Bivalent mRNA vaccine against nipah virus and hendra virus, preparation method therefor and use thereof

Abstract

The present invention relates to an RNA molecule encoding a recombinant Nipah / Hendra virus antigen, a vaccine or immunogenic composition comprising same, and the use thereof in the preparation of a vaccine for preventing and / or treating Nipah virus and / or Hendra virus infections. The RNA vaccine encoding the recombinant Nipah / Hendra virus antigen of the present invention can simultaneously elicit a strong immune response against HeV and NiV, and has great value and broad development prospects in clinical applications.

Description

Bivalent mRNA vaccines against Nipah and Hendra viruses, their preparation methods and applications

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202510200244.2 filed with the China Patent Office on February 21, 2025, entitled "A Bivalent mRNA Vaccine Against Nipah and Hendra Viruses, Its Preparation Method and Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biomedicine, specifically to a bivalent mRNA vaccine against Nipah virus and / or Hendra virus, its preparation method, and its application. Background Technology

[0004] In today's globalized world, emerging and re-emerging infectious diseases have become a serious challenge for global public health. Nipah virus (NiV) and Hendra virus (HeV) are both considered important pathogens that could lead to large-scale epidemics in the future, requiring focused monitoring and research. Both viruses belong to the genus Hennipahvirus in the family Paramyxoviridae, and are negative-sense RNA viruses with extremely high pathogenicity and mortality rates. They are zoonotic infectious pathogens.

[0005] Since its initial discovery in Malaysia in 1998, Nipah virus has caused nearly 300 cases, with over 100 deaths. Over the past two decades, the virus has experienced seasonal outbreaks in Southeast Asian countries such as Bangladesh and India. Unlike the strain prevalent in Malaysia, the strain circulating in Bangladesh is more strongly transmitted from person to person. The natural host of Nipah virus is the fruit bat, and humans are infected by consuming fruit or fruit products (such as raw date juice) contaminated with the urine or saliva of infected fruit bats. Nipah virus infection can cause symptoms such as fever, headache, cough, and difficulty breathing; in severe cases, it can lead to encephalitis and even death, with a high mortality rate.

[0006] Hendra virus shares similar pathological characteristics with Nipah virus and is primarily transmitted across species to horses and humans via flying foxes. As of July 2022, Australia had recorded 66 naturally occurring Hendra virus spillover events, resulting in the deaths of 105 horses and 7 confirmed human cases, including 4 deaths. A Hendra virus vaccine for horses (…) HeV has been approved in Australia, but there is no vaccine available for human use yet.

[0007] Given the high pathogenicity and potential human-to-human transmission of Nipah and Hendra viruses, they pose a serious threat to human health and global public health. Currently, there are no approved treatments or vaccines for human use against either virus; therefore, developing a comprehensive strategy to combat Nipah / Hendra viruses and prevent zoonotic outbreaks is particularly urgent. Currently, there are no effective vaccines simultaneously targeting NiV and HeV. Summary of the Invention

[0008] Purpose of the invention

[0009] In response to the needs of the prior art, the present invention provides a bivalent mRNA vaccine against Nipah / Hendra virus that can effectively elicit a strong immune response against both HeV and NiV, its preparation method, and its application.

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

[0011] In a first aspect, the present invention provides an RNA molecule encoding a Nipah / Hendra virus recombinant antigen having an amino acid sequence arranged as shown in formula (I) or formula (II):

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

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

[0014] In equations (I) to (II):

[0015] (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.

[0016] (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.

[0017] C is either empty or a concatenated subsequence.

[0018] Preferably, in equations (I) to (II) above:

[0019] (AB) represents the G177-T602 segment or L166-T602 segment of the head domain of the Nipah virus G protein;

[0020] (A-B') represents the G177-S604 or L166-S604 segment of the head domain of the Hendra virus G protein.

[0021] C represents either none or a connector (GGS). n , where n is an integer between 1 and 10.

[0022] For equation (I):

[0023] In a feasible implementation, (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:7, or the amino acid sequence shown in SEQ ID NO:7 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it.

[0024] 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:8, or the amino acid sequence as shown in SEQ ID NO:8 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity as it.

[0025] Optionally, C is none;

[0026] In a preferred embodiment, the amino acid sequence of formula (I) encoded by the RNA molecule is shown in SEQ ID NO:9.

[0027] SEQ ID NO:9-NiV / HeV G-head dimer sequence (SEQ ID NO:7+SEQ ID NO:8):

[0028] For equation (II):

[0029] In a feasible implementation, (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 shown in SEQ ID NO:10, or the amino acid sequence shown in SEQ ID NO:10 obtained by substituting, deleting or adding one or more amino acids, and having the same or substantially the same immunogenicity.

