SEQ ID NO:10-NiV-Malaysian strain-G-head dimer sequence (SEQ ID NO:8+SEQ ID NO:9):

[0051] (Among them, the normal font part is SEQ ID NO:8, and the bold font part is SEQ ID NO:9).

[0052] SEQ ID NO:11 - NiV-India-Malaysia-G-head dimer sequence (SEQ ID NO:1 + SEQ ID NO:9):

[0053] (Among them, the normal font part is SEQ ID NO:1, and the bold font part is SEQ ID NO:9).

[0054] In the preferred embodiment, in formula (IV):

[0055] (A-B')1 represents the G177-S604 segment of the head domain of the Hendra virus G protein; preferably, (A-B')1 represents the amino acid sequence as shown in SEQ ID NO:17, or the amino acid sequence as shown in SEQ ID NO:17 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity.

[0056] And / or, (A-B')2 represents the L166-S604 segment of the head domain of the Hendra virus G protein; preferably, (A-B')2 represents the amino acid sequence as shown in SEQ ID NO:18, or the amino acid sequence as shown in SEQ ID NO:18 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it.

[0057] And / or, C is none or is a connector (GGS) n Where n is an integer between 1 and 10; preferably, C is none;

[0058] Optionally, (A-B')1 and (A-B')2 are corresponding segments of the head domain of the G protein of the same or different strains of Hendra virus.

[0059] More preferably, the amino acid sequence of formula (IV) is shown in SEQ ID NO:19.

[0060] SEQ ID NO:19 - HeV-G-head dimer sequence (SEQ ID NO:17 + SEQ ID NO:18):

[0061] (Among them, the normal font part is SEQ ID NO:17, and the bold font part is SEQ ID NO:18).

[0062] Preferably, the N-terminus of the Nipah / Hendra virus recombinant antigen further includes a signal peptide sequence and / or a tag sequence; optionally, the signal peptide sequence is as shown in SEQ ID NO:3, and its DNA coding sequence is as shown in SEQ ID NO:5; optionally, the tag is a His tag, preferably a 6×His tag, the sequence of which is as shown in SEQ ID NO:4, and its DNA coding sequence is as shown in SEQ ID NO:6.

[0063] In a second aspect, the present invention provides a method for preparing the Nipah / Hendra virus recombinant antigen as described in the first aspect above, comprising the following steps: adding a Kozak sequence, a signal peptide and / or a tag coding sequence to the 5' end of the nucleotide sequence encoding the Nipah / Hendra virus recombinant antigen as described in the first aspect above, adding a stop codon to the 3' end, cloning and expressing the antigen, screening for the correct recombinant, transfecting the recombinant antigen into expression system cells for expression, collecting the cell culture supernatant, and isolating the recombinant antigen from the supernatant.

[0064] In a feasible implementation, the expression system cells are mammalian cells, insect cells, yeast cells, or bacterial cells;

[0065] Optionally, the mammalian cells are HEK293T cells, 293F series cells, or CHO cells; more preferably, the 293F series cells are HEK293F cells, Freestyle293F cells, or Expi293F cells.

[0066] Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells, or S2 cells;

[0067] Optionally, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom;

[0068] Optionally, the bacterial cells are Escherichia coli cells.

[0069] Thirdly, the present invention provides a polynucleotide that encodes the Nipah / Hendra virus recombinant antigen as described in the first aspect above.

[0070] The polynucleotide can be DNA or mRNA, preferably a nucleotide sequence optimized with human codons;

[0071] In a preferred embodiment, the polynucleotide has a DNA sequence as shown in one of SEQ ID NO:23, 24, 13, 15, 16, 22 (which encodes the recombinant antigens of the present invention with amino acid sequences as shown in SEQ ID NO:20, 21, 7, 10, 11, 19, respectively), or a corresponding mRNA sequence.

[0072] Fourthly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the third aspect above, and optionally, at least one expression regulatory element operatively linked to the polynucleotide.

[0073] Fifthly, the present invention provides an expression vector comprising the nucleic acid construct as described in the fourth aspect above.

[0074] In a sixth aspect, the present invention provides a host cell wherein it is transformed or transfected with the polynucleotides as described in the third aspect above, the nucleic acid constructs as described in the fourth aspect above, or the expression vectors as described in the fifth aspect above.

[0075] In a seventh aspect, the present invention provides the use of the Nipah / Hendra virus recombinant antigen as described in the first aspect above, the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, the expression vector as described in the fifth aspect above, or the host cell as described in the sixth aspect above in the preparation of a vaccine for the prevention and / or treatment of Nipah virus and / or Hendra virus infection.

[0076] Eighthly, the present invention provides a vaccine or immunogenic composition comprising, as described in the first aspect above, a Nipah / Hendra virus recombinant antigen, as described in the third aspect above, a nucleic acid construct, as described in the fourth aspect above, an expression vector, or a host cell, as described in the sixth aspect above, and physiologically acceptable mediators, adjuvants, excipients, carriers, and / or diluents.

[0077] In some preferred embodiments, the vaccine or immunogenic composition is a Nipah / Hendra virus recombinant protein vaccine, which includes the Nipah / Hendra virus recombinant antigen and adjuvant as described in the first aspect above;

[0078] Optionally, the adjuvant is one or more selected from the following: aluminum adjuvants, MF59 adjuvants, and MF59-like adjuvants. Preferably, the MF59-like adjuvant is AddaVax adjuvant.

[0079] In some other preferred embodiments, the vaccine or immunogenic composition is a Nipah / Hendra virus DNA vaccine, comprising:

[0080] (1) Eukaryotic expression vectors; and

[0081] (2) Constructing a DNA sequence encoding the Nipah / Hendra virus recombinant antigen as described in the first aspect above into the eukaryotic expression vector, preferably a DNA sequence as shown in one of SEQ ID NO: 23, 24, 13, 15, 16, 22;

[0082] Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0083] In some other preferred embodiments, the vaccine or immunogenic composition is a Nipah / Hendra virus mRNA vaccine, the mRNA vaccine comprising:

[0084] (I) The mRNA sequence encoding the Nipah / Hendra virus recombinant antigen as described in the first aspect above; and

[0085] (II) Lipid nanoparticles.

[0086] In some other preferred embodiments, the vaccine or immunogenic composition is a Nipah / Hendra virus-viral vector vaccine, comprising:

[0087] (1) Viral backbone vector; and

[0088] (2) A DNA sequence encoding the Nipah / Hendra virus recombinant antigen as described in the first aspect above is constructed into the viral backbone vector, preferably a DNA sequence as shown in one of SEQ ID NO: 23, 24, 13, 15, 16, 22;

[0089] Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

[0090] In some other preferred embodiments, the vaccine or immunogenic composition is a Nipah / Hendra virus nanoparticle vaccine;

[0091] Preferably, the carrier of the nanoparticles is ferritin, and the Nipah / Hendra virus recombinant antigen described in the first aspect above is covalently linked to ferritin and self-assembled into nanoparticles, so that the Nipah / Hendra virus recombinant antigen is present on the surface of the nanoparticles; more preferably, the Nipah / Hendra virus recombinant antigen and ferritin are linked by a linker or covalently linked by a SpyTag / SpyCatcher linker system.

[0092] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

[0093] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0094] Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0095] More preferably, the tablet is a sublingual tablet;

[0096] More preferably, the granules are fine granules;

[0097] More preferably, the powder is a granule;

[0098] More preferably, the pills are small pills;

[0099] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation.

[0100] Ninthly, the present invention provides a method for preventing and / or treating Nipah virus and / or Hendra virus infection, the method comprising: administering to a subject in need a preventive and / or therapeutically effective amount of the following substances: Nipah / Hendra virus recombinant antigen as described in the first aspect above, polynucleotides as described in the third aspect above, nucleic acid constructs as described in the fourth aspect above, expression vectors as described in the fifth aspect above, host cells as described in the sixth aspect above, and / or vaccines or immunogenic compositions as described in the eighth aspect above.

[0101] The "effective dose for prevention and / or treatment" may vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc. It may be determined based on the doctor's judgment, taking into account factors such as the type of dosage form, the method of administration, the patient's age and weight, and the patient's symptoms. Beneficial effects

[0102] Based on structural biology analysis, this invention designs a tandem structure containing specific fragments of the head domains of the Hendra virus (HeV) and Nipah virus (NiV) G proteins, thereby constructing homodimers or heterodimers of HeV or NiV, as well as heterodimers of HeV and NiV. The inventors have experimentally demonstrated that recombinant protein vaccines based on these dimers or their encoded nucleic acids, as well as various nucleic acid vaccines, can effectively elicit strong immune responses against multiple strains of HeV and / or NiV. These vaccines exhibit broad-spectrum and significant immunogenicity. Therefore, the Nipah / Hendra virus recombinant antigen of this invention has the potential to address potential future HeV and NiV pandemics, indicating its significant value and broad development prospects in the field of clinical application. Attached Figure Description

[0103] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0104] Figure 1 shows the molecular sieve chromatography curve of the NiV-India strain-G-head monomer prepared in Example 2 and the SDS-PAGE identification results of the eluent at the elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0105] Figure 2 shows the molecular sieve chromatography curve of the NiV-India strain-G-head dimer prepared in Example 2 and the SDS-PAGE identification results of the eluent at the elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0106] Figure 3 shows the molecular sieve chromatography curve of the NiV-Malaysia strain-G-head monomer prepared in Example 2 and the SDS-PAGE identification results of the eluent at the elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0107] Figure 4 shows the molecular sieve chromatography curve of the NiV-Malaysia strain-G-head dimer prepared in Example 2 and the SDS-PAGE identification results of the eluent at the elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0108] Figure 5 shows the molecular sieve chromatography curve of the NiV-India strain-Malaysia strain-G-head dimer prepared in Example 2 and the SDS-PAGE identification results of the eluent at the elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0109] Figure 6 is a schematic diagram of the immunization process for BALB / c mice immunized with the vaccine as described in Example 3.

[0110] Figure 7 shows the results of immunogen-specific binding antibody titer determination in serum of immunized mice collected on day 56 by ELISA, as described in Example 4.

[0111] Figure 8 shows the results of neutralizing antibody titer determination against NiV-Malaysia strain pseudovirus in serum collected on day 56 of immunized mice, as described in Example 5, by pseudovirus neutralization assay.

[0112] Figure 9 shows the results of neutralizing antibody titers against NiV-India strain pseudovirus in serum collected on day 56 of immunized mice, as described in Example 5, as tested by a pseudovirus neutralization assay.

[0113] Figure 10 shows the molecular sieve chromatography curve of the HeV-G-head dimer prepared in Example 7 and the SDS-PAGE identification results of the eluent at the elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0114] Figure 11 shows the molecular sieve chromatography curve of the NIV-HeV-G-head dimer prepared in Example 7 and the SDS-PAGE identification results of the eluent at the elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0115] Figure 12 shows the molecular sieve chromatography curve of the HeV-NiV-G-head dimer prepared in Example 7 and the SDS-PAGE identification results of the eluent at the elution peak (under non-reducing (-DTT) or reducing (+DTT) conditions).

[0116] Figure 13 is a schematic diagram of the immunization process for BALB / c mice immunized with the vaccine as described in Example 8.

[0117] Figure 14 shows the titer of antibody specifically binding to NiV-G head monomer protein in immunized mouse serum as determined by ELISA, as described in Example 9.

[0118] Figure 15 shows the titer of antibodies against HeV-G head monomer protein in immunized mouse serum as determined by ELISA, as described in Example 9.

[0119] Figure 16 shows the results of the neutralizing antibody titer against NiV pseudovirus in serum collected on day 56 of immunized mice, as described in Example 10, as tested by the pseudovirus neutralization experiment.

[0120] Figure 17 shows the results of neutralizing antibody titer against HeV pseudovirus in serum collected on day 56 of immunized mice, as described in Example 10, as tested by the pseudovirus neutralization experiment.

[0121] Figure 18 is a schematic diagram of the immunization process for golden hamsters immunized with the vaccine as described in Example 11.

[0122] Figure 19 shows the titer of antibody specifically binding to NiV-G head monomer protein in immunized hamster serum as determined by ELISA, as described in Example 12.

[0123] Figure 20 shows the titer of antibody specifically binding to HeV-G head monomer protein in immunized hamster serum as determined by ELISA, as described in Example 12.

[0124] Figure 21 shows the results of the neutralizing antibody titer against NiV pseudovirus in immunized hamster serum collected on day 56, as described in Example 13, as tested by the pseudovirus neutralization experiment.

[0125] Figure 22 shows the results of the neutralizing antibody titer against HeV pseudovirus in immunized hamster serum collected on day 56, as described in Example 13, as tested by the pseudovirus neutralization experiment. Detailed Implementation

[0126] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0127] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0128] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0129] Example 1: Design of constructs for Nipah virus G protein head domain (G-head) monomer and dimer antigens

[0130] In this embodiment, based on the structural analysis of the NiV G protein, representative circulating NiV strains from India and Malaysia were selected. Constructs of NiV-India strain-G-head monomers, NiV-India strain-G-head homodimers, NiV-Malaysia strain-G-head monomers, NiV-Malaysia strain-G-head homodimers, and NiV-India strain-Malaysia strain-G-head heterodimer antigens were designed, as follows:

[0131] Construct 1: The sequence of the G177-T602 region of the NiV Indian strain G-head (as shown in SEQ ID NO:1) was linked to the N-terminus of a signal peptide (METDTLLLWVLLLWVPGSTG, SEQ ID NO:3) and a tag of 6 histidines (HHHHHH, SEQ ID NO:4) to obtain the NiV-Indian strain-G-head monomer construct (also referred to as "NiV-IG-head" in the following text or figures);

[0132] Construct 2: The sequence of the G177-T602 region of the NiV Indian strain G-head (as shown in SEQ ID NO:1) and the sequence of the L166-T602 region of the NiV Indian strain G-head (as shown in SEQ ID NO:2) are tandemly linked to obtain the sequence of the tandem dimer as shown in SEQ ID NO:7. Then, a signal peptide (METDTLLLWVLLLWVPGSTG, SEQ ID NO:3) and a 6-histidine tag (HHHHHH, SEQ ID NO:4) are linked to its N-terminus to obtain the NiV-Indian strain-G-head dimer construct (also referred to as "NiV-IG-head-dimer" in the following text or in the attached figure);

[0133] Construct 3: The sequence of the G177-T602 region of the NiV Malaysian strain G-head (as shown in SEQ ID NO:8) was linked to the N-terminus of a signal peptide (METDTLLLWVLLLWVPGSTG, SEQ ID NO:3) and a 6-histidine tag (HHHHHH, SEQ ID NO:4) to obtain the NiV-Malaysian strain-G-head monomer construct (also referred to as "NiV-MG-head" in the following text or in the accompanying figures);

[0134] Construct 4: The sequence of the G177-T602 region of the NiV Malaysian strain G-head (as shown in SEQ ID NO:8) and the sequence of the L166-T602 region of the NiV Malaysian strain G-head (as shown in SEQ ID NO:9) are tandemly linked to obtain the sequence of the tandem dimer as shown in SEQ ID NO:10. Then, a signal peptide (METDTLLLWVLLLWVPGSTG, SEQ ID NO:3) and a tag of 6 histidines (HHHHHH, SEQ ID NO:4) are added to its N-terminus to obtain the NiV-Malaysian strain-G-head dimer construct (also referred to as "NiV-MG-head-dimer" in the following text or in the attached figure);

[0135] Construct 5: The sequence of the G177-T602 region of the NiV Indian strain G-head (as shown in SEQ ID NO:1) and the sequence of the L166-T602 region of the NiV Malaysian strain G-head (as shown in SEQ ID NO:9) were tandemly formed, and the sequence of the resulting tandem dimer is shown in SEQ ID NO:11. Then, a signal peptide (METDTLLLWVLLLWVPGSTG, SEQ ID NO:3) and a tag of 6 histidines (HHHHHH, SEQ ID NO:4) were added to its N-terminus to obtain the NiV-India strain-Malaysian strain-G-head dimer construct (also referred to as "NiV-IMG-head dimer" in the following text or in the attached figures).