[0030] 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 shown in SEQ ID NO:11, or the same immunogenic amino acid sequence as SEQ ID NO:11;

[0031] Optionally, C is none;

[0032] In a preferred embodiment, the amino acid sequence of formula (II) encoded by the RNA molecule is shown in SEQ ID NO:12.

[0033] SEQ ID NO:12-HeV / NiV G-head dimer sequence (SEQ ID NO:10+SEQ ID NO:11):

[0034] Most preferably, the RNA molecule has an RNA sequence as shown in SEQ ID NO:17 or 18, which encodes the Nipah / Hendra virus recombinant antigen with an amino acid sequence as shown in SEQ ID NO:9 or 12.

[0035] The mRNA coding sequence of SEQ ID NO:17-NiV / HeV G-head dimer (i.e., SEQ ID NO:9):

[0036] The mRNA coding sequence of SEQ ID NO:18-HeV / NiV G-head dimer (i.e., SEQ ID NO:12):

[0037] Optionally, the N-terminus of the Nipah / Hendra virus recombinant antigen also includes a signal peptide sequence.

[0038] Preferably, the signal peptide sequence is shown in SEQ ID NO:19, and its coding sequence is shown in SEQ ID NO:20.

[0039] In a second aspect, the present invention provides a nucleic acid construct comprising a nucleotide sequence of an RNA molecule as described in the first aspect above, and optionally, at least one expression regulatory element operatively linked to the nucleotide sequence of the RNA molecule.

[0040] Thirdly, the present invention provides an expression vector comprising the nucleic acid construct as described in the second aspect above.

[0041] Fourthly, the present invention provides a host cell wherein the present invention is transformed or transfected with an RNA molecule as described in the first aspect above, a nucleic acid construct as described in the second aspect above, or an expression vector as described in the third aspect above.

[0042] Fifthly, the present invention provides the use of RNA molecules as described in the first aspect above, nucleic acid constructs as described in the second aspect above, expression vectors as described in the third aspect above, or host cells as described in the fourth aspect above in the preparation of medicaments for the prevention and / or treatment of Nipah virus and / or Hendra virus infection.

[0043] Preferably, the drug is a vaccine.

[0044] More preferably, the vaccine is an mRNA vaccine.

[0045] In a sixth aspect, the present invention provides a vaccine or immunogenic composition comprising an RNA molecule as described in the first aspect above, a nucleic acid construct as described in the second aspect above, an expression vector as described in the third aspect above, or a host cell as described in the fourth aspect above, and physiologically acceptable mediators, adjuvants, excipients, carriers, and / or diluents.

[0046] In some preferred embodiments, the vaccine or immunogenic composition is an mRNA vaccine against Nipah / Hendra virus, the mRNA vaccine comprising:

[0047] (I) RNA molecules as described in the first aspect above; and

[0048] (II) Lipid nanoparticles.

[0049] Preferably, the lipid nanoparticles are composed of cationic lipid Dlin-MC3-DMA, distearate phosphatidylcholine (DSPC), cholesterol, and PEGylated lipid DMPE; more preferably, the molar ratio of cationic lipid Dlin-MC3-DMA, distearate phosphatidylcholine (DSPC), cholesterol, and PEGylated lipid DMPE is (40-60):(8-12):(36-40):(1-2), and more preferably 50:10:38.5:1.5.

[0050] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

[0051] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0052] Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0053] More preferably, the tablet is a sublingual tablet;

[0054] More preferably, the granules are fine granules;

[0055] More preferably, the powder is a granule;

[0056] More preferably, the pills are small pills;

[0057] 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.

[0058] In a seventh aspect, 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: RNA molecules as described in the first aspect above, nucleic acid constructs as described in the second aspect above, expression vectors as described in the third aspect above, host cells as described in the fourth aspect above, and / or vaccines or immunogenic compositions as described in the sixth aspect above.

[0059] 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

[0060] Based on structural biology analysis, this invention designs an RNA molecule encoding a Nipah / Hendra virus recombinant antigen. This RNA molecule encodes a Nipah / Hendra virus recombinant antigen that is a dimer structure formed by the tandem linking specific fragments of the head domains of the Nipah virus (NiV) and Hendra virus (HeV) G proteins. The inventors have experimentally demonstrated that the mRNA vaccine based on this RNA molecule can simultaneously elicit a strong immune response against both HeV and NiV, thus possessing the potential to address potential future HeV and NiV pandemics, indicating its significant value and broad development prospects in the clinical application field. Attached Figure Description

[0061] 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.

[0062] Figure 1 is a schematic diagram of the experimental procedure for immunizing BALB / c mice with mRNA vaccine as described in Example 2 of the present invention.