[0136] Example 2: Expression and purification of NiV Indian strain G-head monomers, NiV Indian strain G-head dimers, NiV Malaysian strain G-head monomers, NiV Malaysian strain G-head dimers, and NiV-India-Malaysia-G-head dimer proteins.

[0137] In this embodiment, following the construct design of Example 1, NiV Indian strain G-head monomer, NiV Indian strain G-head dimer, NiV Malaysian strain G-head monomer, NiV Malaysian strain G-head dimer, and NiV-India-Malaysia-G-head dimer proteins were constructed and expressed, respectively. The specific procedures are as follows:

[0138] Construction of expression plasmids

[0139] The antigen amino acid sequences of the five constructs designed in Example 1 above were optimized using human codons, and the corresponding DNA coding sequences are shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively. The Kozak sequence gccacc, the coding sequence for the signal peptide (as shown in SEQ ID NO:5), and the coding sequence for the 6×His tag (as shown in SEQ ID NO:6) were added upstream of the 5' end of these DNA coding sequences, and a stop codon was added to the 3' end. These sequences were synthesized by Nanjing Genscript Biotech Co., Ltd. The five synthesized DNA sequences were cloned into the pCAGGS plasmid using EcoRI and XhoI restriction sites to obtain pCAGGS expression plasmids expressing NiV-India strain-G-head monomers, NiV-India strain-G-head dimers, NiV-Malaysia strain-G-head monomers, NiV-Malaysia strain-G-head dimers, and NiV-India strain-Malaysia strain-G-head dimers, respectively.

[0140] Protein expression and purification

[0141] The NiV-India strain-G-head monomer, NiV-India strain-G-head dimer, NiV-Malaysia strain-G-head monomer, NiV-Malaysia strain-G-head dimer, and NiV-India strain-Malaysia strain-G-head dimer proteins were expressed using Expi 293F cells.

[0142] Specifically, the expression plasmids pCAGGS-NiV-India strain-G-head monomer, pCAGGS-India strain-G-head dimer, pCAGGS-NiV-Malaysia strain-G-head monomer, pCAGGS-NiV-Malaysia strain-G-head dimer, and pCAGGS-NiV-India strain-Malaysia strain-G-head dimer constructed above were transfected into Expi293F cells. After 5 days, the supernatant was collected, centrifuged to remove the precipitate, and then filtered through a 0.22 μm filter membrane to further remove impurities. The filtered cell supernatant was purified by nickel affinity column chromatography. Specifically, at 4°C, the cell supernatant was passed through a nickel affinity column (Histrap, GE Healthcare) and washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specifically binding proteins. Then, impurities were eluted with a low concentration of imidazole (20 mM Tris, 150 mM NaCl, pH 8.0, 20 mM imidazole). The target protein was eluted from the HisTrap column with buffer B (20 mM Tris, 150 mM NaCl, pH 8.0, 300 mM imidazole), and the eluent was concentrated more than 30 times to buffer A using a 10 kDa concentrator, resulting in a final volume of less than 1 mL. Finally, molecular sieve chromatography was performed using a Superdex™ 200 Increase 10 / 300GL column (GE Healthcare) to further purify the target protein. The molecular sieve chromatography buffer was PBS buffer (8mM Na2HPO4, 136mM NaCl, 2mM KH2PO4, 2.6mM KCl, pH 7.4).

[0143] The molecular sieve chromatography curves of NiV-India strain-G-head monomer, NiV-India strain-G-head dimer, NiV-Malaysia strain-G-head monomer, NiV-Malaysia strain-G-head dimer, and NiV-India strain-Malaysia strain-G-head dimer proteins and the SDS-PAGE identification results of the eluent at the elution peaks are shown in Figures 1-5.

[0144] Figures 1 and 3 show that the NiV-Indian strain-G-head monomer and the NiV-Malaysian strain-G-head monomer have an elution peak at around 15 mL. The eluent from the elution peak was collected and analyzed by SDS-PAGE. The results showed that the molecular weight of the eluted protein was around 55 kDa, which is consistent with the expected molecular weight of the protein.

[0145] In addition, Figures 2, 4, and 5 show the molecular sieve chromatography curves of NiV-India strain-G-head dimer protein, NiV-Malaysia strain-G-head dimer protein, and NiV-India strain-Malaysia strain-G-head dimer protein, respectively, and the SDS-PAGE identification results of the eluent at the elution peak. It shows that there is an elution peak at about 13 mL. SDS-PAGE analysis of the eluent at this elution peak shows that the size of the eluted protein is about 110 kDa, which is consistent with the molecular weight of the protein.

[0146] These results indicate that, through the above expression and purification, NiV-India strain-G-head monomer, NiV-India strain-G-head dimer, NiV-Malaysia strain-G-head monomer, NiV-Malaysia strain-G-head dimer, and NiV-India strain-Malaysia strain-G-head dimer proteins were obtained, and the electrophoretic bands were single, indicating that the purified proteins had high purity.

[0147] Example 3: Immunization and Sample Collection of Laboratory Animals

[0148] To test the immunogenicity of the five Nipah virus construct recombinant subunit vaccines designed in Example 1, we used the purified NiV-India strain-G-head monomer, NiV-India strain-G-head dimer, NiV-Malaysia strain-G-head monomer, NiV-Malaysia strain-G-head dimer, and NiV-India strain-Malaysia strain-G-head dimer proteins obtained in Example 2 as immunogens to immunize 6-8 week old female BALB / c mice. The negative control (Sham group) was immunized with PBS solution. Eight mice were used in each group. The BALB / c mice used were purchased from Vital River Pharmaceuticals. The immunization flowchart is shown in Figure 6; the mouse immunization grouping and immunization dosage are shown in Table 1 below.

[0149] Table 1. Immunization groups and dosages in mice

[0150] The specific experimental procedure is as follows:

[0151] Immunogens NiV-India strain-G-head monomer, NiV-India strain-G-head dimer, NiV-Malaysia strain-G-head monomer, NiV-Malaysia strain-G-head dimer, and NiV-India strain-Malaysia strain-G-head dimer protein were diluted with PBS to 4 μg / mL and 40 μg / mL, respectively. The diluted immunogens were then mixed with AddaVax adjuvant at a 1:1 volume ratio and emulsified to prepare the vaccine. The negative control group consisted of a mixture of PBS solution and AddaVax adjuvant.

[0152] The vaccine obtained according to the above method was administered to BALB / c mice via intramuscular injection in the leg. All mice received their first, second, and third immunizations on days 0, 21, and 42, respectively, with each immunization using a 100 μL inoculation volume (containing 0.2 μg or 2 μg of antigen protein), and 50 μL injected into each leg. On day 56 of the immunization program, blood was collected from the orbital venous plexus of the mice. Serum was collected by centrifugation, inactivated at 56°C for 30 min, and then stored at -80°C for the detection of binding antibody titers and pseudovirus neutralizing antibody titers.

[0153] Example 4: Detection of antigen-specific antibody titers in the serum of immunized mice using enzyme-linked immunosorbent assay (ELISA).

[0154] In this embodiment, the antigen-specific antibody titer levels in the serum of mice in each immunization group collected on day 56 of the immunization program in Example 3 were detected by enzyme-linked immunosorbent assay (ELISA). The specific procedure is as follows:

[0155] (1) NiV-India strain-G-head monomer, NiV-India strain-G-head dimer, NiV-Malaysia strain-G-head monomer, NiV-Malaysia strain-G-head dimer, and NiV-India strain-Malaysia strain-G-head dimer were diluted to 3 μg / mL with ELISA coating buffer (Solepro, C1050), and 100 μL of the above dilution buffer was added to each well of a 96-well ELISA plate (Coring, 3590). The plate was incubated at 4°C overnight (or more than 12 h) for coating.

[0156] (2) Discard the coating solution, add PBS, and wash once; use 5% skim milk prepared with PBS as the blocking solution, add 100 μL to each well of a 96-well plate, let stand at room temperature for 1 h to block, and then wash once with PBS solution.

[0157] (3) During the blocking period, the serum samples of immunized mice were diluted with blocking buffer. The serum samples were diluted sequentially in a 4-fold gradient, starting from 20-fold. Specifically, 152 μL of blocking buffer and 8 μL of serum sample were added to the first well and mixed. The second dilution was 120 μL of blocking buffer and 40 μL of the solution from the first well. After dilution, 100 μL of blocking buffer was added to each well of the ELISA plate. The negative control group was added to the blocking buffer. The plates were incubated at 37°C for 2 hours and then washed 4 times with PBST.

[0158] (4) Add HRP-conjugated goat anti-mouse secondary antibody (BE0102-100) diluted 1:2000 with blocking buffer to each well, incubate at 37°C for 1.5 hours, then wash 3 times with PBST; then add 60 μL of TMB chromogenic solution for color development, and after an appropriate reaction time, add 60 μL of 2M hydrochloric acid to terminate the reaction, and detect the OD450 value on a microplate reader.

[0159] Antibody titer is defined as the highest dilution of serum with a reaction value greater than 2.5 times that of the negative control. When the reaction value at the lowest dilution (limit of detection) is still less than 2.5 times the background value, the titer of the sample is defined as half of the lowest dilution, i.e., 1:10.

[0160] Figure 7 shows the ELISA results of the serum collected from each immunized group on day 56. Figure 7 shows that after three immunizations, high titers of immunogen-specific binding antibodies could be detected in the serum of each immunized group. The antibody titer of the NiV-India strain-G-head dimer group was higher than that of the NiV-India strain-G-head monomer group, the antibody titer of the NiV-Malaysia strain-G-head dimer group was higher than that of the NiV-Malaysia strain-G-head monomer group, and the antibody titer of the NiV-India strain-Malaysia strain-G-head dimer group was similar to that of the NiV-India strain-G-head dimer group, but higher than that of the NiV-Malaysia strain-G-head dimer group.

[0161] The results indicate that the immunogenicity of each dimer construct is higher than that of the monomer construct; and that the immunogenicity of the heterodimer is comparable to or higher than that of the homodimer.

[0162] Example 5: Detection of neutralizing antibody titers induced by recombinant subunit vaccine using a sham virus neutralization experiment.

[0163] In this embodiment, Nipah virus pseudovirus was used to detect the neutralizing titers (pVNT) of pseudoviruses against NiV Malaysian strain and NiV Indian strain in the serum of mice in each immunization group collected on day 56 of the immunization program in Example 3. 50 ).

[0164] The Nipah virus pseudovirus used in this embodiment is a pseudovirus displaying the Nipah virus G and F proteins, prepared based on the vesicular stomatitis virus (VSV) backbone. The preparation method is described in the method section of our published paper (Chen, L., Sun, M., Zhang, H. et al. Potent human neutralizing antibodies against Nipah virus derived from two ancestral antibody heavy chains. Nat Commun 15, 2987 (2024). https: / / doi.org / 10.1038 / s41467-024-47213-8).

[0165] The method for detecting neutralizing antibody titers against Nipah virus pseudovirus (hereinafter referred to as pseudovirus) is as follows:

[0166] In 96-well plates, immunized mouse serum was serially diluted 3-fold to an initial concentration of 1:30, with a total of 11 concentration gradients. The diluted immunized mouse serum was then mixed with pseudovirus separately (blank medium mixed with pseudovirus served as a negative control (NC), and blank medium not mixed with pseudovirus served as a blank control (MOCK)). The mixtures were incubated at 37°C for 1 hour. The immunized mouse serum-pseudovirus mixture was then transferred to 96-well plates already plated with 293T cells and incubated at 37°C for 24 hours. The number of positive cells was then detected using a CQ1 confocal cell imaging system (Yokogawa). A fitted curve was then plotted in GraphPad Prism software, and the reciprocal of the serum dilution corresponding to 50% neutralization was calculated, which is the 50% pseudovirus neutralization titer (pVNT). 50 ).

[0167] Figure 8 shows the results of the neutralization experiment of serum collected on day 56 against the pseudovirus of the Malaysian strain of NiV. Figure 8 shows:

[0168] (1) pVNT of NiV-Malaysian strain-G-head monomer at a dose of 0.2 μg 50 It is 6009, the pVNT of the NiV-Malaysia strain-G-head dimer. 50 It is 52826, the pVNT of the NiV-Indian strain-G-head monomer. 50 It is 17489, the pVNT of the NiV-India strain-G-head dimer. 50 It is 49615, the pVNT of the NiV-India-Malaysia-G-head dimer. 50The answer is 139018; that is, the neutralizing titer of pseudovirus induced by NiV-Malaysia strain-G-head dimer is significantly higher than that of NiV-Malaysia strain-G-head monomer (***), the neutralizing titer of pseudovirus induced by NiV-India strain-G-head dimer is significantly higher than that of NiV-India strain-G-head monomer (***), and the neutralizing titer of pseudovirus induced by NiV-India strain-Malaysia strain-G-head dimer is significantly higher than that of NiV-Malaysia strain-G-head dimer (**) and NiV-India strain-G-head dimer (***).

[0169] (2) pVNT of NiV-Malaysian strain-G-head monomer at a dose of 2 μg 50 It is 58153, the pVNT of the NiV-Malaysia strain-G-head dimer. 50 It is 91682, the pVNT of the NiV-Indian strain-G-head monomer. 50 It is 30044, the pVNT of the NiV-India strain-G-head dimer. 50 It is 82386, the pVNT of the NiV-India-Malaysia strain G-head dimer. 50 It is 225353; that is, the neutralizing titer of pseudovirus induced by NiV-Malaysia strain-G-head dimer is higher than that of NiV-Malaysia strain-G-head monomer, the neutralizing titer of pseudovirus induced by NiV-India strain-G-head dimer is significantly higher than that of NiV-India strain-G-head monomer immunization group (*), and the neutralizing titer of pseudovirus induced by NiV-India strain-Malaysia strain-G-head dimer is significantly higher than that of NiV-Malaysia strain-G-head dimer (**) and NiV-India strain-G-head dimer (*).

[0170] Figure 9 shows the results of the neutralization experiment of serum collected on day 56 against the pseudovirus of NiV Indian strain. Figure 9 shows:

[0171] (I) pVNT of NiV-Malaysian strain-G-head monomer at a dose of 0.2 μg 50 It is 4046, the pVNT of the NiV-Malaysia strain-G-head dimer. 50 It is 38466, the pVNT of the NiV-Indian strain-G-head monomer. 50 It is 50880, the pVNT of the NiV-India strain-G-head dimer. 50 It is 144673, the pVNT of the NiV-India-Malaysia-G-head dimer. 50The value is 150890; that is, the neutralizing titer of pseudovirus induced by NiV-Malaysia strain-G-head dimer is significantly higher than that of NiV-Malaysia strain-G-head monomer (***), the neutralizing titer of pseudovirus induced by NiV-India strain-G-head dimer is significantly higher than that of NiV-India strain-G-head monomer (***), and the neutralizing titer of pseudovirus induced by NiV-India strain-Malaysia strain-G-head dimer is not significantly different from that of NiV-India strain-G-head dimer, but is significantly higher than that of NiV-Malaysia strain-G-head dimer (***).

[0172] (II) pVNT of NiV-Malaysian strain-G-head monomer at a dose of 2 μg 50 It is 37431, the pVNT of the NiV-Malaysia strain-G-head dimer. 50 It is 60098, pVNT of NiV-India strain-G-head monomer. 50 It is 88871, the pVNT of the NiV-India strain-G-head dimer. 50 It is 146130, the pVNT of the NiV-India-Malaysia-G-head dimer. 50 The answer is 160582; that is, the neutralizing titer of pseudovirus induced by NiV-Malaysia strain-G-head dimer is higher than that of NiV-Malaysia strain-G-head monomer, the neutralizing titer of pseudovirus induced by NiV-India strain-G-head dimer is higher than that of NiV-India strain-G-head monomer, the neutralizing titer of pseudovirus induced by NiV-India strain-Malaysia strain-G-head dimer is not significantly different from that of NiV-India strain-G-head dimer, but is significantly higher than that of NiV-Malaysia strain-G-head dimer (**).