[0063] Figure 2 shows the results of humoral immunity experiments induced by immunization of mice with mRNA vaccine according to the experimental procedure in Figure 1; Figure A shows the titer of IgG binding antibody against the extracellular domain (G-ecto) of NiV G protein in the serum of immunized mice collected on day 28; Figure B shows the titer of neutralizing antibody against NiV pseudovirus in the serum of immunized mice collected on day 28.

[0064] Figure 3 is a schematic diagram of the experimental procedure for immunizing BALB / c mice with mRNA vaccine as described in Example 4 of the present invention.

[0065] Figure 4 shows the results of antigen-specific binding antibody titers induced by mRNA vaccine immunization of mice following the experimental procedure in Figure 3; Figures A and B show the results of IgG antibody titers against NiV G-head and HeV G-head proteins in the serum of immunized mice collected on day 35, respectively.

[0066] Figure 5 shows the results of neutralizing antibody titer detection induced by mRNA vaccine immunization of mice according to the experimental procedure in Figure 3; Figure A and Figure B show the results of neutralizing antibody titer detection against NiV and HeV pseudoviruses in the serum of immunized mice collected on day 35, respectively.

[0067] Figure 6 is a schematic diagram of the experimental procedure for immunizing golden hamsters with mRNA vaccine as described in Example 5 of the present invention.

[0068] Figure 7 shows the results of antigen-specific binding antibody titer detection in serum collected from immunized hamsters on day 19, following the experimental procedure shown in Figure 6. Figures A and B show the results of IgG antibody titer detection in serum against NiV G-head protein and HeV G-head protein, respectively.

[0069] Figure 8 shows the results of antigen-specific binding antibody titer detection in serum collected from immunized hamsters on day 35, following the experimental procedure in Figure 6; Figure A and Figure B show the results of serum IgG binding antibody titer detection against NiV G-head protein and HeV G-head protein, respectively.

[0070] Figure 9 shows the results of neutralizing antibody titer detection in serum collected from immunized hamsters on day 19, following the experimental procedure in Figure 6; Figures A and B show the results of neutralizing antibody titer detection in serum against NiV and HeV pseudoviruses, respectively.

[0071] Figure 10 shows the results of neutralizing antibody titer detection in serum collected from immunized hamsters on day 35, following the experimental procedure shown in Figure 6; Figures A and B show the results of neutralizing antibody titer detection in serum against NiV and HeV pseudoviruses, respectively. Detailed Implementation

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Example 1: Design of mRNA constructs with different NiV G protein domains and preparation of mRNA vaccines

[0076] In this embodiment, based on the structural analysis of the NiV G protein, the following mRNA construct was designed and constructed:

[0077] (1) The mRNA construct encoding the full-length (G-full length) NiV G protein,

[0078] (2) mRNA construct encoding the extracellular domain (G-ecto) of the NiV G protein, and

[0079] (3) mRNA construct encoding the head domain (G-head) of NiV G protein.

[0080] The specific construction method is as follows:

[0081] 1) Sequence design of the mRNA coding region of the mRNA construct

[0082] Based on the NiV Bangladeshi strain (GenBank: MK673566.1), the full-length NiV G protein antigen was obtained, and its amino acid sequence is shown in SEQ ID NO: 1; the extracellular domain (G-ecto) of the NiV G protein, and its amino acid sequence are shown in SEQ ID NO: 2; the head domain (G-head) of the NiV G protein, and its amino acid sequence are shown in SEQ ID NO: 3.

[0083] 2) Expression plasmid construction, linearization, in vitro transcription and construction

[0084] First, pcDNA3.1 was selected as the plasmid vector. Using conventional molecular biology techniques, DNA coding sequences of different domains of the NiV G protein (as shown in SEQ ID NO:4, 5, and 6, respectively) and other DNA expression elements were introduced. These included, in order from upstream to downstream: the T7 promoter, the 5' UTR sequence upstream of the coding region, the signal peptide coding sequence (as shown in SEQ ID NO:19, and its coding sequence in SEQ ID NO:20), the coding sequence of a 6-histidine tag, the DNA coding sequence of the vaccine antigen, the downstream 3' UTR sequence, and the poly-A-tail. The resulting expression plasmids for the three mRNA constructs were named pcDNA3.1-G-full length, pcDNA3.1-G-ecto, and pcDNA3.1-G-head, respectively.

[0085] Then, the plasmid was digested with the restriction endonuclease EcoR321I to linearize it, and then purified using conventional DNA purification methods to obtain a template for in vitro transcription.