[0173] In summary, the experimental results show that the immunogenicity of the G-head dimer is superior to that of the monomeric G-head monomer, proving the feasibility of this dimer design strategy. Furthermore, the serum of mice immunized with the NiV-India strain-Malaysia strain-G-head dimer has a broader spectrum of pseudovirus neutralizing titers. In addition, among monovalent vaccines, the immunogenicity of the NiV India strain G-head monomer is higher than that of the NiV Malaysia strain G-head monomer.

[0174] Example 6: Design of constructs for HeV-G-head homodimer, NiV-HeV-G-head heterodimer, and HeV-NiV-G-Head heterodimer

[0175] In this embodiment, constructs of HeV-G-head homodimer, NiV-HeV-G-head heterodimer, and HeV-NiV-G-Head heterodimer were further designed, and the specific schemes are as follows:

[0176] Construct I: The sequence of the HeV G-head G177-S604 region (as shown in SEQ ID NO:17) and the sequence of the HeV G-head L166-S604 region (as shown in SEQ ID NO:18) are tandemly linked to obtain the sequence of the tandem dimer as shown in SEQ ID NO:19. Then, a signal peptide (as shown in SEQ ID NO:3) and a His tag (as shown in SEQ ID NO:4) are added to its N-terminus to construct the HeV-G-head dimer construct (hereinafter referred to as "HeV-G-head-dimer" in the figures or figures).

[0177] Construct II: The sequence of the NiV-India strain-G-head G177-T602 region (as shown in SEQ ID NO:1) and the sequence of the HeV-G-head L166-S604 region (as shown in SEQ ID NO:18) were tandemly connected to obtain the sequence of the tandem dimer as shown in SEQ ID NO:20. Then, a signal peptide (as shown in SEQ ID NO:3) and a His tag (as shown in SEQ ID NO:4) were added to its N-terminus to construct the NiV-HeV-G-head dimer construct (also referred to as "NiV-HeV-G-head-dimer" in the following text or figures).

[0178] Construct III: The sequence of the HeV-G-head G177-S604 region (as shown in SEQ ID NO:17) and the sequence of the NiV Indian strain G-head L166-T602 region (as shown in SEQ ID NO:2) were tandemly formed, and the sequence of the resulting tandem dimer is shown in SEQ ID NO:21. Then, a signal peptide (as shown in SEQ ID NO:3) and a His tag (as shown in SEQ ID NO:4) were added to its N-terminus to construct the HeV-NiV-G-head dimer construct (also referred to as "HeV-NiV-G-head-dimer" in the following text or figures).

[0179] Example 7: Expression and purification of HeV-G-head homodimer, NiV-HeV-G-head heterodimer, and HeV-NiV-G-Head heterodimer proteins

[0180] In this embodiment, following the construct design of Example 6, HeV-G-head homodimer, NiV-HeV-G-head heterodimer, and HeV-NiV-G-Head heterodimer proteins were constructed and expressed, respectively. The specific procedure is as follows:

[0181] Construction of expression plasmids

[0182] The amino acid sequences of the three constructs designed in Example 6 above were optimized using human codons, and the corresponding DNA coding sequences are shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively. A Kozak sequence (gccacc) was added upstream of the 5' end of these DNA coding sequences, and a stop codon was added to the 3' end. These sequences were then synthesized by Nanjing Genscript Biotech Co., Ltd. The three synthesized DNA sequences were cloned into the pCAGGS plasmid using EcoRI and XhoI restriction sites to obtain pCAGGS expression plasmids expressing HeV-G-head dimers, NiV-HeV-G-head dimers, and HeV-NiV-G-head dimers, respectively.

[0183] Protein expression and purification

[0184] HeV-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer proteins were expressed using Expi 293F cells.

[0185] Specifically, the expression plasmids pCAGGS-HeV-G-head dimer, pCAGGS-NiV-HeV-G-head dimer, and pCAGGS-HeV-NiV-G-head dimer constructed above were transfected into Expi293F cells. After 5 days, the supernatant was collected, centrifuged to remove the precipitate, and then filtered through a 0.22 μm filter membrane to further remove impurities. The filtered cell supernatant was purified by nickel affinity column chromatography. Specifically, at 4°C, the cell supernatant was passed through a nickel affinity column (Histrap, GE Healthcare) and washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specifically binding proteins. Then, impurities were eluted with a low concentration of imidazole (20 mM Tris, 150 mM NaCl, pH 8.0, 20 mM imidazole). The target protein was eluted from the HisTrap column with buffer B (20 mM Tris, 150 mM NaCl, pH 8.0, 300 mM imidazole), and the eluent was concentrated more than 30 times to buffer A using a 10 kDa concentrator, resulting in a final volume of less than 1 mL. Finally, molecular sieve chromatography was performed using a Superdex™ 200 Increase 10 / 300GL column (GE Healthcare) to further purify the target protein. The molecular sieve chromatography buffer was PBS buffer (8mM Na2HPO4, 136mM NaCl, 2mM KH2PO4, 2.6mM KCl, pH 7.4).

[0186] The molecular sieve chromatography curves of HeV-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer proteins, and the SDS-PAGE identification results of the eluent at the elution peaks are shown in Figures 10, 11, and 12, respectively. Figures 10, 11, and 12 show that HeV-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer all have an elution peak at approximately 13 mL. SDS-PAGE analysis of the eluent at this elution peak shows that the eluted proteins are all around 110 kDa, which is consistent with the molecular weight of the proteins.

[0187] The results indicate that HeV-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer proteins were obtained through the above expression and purification methods, and the electrophoretic bands were single, indicating that the purified proteins had high purity.

[0188] Example 8: Immunization and Sample Collection of Laboratory Animals

[0189] To test the immunogenicity of the HeV-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer protein vaccines designed in Example 6, we used the purified HeV-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer proteins obtained in Example 7, as well as the purified NiV-India-Malaysia-G-head dimer protein obtained in Example 2, as immunogens to immunize female BALB / c mice at 6-8 weeks of age. The negative control (Sham group) was immunized with PBS solution. Eight mice were used in each group. The BALB / c mice used were purchased from Vital River Pharmaceuticals. The immunization flowchart is shown in Figure 13; the mouse immunization grouping and dosage are shown in Table 2.

[0190] Table 2. Mouse immunization groups and immunization doses

[0191] The specific experimental procedure is as follows:

[0192] Immunogens HeV-G-head dimer, NiV-India-Malaysia-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer proteins were diluted to 40 μg / mL with PBS. The diluted immunogens were then mixed with AddaVax adjuvant at a 1:1 volume ratio and emulsified to prepare the vaccine. The negative control group consisted of a mixture of PBS solution and AddaVax adjuvant.

[0193] The vaccine obtained according to the above method was administered to BALB / c mice via intramuscular injection in the leg. All mice received their first, second, and third immunizations on days 0, 21, and 42, respectively, with each immunization using a 100 μL inoculation volume (containing 2 μg of antigen protein), and 50 μL injected into each leg. Blood was collected from the orbital venous plexus of the mice on days 19, 35, and 56 of the immunization schedule. Serum was collected by centrifugation, inactivated at 56°C for 30 min, and stored at -80°C for the detection of binding antibody titers and pseudovirus neutralizing antibody titers.

[0194] Example 9: The titers of antibodies against NiV-G-head and HeV-G-head binding in mice induced by four immunogens were detected by ELISA.

[0195] In this embodiment, the titer of antigen-specific binding antibodies in the serum of mice from each immunized group collected in Example 8 was detected by ELISA. The specific procedure is as follows:

[0196] (1) NiV-Indian strain-G-head monomer protein and HeV-G-head monomer protein were diluted to 3 μg / mL with ELISA coating buffer (Solepro, C1050), and 100 μL of the above dilution was added to each well of a 96-well ELISA plate (Coring, 3590). The plate was incubated at 4°C overnight (more than 12 h) for coating.

[0197] (2) Discard the coating solution, add PBS, and wash once; use 5% skim milk prepared with PBS as the blocking solution, add 100 μL to each well of a 96-well plate, let stand at room temperature for 1 h to block, and then wash once with PBS solution.

[0198] (3) During the blocking period, the serum samples of immunized mice were diluted with blocking buffer. The serum samples were diluted sequentially in a 4-fold gradient, starting from 20-fold. Specifically, 152 μL of blocking buffer and 8 μL of serum sample were added to the first well and mixed. The second dilution was 120 μL of blocking buffer and 40 μL of the solution from the first well. After dilution, 100 μL of blocking buffer was added to each well of the ELISA plate. The negative control group was added to the blocking buffer. The plates were incubated at 37°C for 2 hours and then washed 4 times with PBST.

[0199] (4) Add HRP-conjugated goat anti-mouse secondary antibody (BE0102-100) diluted 1:2000 with blocking buffer to each well, incubate at 37°C for 1.5 hours, then wash 3 times with PBST; then add 60 μL of TMB chromogenic solution for color development, and after an appropriate reaction time, add 60 μL of 2M hydrochloric acid to terminate the reaction, and detect the OD450 value on a microplate reader.

[0200] Antibody titer is defined as the highest dilution of serum with a reaction value greater than 2.5 times that of the negative control. When the reaction value at the lowest dilution (limit of detection) is still less than 2.5 times the background value, the titer of the sample is defined as half of the lowest dilution, i.e., 1:10.

[0201] Figure 14 shows the results of antibody titer detection against NiV-India strain-G-head monomer in serum collected on days 19, 35, and 56. The results in Figure 14 indicate that NiV-India strain-Malaysia strain-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer vaccines can induce high antibody titers against NiV-G-head, but the cross-reactivity against NiV-G-head produced by HeV-G-head dimer is weak.

[0202] Figure 15 shows the results of antibody titer detection against HeV-G-head monomers in serum collected on days 19, 35, and 56. The results in Figure 15 indicate that HeV-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer vaccines can induce high antibody titers against HeV-G-head, but the cross-reactivity against HeV-G-head produced by NiV-India strain-Malaysia strain-G-head dimer is not high.

[0203] Example 10: Detection of neutralizing antibody titers against NiV and HeV pseudoviruses induced by recombinant subunit vaccine in mice using a pseudovirus neutralization experiment.

[0204] In this embodiment, Nipah virus pseudovirus and Hendra virus pseudovirus were used to detect the neutralizing titers (pVNT) of immunized mouse serum collected on day 56 of Example 8 against NiV-India strain and HeV pseudoviruses, respectively. 50 ).

[0205] The Nipah virus and Hendra virus pseudoviruses used in this embodiment are pseudoviruses based on the vesicular stomatitis virus (VSV) backbone, displaying the G and F proteins of Nipah virus and Hendra virus. The preparation method is described in the method section of our published paper (Chen, L., Sun, M., Zhang, H. et al. Potent human neutralizing antibodies against Nipah virus derived from two ancestral antibody heavy chains. Nat Commun 15, 2987 (2024). https: / / doi.org / 10.1038 / s41467-024-47213-8).

[0206] The method for detecting neutralizing antibody titers against Nipah virus Indian strain pseudovirus and Hendra virus pseudovirus (hereinafter referred to as pseudovirus) is as follows:

[0207] In 96-well plates, immunized mouse serum was serially diluted 3-fold to an initial concentration of 1:30, with a total of 11 concentration gradients. The diluted immunized mouse serum was then mixed with pseudovirus separately (blank medium mixed with pseudovirus served as a negative control (NC), and blank medium not mixed with pseudovirus served as a blank control (MOCK)). The mixtures were incubated at 37°C for 1 hour. The immunized mouse serum-pseudovirus mixture was then transferred to 96-well plates already plated with 293T cells and incubated at 37°C for 24 hours. The number of positive cells was then detected using a CQ1 confocal cell imaging system (Yokogawa). A fitted curve was then plotted in GraphPad Prism software, and the reciprocal of the serum dilution corresponding to 50% neutralization was calculated, which is the 50% pseudovirus neutralization titer pVNT. 50 .

[0208] Figure 16 shows the results of the neutralization experiment of mouse serum collected on day 56 against the NiV-India strain pseudovirus. Figure 16 shows: pVNT of HeV-G-head dimer 50 It is 2828, the pVNT of the NiV-India strain-Malaysia-G-head dimer. 50 It is 172559, the pVNT of the NiV-HeV-G-head dimer. 50 It is 159945, the pVNT of the HeV-NiV-G-head dimer. 50 The result is 118776; that is, the titer of the HeV-G-head dimer immunization group was significantly lower than that of the other three vaccine groups (***), indicating that the immune response induced by the HeV immunogen had a low cross-neutralization response to NiV; there were no significant differences among NiV-India strain-Malaysia-G-head dimer, NiV-HeV-G-head dimer, and HeV-NiV-G-head dimer.

[0209] Figure 17 shows the results of the neutralization experiment of mouse serum against HeV pseudovirus collected on day 56. Figure 17 shows: pVNT of HeV-G-head dimer. 50 It is 52152, the pVNT of the NiV-India strain-Malaysia-G-head dimer. 50 It is 1199, the pVNT of the NiV-HeV-G-head dimer. 50 It is 27665, the pVNT of the HeV-NiV-G-head dimer. 50The result is 23482; that is, the titer of the NiV-India strain-Malaysia strain-G-head dimer immunization group was significantly lower than that of the other three vaccine groups (***), indicating that the immune response induced by the NiV immunogen was not highly effective in neutralizing HeV; there was no significant difference between NiV-HeV-G-head dimer and HeV-NiV-G-head dimer.

[0210] The results in summary indicate that the cross-reactivity between NiV and HeV is not high. However, NiV-HeV-G-head and HeV-NiV-G-head heterodimers can simultaneously induce a broader and more efficient neutralization reaction against both NiV and HeV.

[0211] Example 11: Immunization and Sample Collection of Laboratory Animals

[0212] To further investigate the immunogenicity of NiV-HeV-G-head heterodimer and HeV-NiV-G-head heterodimer in other animal models, we used the purified NiV-HeV-G-head dimer and HeV-NiV-G-head dimer obtained in Example 7 as immunogens to immunize female Syrian golden hamsters at 6-8 weeks of age. The negative control (Sham group) was immunized with PBS solution. Each group consisted of 12 hamsters. The Syrian golden hamsters used were purchased from Vital Rivers. The immunization procedure is shown in Figure 18; the hamster immunization grouping and dosage are shown in Table 3 below.

[0213] Table 3. Hamster Immunization Groups and Doses

[0214] The specific experimental procedure is as follows:

[0215] Immunogens NiV-HeV-G-head dimer and HeV-NiV-G-head dimer protein were diluted to 200 μg / mL with PBS. The diluted immunogens were then mixed with AddaVax adjuvant at a 1:1 volume ratio and emulsified to prepare the vaccine. The negative control group consisted of a mixture of PBS solution and AddaVax adjuvant.

[0216] Specifically, the vaccine obtained according to the above method was administered to Syrian golden hamsters via intramuscular injection in the leg. The first, second, and third immunizations were given on days 0, 21, and 42, respectively, with each injection consisting of a 100 μL inoculation volume (containing 10 μg of antigen protein), injected into each hamster. Blood was collected from the orbital venous plexus of the Syrian golden hamsters on days 19, 35, and 56 of the immunization schedule. Serum was collected by centrifugation, inactivated at 56°C for 30 min, and then stored at -80°C for the detection of binding antibody titers and neutralizing antibody titers.

[0217] Example 12: Detection of antibody titers binding to NiV-HeV-G-head dimers and HeV-NiV-G-head dimer vaccines in Syrian golden hamsters using ELISA assay.

[0218] In this embodiment, the titer of antigen-specific binding antibodies in the serum of Syrian golden hamsters immunized with NiV-HeV-G-head dimer and HeV-NiV-G-head dimer proteins collected in Example 11 was detected by ELISA. The specific procedure is as follows:

[0219] (1) NiV-Indian strain G-head monomer and HeV-G-head monomer were diluted to 3 μg / mL with ELISA coating buffer (Solepro, C1050), and 100 μL of the above dilution was added to each well of a 96-well ELISA plate (Coring, 3590). The plate was incubated at 4°C overnight (more than 12 h) for coating.