[0086] Next, based on this template, in vitro transcription was performed using the T7RNA in vitro transcription kit (E131-01A, Suzhou Nearshore Protein Technology Co., Ltd.) to obtain in vitro transcribed mRNA; then, the mRNA was purified by lithium chloride precipitation using the lithium chloride recovery kit (S125, Suzhou Nearshore Protein Technology Co., Ltd.) to obtain purified in vitro transcribed mRNA.

[0087] Finally, the purified in vitro transcribed mRNA was capped at the 5' end using a Cap1 capping enzyme kit (M082-01B, Suzhou Nearshore Protein Technology Co., Ltd.) to meet the conditions for translation in eukaryotic cells. Subsequently, the mRNA was purified again using the same lithium chloride precipitation method as above to obtain purified mRNA with 5' capping modification.

[0088] 3) Liposome packaging of mRNA

[0089] The cationic lipid Dlin-MC3-DMA, distearate phosphatidylcholine (DSPC), cholesterol, and PEGylated lipid DMPE were mixed in a molar ratio of 50:10:38.5:1.5, and then the mixture was prepared using the INano nanomedicine manufacturing system manufactured by Myannah Pharmaceuticals. TM E, is mixed with the 5' capped mRNA described above and packaged to form nanoparticles.

[0090] After packaging, the buffer solution was replaced with D-PBS by centrifugation for use as an mRNA vaccine.

[0091] Example 2: BALB / c mice were immunized once with mRNA vaccines containing different NiV G protein domains to evaluate their immunogenicity.

[0092] To compare the immunogenicity of the three mRNA vaccines encoding NiV G-full length, G-ecto, and G-head constructed in Example 1, animal immunization experiments were conducted using 6-8 week old female BALB / c mice (purchased from Vital River). The experimental groups, immunization doses, and procedures are shown in Figure 1.

[0093] Specifically, BALB / c mice were immunized with the mRNA vaccines prepared in Example 1, and their binding antibody titers and neutralizing antibody titers were detected.

[0094] The immunization experiment was divided into the following groups: an mRNA vaccine immunization group and a negative control group. The mRNA vaccine immunization group included a G-full-length mRNA vaccine immunization group, a G-ecto mRNA vaccine immunization group, and a G-head mRNA vaccine immunization group. The negative control group was a GFP mRNA immunization group. All mice in the mRNA vaccine immunization group were immunized with the respective mRNA vaccines (i.e., G-full-length, G-ecto, and G-head vaccines) on day 0. Each mRNA vaccine was administered at two doses: 0.1 μg mRNA and 1 μg mRNA per mouse. The immunization procedure for the negative control group was the same as that for the vaccine groups, with an immunization dose of 1 μg mRNA per mouse. All doses were administered via intramuscular injection. Serum samples were collected on day 28 post-immunization to detect the levels of antigen-specific binding antibodies and neutralizing antibodies in the mouse serum.

[0095] ELISA method for detecting antigen-specific binding antibody levels

[0096] The specific method for detecting the titer of IgG-binding antibodies in the serum of immunized BALB / c mice using an ELISA assay is as follows: NiV G-ecto protein (amino acid sequence as shown in SEQ ID NO: 2) was diluted to 3 μg / ml with ELISA coating buffer (sodium carbonate-sodium bicarbonate buffer, pH 9.6), and 100 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The coating buffer was discarded the next day, and the ELISA plate was blocked with 5% skim milk prepared with PBS and incubated at room temperature for 1 hour. Serum samples from mice in each experimental group in Example 2 were serially diluted four times starting from 1:20 in 96-well round plates, and then the diluted solutions were transferred to ELISA plates. The ELISA plates were then incubated at 37°C for 1.5 hours. The blocking solution was discarded, and the plates were washed four times with PBST. Then, Goat Anti-Mouse HRP secondary antibody (purchased from Jinpulai) diluted 1:2000 with blocking solution was added, and the plates were incubated at 37°C for 1.5 hours and washed five times with PBST. Then, 60 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added for color development. After 10 minutes, 60 μL of 2M hydrochloric acid was added to terminate the reaction. The OD was measured using a PerkinElmer microplate reader. 450 Reading value. The endpoint titer is defined as the serum dilution factor corresponding to a reaction absorbance greater than 2.5 times the background value. Antibody titers below the detection limit are defined as half of the detection limit.

[0097] The titer of IgG antibodies binding to the G-ecto protein antigen in the serum of mice that underwent a single immunization is shown in Figure 2A. It indicates that mice in the 1 μg and 0.1 μg dose groups of the G-head mRNA vaccine were induced to produce G-ecto protein-specific binding antibodies on day 28, with mean antibody titers of 9.2 × 10⁻⁶ and 9.1 μg, respectively. 4 and 4.56×10 3 The mean titers of binding antibodies induced by the 1 μg and 0.1 μg doses of the G-full length mRNA vaccine were 4.6 × 10⁻⁶ and 4.6 × 10⁻⁶, respectively. 4 The mean titers of binding antibodies induced by the 1 μg and 0.1 μg doses of the G-ecto mRNA vaccine were 4.06 × 10⁻⁶ and 226, respectively. 3 And 12. Comparative analysis shows that the G-head mRNA vaccine induced the highest titer of antigen-specific binding antibodies.