[0220] (2) Discard the coating solution, add PBS, and wash once; use 5% skim milk prepared with PBS as the blocking solution, add 100 μL to each well of a 96-well plate, let stand at room temperature for 1 h to block, and then wash once with PBS solution.

[0221] (3) During the blocking period, the serum samples of the immunized Syrian golden hamsters were diluted with blocking buffer. The initial concentration of the serum samples was 20-fold, and the dilution was carried out in a 4-fold gradient. Specifically, 152 μL of blocking buffer and 8 μL of serum sample were added to the first well and mixed. The second dilution was 120 μL of blocking buffer and 40 μL of the solution from the first well. After dilution, 100 μL of blocking buffer was added to each well of the ELISA plate, and the negative control group was added to the blocking buffer. The plates were incubated at 37°C for 2 hours, and then washed 4 times with PBST.

[0222] (4) Add goat anti-Syrian hamster IgG H&L (Abcam, Ab6892) conjugated with HRP to each well, diluted 1:7000 with blocking buffer, incubate at 37°C for 1.5 hours, then wash 3 times with PBST; then add 60 μL of TMB chromogenic solution for color development, and after an appropriate reaction time, add 60 μL of 2M hydrochloric acid to terminate the reaction, and detect the OD450 value on a microplate reader.

[0223] Antibody titer is defined as the highest dilution of serum with a reaction value greater than 2.5 times that of the negative control. When the reaction value at the lowest dilution (limit of detection) is still less than 2.5 times the background value, the titer of the sample is defined as half of the lowest dilution, i.e., 1:10.

[0224] Figure 19 shows the titer of binding antibodies against NiV-India strain G-head monomer protein in serum collected from each immunization group on days 19, 35, and 56. Figure 19 shows that both the NiV-HeV-G-head dimer and the HeV-NiV-G-head dimer vaccines produced high levels of binding antibodies against NiV-G-head protein after immunization.

[0225] Figure 20 shows the titer of binding antibodies against HeV-G-head monomers in serum collected from each immunization group on days 19, 35, and 56. Figure 20 shows that both the NiV-HeV-G-head dimer and the HeV-NiV-G-head dimer vaccines produced high levels of binding antibodies against the HeV-G-head protein after immunization.

[0226] Example 13: Detection of neutralizing antibody titers against NiV and HeV produced in Syrian golden hamsters stimulated by NiV-HeV-G-head dimer and HeV-NiV-G-head dimer vaccines using a pseudovirus neutralization experiment.

[0227] In this embodiment, Nipah virus pseudovirus and Hendra virus pseudovirus were used to detect the neutralizing antibody titers (pVNTs) against NiV and HeV pseudoviruses in immunized hamster serum collected on day 56 of Example 11. 50 ).

[0228] The Nipah virus and Hendra virus pseudoviruses used in this embodiment are pseudoviruses based on the vesicular stomatitis virus (VSV) backbone, displaying the G and F proteins of Nipah virus and Hendra virus. The preparation method is described in the method section of a paper published by our research group (Chen, L., Sun, M., Zhang, H. et al. Potent human neutralizing antibodies against Nipah virus derived from two ancestral antibody heavy chains. Nat Commun 15, 2987 (2024). https: / / doi.org / 10.1038 / s41467-024-47213-8).

[0229] The method for detecting neutralizing antibody titers against Nipah virus pseudovirus and Hendra virus pseudovirus (hereinafter referred to as pseudoviruses) is as follows:

[0230] In 96-well plates, immunized Syrian golden hamster serum was serially diluted 3-fold, with an initial dilution of 1:30, resulting in 11 concentration gradients. The diluted serum was then mixed with pseudovirus (blank medium mixed with pseudovirus served as a negative control (NC), and blank medium not mixed with pseudovirus served as a blank control (MOCK)). The mixtures were incubated at 37°C for 1 hour. The immunized Syrian golden hamster serum-pseudovirus mixture was then transferred to 96-well plates already plated with 293T cells and incubated at 37°C for 24 hours. Positive cell counts were then detected using a CQ1 confocal cell imaging system (Yokogawa). A fitted curve was plotted in GraphPad Prism software, and the reciprocal of the serum dilution corresponding to 50% neutralization was calculated, representing the 50% pseudovirus neutralization titer pVNT. 50 .

[0231] Figure 21 shows the results of the pseudovirus neutralization experiment of the NiV Indian strain in serum collected on day 56 from immunized hamsters. Figure 21 shows the pVNT of the NiV-HeV-G-head dimer. 50 It is 1526, the pVNT of the HeV-NiV-G-head dimer. 50 The titer of neutralizing antibodies against the NiV Indian strain pseudovirus induced by the HeV-NiV-G-head dimer was significantly higher than that of the NiV-HeV-G-head dimer immunization group (*).

[0232] Figure 22 shows the results of the pseudovirus neutralization experiment of immunized hamster serum collected on day 56 against HeV. Figure 22 shows: pVNT of NiV-HeV-G-head dimer 50 It is 203, the pVNT of the HeV-NiV-G-head dimer. 50 It is 533. The neutralizing antibody titer against HeV pseudovirus induced by HeV-NiV-G-head dimer was significantly higher than that of the NiV-HeV-G-head dimer immunization group (*).

[0233] In summary, the results indicate that both the NiV-HeV-G-head dimer and the HeV-NiV-G-head dimer vaccines induced strong immune responses in hamsters, producing high levels of neutralizing antibody titers against NiV and HeV. Furthermore, the neutralizing antibody titer induced by the HeV-NiV-G-head dimer vaccine was higher than that induced by the NiV-HeV-G-head dimer vaccine.

[0234] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the present invention. Industrial applicability

[0235] This application provides a Nipah / Hendra virus recombinant antigen encoding a nucleic acid, a vaccine or immunogenic composition based on the recombinant antigen or its encoded nucleic acid, and the use of the aforementioned products in the preparation of vaccines for the prevention and / or treatment of Nipah virus and / or Hendra virus infection. The Nipah / Hendra virus recombinant antigen of this invention can effectively elicit a strong immune response against multiple strains of HeV and / or NiV, and has great value and broad development prospects in the field of clinical application.

Claims

1. A Nipah / Hendra virus recombinant antigen, characterized in that, The recombinant antigen has an amino acid sequence arranged according to one of the structures shown in formulas (I) to (IV) below: (AB)-C-(A-B') (I) (A-B')-C-(AB) (II) (AB)1-C-(AB)2 (III) (A-B')1-C-(A-B')2 (IV) In equations (I) to (II): (AB) represents an amino acid sequence of the head domain or a portion thereof of the Nipah virus G protein, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to it and has the same or substantially the same immunogenicity as it. (A-B') represents the amino acid sequence of the head domain or a portion thereof of the Hendra virus G protein, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and has the same or substantially the same immunogenicity as it. C is either empty or a connected subsequence; In equations (III) to (IV): (AB)1 and (AB)2 each independently represent the amino acid sequence of the head domain or a portion thereof of the Nipah virus G protein, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it and has the same or substantially the same immunogenicity as it. (A-B')1 and (A-B')2 each independently represent an amino acid sequence of the head domain or a portion thereof of the Hendra virus G protein, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity as it. C represents a sequence of no or no connected subsequences.

2. The Nipah / Hendra virus recombinant antigen according to claim 1, characterized in that, In equations (I) to (II): (AB) represents the G177-T602 segment or L166-T602 segment of the head domain of the Nipah virus G protein; And / or, (A-B') represents the G177-S604 segment or L166-S604 segment of the Hendra virus G protein head domain; And / or, C is none or is a connector (GGS) n , where n is an integer between 1 and 10.

3. The Nipah / Hendra virus recombinant antigen according to claim 2, characterized in that, In formula (I): (AB) represents the G177-T602 segment of the head domain of the Nipah virus G protein; preferably, (AB) represents the amino acid sequence shown in SEQ ID NO:1, or the amino acid sequence shown in SEQ ID NO:1 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it. And / or, (A-B') represents the L166-S604 segment of the head domain of the Hendra virus G protein; preferably, (A-B') represents the amino acid sequence as shown in SEQ ID NO:18, or the amino acid sequence as shown in SEQ ID NO:18 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity. Optionally, C is none; Preferably, the amino acid sequence of formula (I) is as shown in SEQ ID NO:

20.