[0098] The pseudovirus neutralization assay detects the level of neutralizing antibody titers.

[0099] The NiV pseudovirus used in this embodiment is a pseudovirus displaying NiV surface glycoproteins F and G, prepared based on the vesicular stomatitis virus (VSV) backbone. The preparation method is described in the method section of our published paper (Effects of a Prolonged Booster Interval on Neutralization of Omicron Variant, N Engl J Med, 2022, PMID:35081296).

[0100] The method for detecting neutralizing antibody titers in pseudoviruses is as follows:

[0101] In 96-well plates, immunized mouse serum was serially diluted 4-fold to an initial concentration of 1:20, 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 pre-coated with HEK 293T cells and incubated at 37°C for 24 hours. The number of positive cells was detected using a CQ1 confocal cell imaging system (Yokogawa), and a fitted curve was plotted in GraphPad Prism software. The reciprocal of the serum dilution corresponding to 50% neutralization was calculated as the pseudovirus neutralizing titer.

[0102] The results of neutralizing antibody titers against NiV pseudovirus in the serum of mice immunized as described above are shown in Figure 2B. The figures show that the mean neutralizing antibody titers induced by the 1 μg and 0.1 μg doses of the G-head mRNA vaccine were 3033 and 38, respectively; the mean neutralizing antibody titers induced by the 1 μg and 0.1 μg doses of the G-full length mRNA vaccine were 1476 and 20, respectively; and the mean neutralizing antibody titers induced by the 1 μg and 0.1 μg doses of the G-ecto mRNA vaccine were 22 and 20, respectively. Comparative analysis indicates that the G-head mRNA vaccine induced the highest level of neutralizing antibody titers.

[0103] Example 3: Design of NiV and HeV heterodimer mRNA constructs and preparation of mRNA vaccines

[0104] The results of Example 2 showed that the antibody titer induced by the G-head was the highest. In order to obtain a bivalent mRNA vaccine that can induce a high level of immune response against both NiV and HeV, the inventors selected the G-head portions of NiV and HeV for tandem fusion to construct a NiV and HeV heterodimer.

[0105] In this embodiment, two NiV and HeV heterodimer mRNA constructs were designed: NiV / HeV G-head dimer and HeV / NiV G-head dimer. The design and preparation process is as follows:

[0106] Based on the NiV Indian strain (Genbank: NP_047112.2), the sequences of the G177-T602 and L166-T602 segments of the NiV G protein head domain were obtained, and their amino acid sequences are shown in SEQ ID NO: 7 and 11, respectively.

[0107] Based on the Australian HeV strain (Genbank: NP_047112.2), the sequences of the G177-S604 region or the L166-S604 region of the HeV G protein head domain were obtained, and their amino acid sequences are shown in SEQ ID NO: 10 and 8, respectively.

[0108] The NiV G protein head domain fragment shown in SEQ ID NO: 7 and the HeV G protein head domain fragment shown in SEQ ID NO: 8 were directly tandemly connected in order from N-terminus to C-terminus to obtain the tandem dimer of the two to construct the NiV / HeV G-head dimer, the amino acid sequence of which is shown in SEQ ID NO: 9.

[0109] The HeV G protein head domain fragment shown in SEQ ID NO: 10 and the NiV G protein head domain fragment shown in SEQ ID NO: 11 were directly tandemly connected in order from the N-terminus to the C-terminus to obtain the tandem dimer of the two to construct the HeV / NiV G-head dimer, the amino acid sequence of which is shown in SEQ ID NO: 12.

[0110] Following the method described in Example 1, mRNA expression plasmids and mRNA vaccines encoding the following antigens were prepared: NiV G protein head domain G177-T602 region (as shown in SEQ ID NO: 7, its DNA coding sequence is shown in SEQ ID NO: 13), HeV G protein head domain G177-S604 region (as shown in SEQ ID NO: 10, its DNA coding sequence is shown in SEQ ID NO: 14), NiV / HeV G-head dimer (as shown in SEQ ID NO: 9, its DNA coding sequence is shown in SEQ ID NO: 15, and the transcribed RNA sequence is shown in SEQ ID NO: 17), and HeV / NiV G-head dimer (as shown in SEQ ID NO: 12, its DNA coding sequence is shown in SEQ ID NO: 16, and the transcribed RNA sequence is shown in SEQ ID NO: 18). The resulting mRNA vaccines were named NiV-G-head, HeV-G-head, NiV / HeV G-head dimer, and HeV / NiV G-head dimer, respectively.

[0111] Example 4: Levels of binding and neutralizing antibodies against NiV and HeV induced by the NiV / HeV bivalent mRNA vaccine in BALB / c mice.

[0112] To evaluate the levels of binding and neutralizing antibodies against both NiV and HeV induced by the NiV / HeV heterodimeric mRNA vaccine prepared in Example 3 in BALB / c mice, animal immunization experiments were conducted using 6-8 week old female BALB / c mice (purchased from Vital River). The experimental groups, immunization doses, and procedures are shown in Figure 3.

[0113] The immunization experiment was divided into the following groups: an mRNA vaccine immunization group and a negative control group. The mRNA vaccine groups consisted of NiV G-head, HeV G-head, NiV / HeV G-head dimer, and HeV / NiV G-head dimer, respectively; the negative control group consisted of a GFP mRNA immunization group. All mice in the mRNA vaccine immunization group were immunized with the mRNA vaccine (i.e., NiV G-head, HeV G-head, NiV / HeV G-head dimer, and HeV / NiV G-head dimer) on days 0 and 21. The immunization procedure and dosage for the negative control group were the same as for the vaccine groups. All vaccinations were administered intramuscularly. The dosage for both the mRNA vaccine group and the negative control group was 10 μg mRNA per mouse. Serum samples were collected on day 35. The levels of antigen-specific binding IgG antibodies and neutralizing antibodies in the serum of the immunized mice were measured.

[0114] ELISA method for detecting antigen-specific binding antibody levels

[0115] The specific method for detecting the titer of IgG-binding antibodies in the serum of immunized BALB / c mice using ELISA was as follows: NiV G-head protein (amino acid sequence as shown in SEQ ID NO: 7) and HeV G-head protein (amino acid sequence as shown in SEQ ID NO: 10) were diluted to 3 μg / ml with ELISA coating buffer (sodium carbonate-sodium bicarbonate buffer, pH 9.6), and 100 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The coating buffer was discarded the next day, and the ELISA plate was blocked with 5% skim milk prepared with PBS and incubated at room temperature for 1 hour. Serum samples from mice in each experimental group in Example 2 (Figure 4) were serially diluted four times starting from 1:40 in 96-well plates, and then the diluted solutions were transferred to ELISA plates. The ELISA plates were then incubated at 37°C for 1.5 hours; the blocking solution was discarded, and the plates were washed four times with PBST. Next, add Goat Anti-Mouse HRP secondary antibody (purchased from Jinpulai) diluted 1:2000 with blocking buffer, incubate at 37°C for 1.5 hours, and wash 5 times with PBST. Then, add 60 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate for color development, and stop the reaction with 60 μL of 2M hydrochloric acid after 10 minutes. OD is measured on a PerkinElmer microplate reader. 450 Reading value. The endpoint titer is defined as the serum dilution factor corresponding to a reaction absorbance greater than 2.5 times the background value. Antibody titers below the detection limit are defined as half of the detection limit.

[0116] The titers of IgG antibodies against the NiV G-head protein antigen in the serum of immunized mice collected on day 35 are shown in Figure 4A. The results show that the NiV G-head, NiV / HeV G-head dimer, and HeV / NiV G-head dimer mRNA vaccines all induced high levels of NiV antigen-specific binding antibodies, with no statistically significant differences among the groups. The mean antibody titers induced were 6.6 × 10⁻⁶. 5 4.26.6×10 5 and 1.62×10 5 The titer of NiV antigen-specific binding antibodies induced by the HeV G-head mRNA vaccine was relatively low, with an average value of 2.57 × 10⁻⁶. 3 .

[0117] The titer of antibodies against HeV G-head protein antigen in the serum of immunized mice collected on day 35 is shown in Figure 4B. It shows that HeV G-head, NiV / HeV G-head dimer, and HeV / NiV G-head dimer mRNA vaccines all induced high levels of HeV antigen-specific binding antibodies, with mean antibody titers of 5.2 × 10⁻⁶ respectively. 5 6.56×10 5 and 5.2×10 5 All titers were significantly higher than those in the NiV G-head group (mean titer was 5.1 × 10⁻⁶). 3 ).

[0118] The results in Figure 4 show that: NiV G-head induced a high binding antibody titer against NiV, but a weak cross-reactivity against HeV; HeV G-head induced a high binding antibody titer against HeV, but a weak cross-reactivity against NiV; while NiV / HeV G-head dimer and HeV / NiV G-head dimer induced high levels of binding antibodies against both NiV and HeV, showing broad-spectrum activity.