4. The Nipah / Hendra virus recombinant antigen according to claim 2, characterized in that, In formula (II): (A-B') represents the G177-S604 segment of the head domain of the Hendra virus G protein; preferably, (A-B') represents the amino acid sequence as shown in SEQ ID NO:17, or the amino acid sequence as shown in SEQ ID NO:17 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity. And / or, (AB) represents the L166-T602 segment of the head domain of the Nipah virus G protein; preferably, (AB) represents the amino acid sequence as shown in SEQ ID NO:2, or the amino acid sequence as shown in SEQ ID NO:2 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it. Optionally, C is none; Preferably, the amino acid sequence of formula (II) is shown in SEQ ID NO:

21.

5. The Nipah / Hendra virus recombinant antigen according to claim 1, characterized in that, In formula (III): (AB)1 represents the G177-T602 segment of the head domain of the Nipah virus G protein; preferably, (AB)1 represents the amino acid sequence as shown in SEQ ID NO:1 or 8, or the amino acid sequence as shown in SEQ ID NO:1 or 8 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity. And / or, (AB)2 represents the L166-T602 segment of the head domain of the Nipah virus G protein; preferably, (AB)2 represents the amino acid sequence as shown in SEQ ID NO:2 or 9, or the amino acid sequence as shown in SEQ ID NO:2 or 9 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it. And / or, C is none or is a connector (GGS) n , where n is an integer between 1 and 10; preferably, C is none.

6. The Nipah / Hendra virus recombinant antigen according to claim 5, characterized in that, (AB)1 and (AB)2 are the corresponding segments of the head domain of the Nipah virus G protein from the same or different strains, preferably the corresponding segments of the head domain of the Nipah virus G protein from different strains. Preferably, the amino acid sequence of formula (III) is shown in one of SEQ ID NO:7, 10, 11.

7. The Nipah / Hendra virus recombinant antigen according to claim 1, characterized in that, In formula (IV): (A-B')1 represents the G177-S604 segment of the head domain of the Hendra virus G protein; preferably, (A-B')1 represents the amino acid sequence as shown in SEQ ID NO:17, or the amino acid sequence as shown in SEQ ID NO:17 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity. And / or, (A-B')2 represents the L166-S604 segment of the head domain of the Hendra virus G protein; preferably, (A-B')2 represents the amino acid sequence as shown in SEQ ID NO:18, or the amino acid sequence as shown in SEQ ID NO:18 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it. And / or, C is none or is a connector (GGS) n Where n is an integer between 1 and 10; preferably, C is none; Optionally, (A-B')1 and (A-B')2 are corresponding segments of the head domain of the G protein of the same or different strains of Hendra virus. Preferably, the amino acid sequence of formula (IV) is shown in SEQ ID NO:

19.

8. The Nipah / Hendra virus recombinant antigen according to any one of claims 1-7, characterized in that, The N-terminus of the Nipah / Hendra virus recombinant antigen also includes a signal peptide sequence and / or a tag sequence; Preferably, the signal peptide sequence is as shown in SEQ ID NO:3; Preferably, the label is a His label, more preferably a 6×His label.

9. A method for preparing the Nipah / Hendra virus recombinant antigen as described in any one of claims 1-7, comprising the following steps: By adding a Kozak sequence, a signal peptide, and / or a tag coding sequence to the 5' end of the nucleotide sequence encoding the Nipah / Hendra virus recombinant antigen as described in any one of claims 1-7, and adding a stop codon to the 3' end, cloning and expression are performed. Correct recombinants are screened, and then they are transfected into expression system cells for expression. The cell culture supernatant is collected, and the recombinant antigen is isolated from it.

10. The preparation method according to claim 9, characterized in that: The expression system cells are mammalian cells, insect cells, yeast cells, or bacterial cells; Optionally, the mammalian cells are HEK293T cells, 293F series cells, or CHO cells; more preferably, the 293F series cells are HEK293F cells, Freestyle293F cells, or Expi293F cells. Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells, or S2 cells; Optionally, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom; Optionally, the bacterial cells are Escherichia coli cells.

11. A polynucleotide encoding the Nipah / Hendra virus recombinant antigen as described in any one of claims 1-8.

12. The polynucleotide according to claim 11, characterized in that, The polynucleotide is DNA or mRNA; Preferably, the polynucleotide has a DNA sequence as shown in one of SEQ ID NO:23, 24, 13, 15, 16, 22, or a corresponding mRNA sequence.

13. A nucleic acid construct comprising a polynucleotide as described in claim 11 or 12, and optionally, at least one expression regulatory element operatively linked to said polynucleotide.

14. An expression vector comprising the nucleic acid construct as described in claim 13.

15. A host cell wherein it is transformed or transfected with the polynucleotide of claim 11 or 12, the nucleic acid construct of claim 13, or the expression vector of claim 14.

16. The use of the Nipah / Hendra virus recombinant antigen as described in any one of claims 1-8, the polynucleotide as described in claim 11 or 12, the nucleic acid construct as described in claim 13, the expression vector as described in claim 14, or the host cell as described in claim 15 in the preparation of a vaccine for the prevention and / or treatment of Nipah virus and / or Hendra virus infection.

17. A vaccine or immunogenic composition comprising a Nipah / Hendra virus recombinant antigen as claimed in any one of claims 1-8, a polynucleotide as claimed in claim 11 or 12, a nucleic acid construct as claimed in claim 13, an expression vector as claimed in claim 14, or a host cell as claimed in claim 15, and a physiologically acceptable medium, adjuvant, excipient, carrier, and / or diluent.

18. The vaccine or immunogenic composition according to claim 17, which is a Nipah / Hendra virus recombinant protein vaccine, comprising the Nipah / Hendra virus recombinant antigen and adjuvant as described in any one of claims 1-8; Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, and MF59-like adjuvant; preferably, the MF59-like adjuvant is AddaVax adjuvant.

19. The vaccine or immunogenic composition according to claim 17, which is a Nipah / Hendra virus DNA vaccine, said DNA vaccine comprising: (i) Eukaryotic expression vectors; and (ii) Constructing a DNA sequence encoding the Nipah / Hendra virus recombinant antigen as described in any one of claims 1-8 into the eukaryotic expression vector, preferably a DNA sequence as shown in any one of SEQ ID NO: 23, 24, 13, 15, 16, 22; Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

20. The vaccine or immunogenic composition according to claim 17, wherein it is a Nipah / Hendra virus mRNA vaccine, said mRNA vaccine comprising: (I) An mRNA sequence encoding the Nipah / Hendra virus recombinant antigen as described in any one of claims 1-8; and (II) Lipid nanoparticles.

21. The vaccine or immunogenic composition according to claim 17, which is a Nipah / Hendra virus-viral vector vaccine, comprising: (1) Viral backbone vector; and (2) A DNA sequence encoding the recombinant Nipah / Hendra virus antigen as described in any one of claims 1-8, preferably a DNA sequence as shown in any one of SEQ ID NO: 23, 24, 13, 15, 16, 22, is constructed into the viral backbone vector; Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

22. The vaccine or immunogenic composition according to claim 17, wherein it is a Nipah / Hendra virus nanoparticle vaccine; Preferably, the carrier of the nanoparticles is ferritin, and the Nipah / Hendra virus recombinant antigen as described in any one of claims 1-8 is covalently linked to ferritin and self-assembled into nanoparticles, so that the Nipah / Hendra virus recombinant antigen is present on the surface of the nanoparticles; more preferably, the Nipah / Hendra virus recombinant antigen and ferritin are linked by a linker or covalently linked by a SpyTag / SpyCatcher linker system.

23. The vaccine or immunogenic composition according to any one of claims 17-22, characterized in that, The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation; Preferably, the nasal spray is selected from aerosols, sprays, and powders; Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments; More preferably, the tablet is a sublingual tablet; More preferably, the granules are fine granules; More preferably, the powder is a granule; More preferably, the pills are small pills; Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation.

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