[0119] In addition, the neutralizing antibody titers against NiV and HeV pseudoviruses in serum collected on day 35 of immunized mice were detected according to the pseudovirus neutralizing antibody detection method described in Example 2. The results are shown in Figures A and B of Figure 5, respectively. Figure A of Figure 5 shows that both NiV / HeV G-head dimer and HeV / NiV G-head dimer induced high levels of NiV pseudovirus neutralizing antibodies, with mean neutralizing antibody titers of 2.64 × 10⁻⁶. 5 2.76×10 5Compared with the NiV G-head group (mean titer: 2.97 × 10⁻⁶), 5 Similar to, but significantly higher than the HeV G-head group.

[0120] Figure 5B shows that the HeV / NiV G-head dimer group induced the highest level of neutralizing antibodies against HeV pseudoviruses, with a mean titer of 2.3 × 10⁻⁶. 5 The mean neutralizing antibody titers of HeV pseudoviruses induced by HeV G-head and NiV / HeV G-head drimer groups were 7.71 × 10⁻⁶. 4 and 7.22×10 4 The NiV G-head group had the lowest titer, with a mean titer of 9.71 × 10⁻⁶. 3 .

[0121] The results in Figure 5 show that: NiV G-head induced a high neutralizing antibody titer against NiV, but a relatively weak cross-reactivity against HeV; HeV G-head induced a high neutralizing antibody titer against HeV, but a relatively weak cross-reactivity against NiV; while NiV / HeV G-head dimer and HeV / NiV G-head dimer induced high neutralizing antibody titers against both NiV and HeV, showing broad-spectrum activity.

[0122] Example 5: Levels of binding and neutralizing antibodies against NiV and HeV induced by the NiV / HeV bivalent mRNA vaccine in golden hamsters.

[0123] To evaluate the levels of binding and neutralizing antibodies against both NiV and HeV induced by the NiV / HeV heterodimeric mRNA vaccine prepared in Example 3 in golden hamsters, animal immunization experiments were conducted using 6-week-old female hamsters (purchased from Vital River). The experimental groups, immunization doses, and procedures are shown in Figure 6.

[0124] The immunization experiment was divided into the following groups: an mRNA vaccine immunization group and a negative control group. The mRNA vaccine group received HeV / NiV G-head dimer, and the negative control group received GFP mRNA immunization. All hamsters in the mRNA vaccine immunization group were immunized with the mRNA vaccine (i.e., HeV / NiV G-head dimer) on days 0 and 21. The immunization procedure and dosage for the negative control group hamsters were the same as those in the vaccine group. All hamsters were immunized via intramuscular injection. The dose for both the mRNA vaccine group and the negative control group was 20 μg mRNA per hamster. Serum samples were collected on days 19 and 35, respectively. The levels of antigen-specific binding antibodies and neutralizing antibodies in the serum of the immunized hamsters were measured.

[0125] First, the titer of IgG-binding antibodies in immunized hamster serum was detected by ELISA. Specifically, NiV G-head protein (amino acid sequence as shown in SEQ ID NO: 7) and HeV G-head protein (amino acid sequence as shown in SEQ ID NO: 10) were diluted to 3 μg / ml with ELISA coating buffer (sodium carbonate-sodium bicarbonate buffer, pH 9.6). 100 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the ELISA plates were blocked with 5% skim milk prepared with PBS and incubated at room temperature for 1 hour. Serum samples from each experimental group of mice in Example 2 were serially diluted four times starting at 1:20 in 96-well round plates. The diluted solutions were then transferred to ELISA plates, and the plates were incubated at 37°C for 1.5 hours. The blocking solution was discarded, and the plates were washed four times with PBST. Next, add Goat Antihamster HRP secondary antibody diluted 1:15000 with blocking buffer, incubate at 37°C for 1.5 hours, and wash 5 times with PBST. Then, add 60 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate for color development, and stop the reaction with 60 μL of 2M hydrochloric acid after 10 minutes. OD is then measured using a PerkinElmer microplate reader. 450 Reading value. The endpoint titer is defined as the serum dilution factor corresponding to a reaction absorbance greater than 2.5 times the background value. Antibody titers below the detection limit are defined as half of the detection limit.

[0126] The titers of binding IgG antibodies against NiV-head and HeV-head protein antigens in immunized hamster serum collected on day 19 are shown in Figures A and B of Figure 7, respectively. This shows that the HeV / NiV G-head dimer mRNA vaccine can simultaneously induce binding antibodies against both NiV-head and HeV-head protein antigens, with mean binding antibody titers of 245 and 136, respectively. The titers of binding IgG antibodies against NiV-head and HeV-head protein antigens in immunized hamster serum collected on day 35 are shown in Figures A and B of Figure 8, respectively. This shows that the HeV / NiV G-head drimer mRNA vaccine can simultaneously induce high levels of binding antibodies against both NiV and HeV protein antigens, with mean binding antibody titers of 3221 and 3620, respectively.

[0127] Next, following the pseudovirus neutralizing antibody assay method described in Example 2, the neutralizing antibody titers against NiV and HeV pseudoviruses were detected in immunized hamster serum collected on day 19 and day 35, respectively.

[0128] The results of neutralizing antibody titers against NiV and HeV pseudoviruses in immunized hamster serum collected on day 19 are shown in Figures 9A and 9B, respectively. These figures show that the HeV / NiV G-head dimer vaccine can simultaneously induce neutralizing antibodies against both NiV and HeV pseudoviruses, with mean neutralizing antibody titers of 822 and 132, respectively. The results of neutralizing antibody titers against NiV and HeV pseudoviruses in immunized hamster serum collected on day 35 are shown in Figures 10A and 10B, respectively. These figures show that the HeV / NiV G-head dimer vaccine can simultaneously induce high levels of neutralizing antibodies against both NiV and HeV pseudoviruses, with mean neutralizing antibody titers of 7.16 × 10⁻⁶, respectively. 4 and 9.5×10 3 .

[0129] The results in Figures 7-10 indicate that the HeV / NiV G-head dimer vaccine can effectively induce golden hamsters to produce high levels of binding antibodies against NiV-head and HeV-head protein antigens, while also inducing high levels of neutralizing antibodies against NiV and HeV pseudoviruses.

[0130] 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

[0131] This invention relates to an RNA molecule encoding a recombinant Nipah / Hendra virus antigen, a vaccine or immunogenic composition comprising the same, and its use in the preparation of vaccines for the prevention and / or treatment of Nipah virus and / or Hendra virus infection. The RNA vaccine encoding the recombinant Nipah / Hendra virus antigen of this invention can simultaneously elicit a strong immune response against both HeV and NiV, and has significant value and broad prospects for clinical application.

Claims

1. An RNA molecule encoding a Nipah / Hendra virus recombinant antigen, said Nipah / Hendra virus recombinant antigen having an amino acid sequence arranged as shown in formula (I) or formula (II): (AB)-C-(A-B') (I) (A-B')-C-(AB) (II) 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 concatenated subsequence.

2. The RNA molecule 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 RNA molecule 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:7, or the amino acid sequence shown in SEQ ID NO:7 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:8, or the amino acid sequence as shown in SEQ ID NO:8 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 (I) is shown in SEQ ID NO:

9.

4. The RNA molecule 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:10, or the amino acid sequence as shown in SEQ ID NO:10 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:11, or the amino acid sequence as shown in SEQ ID NO:11 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:

12.

5. The RNA molecule according to any one of claims 1-4, characterized in that, The RNA molecule has an RNA sequence as shown in SEQ ID NO:17 or 18.

6. The RNA molecule according to any one of claims 1-4, characterized in that, The N-terminus of the Nipah / Hendra virus recombinant antigen also includes a signal peptide sequence.

7. The RNA molecule according to claim 6, characterized in that, The signal peptide sequence is shown in SEQ ID NO:19, and its coding sequence is shown in SEQ ID NO:

20.

8. A nucleic acid construct comprising the nucleotide sequence of an RNA molecule as described in any one of claims 1-7, and optionally, at least one expression regulatory element operatively linked to the nucleotide sequence of the RNA molecule.

9. An expression vector comprising the nucleic acid construct as described in claim 8.

10. A host cell wherein the cell is transformed or transfected with an RNA molecule as described in any one of claims 1-7, a nucleic acid construct as described in claim 8, or an expression vector as described in claim 9.

11. The use of the RNA molecule as described in any one of claims 1-7, the nucleic acid construct as described in claim 8, the expression vector as described in claim 9, or the host cell as described in claim 10 in the preparation of a medicament for the prevention and / or treatment of Nipah virus and / or Hendra virus infection.

12. The application according to claim 11, characterized in that, The drug in question is a vaccine.

13. The application according to claim 12, characterized in that, The vaccine in question is an mRNA vaccine.

14. A vaccine or immunogenic composition comprising an RNA molecule as described in any one of claims 1-7, a nucleic acid construct as described in claim 8, an expression vector as described in claim 9, or a host cell as described in claim 10, and a physiologically acceptable medium, adjuvant, excipient, carrier, and / or diluent.

15. The vaccine or immunogenic composition according to claim 14, wherein it is an mRNA vaccine, the mRNA vaccine comprising: (I) The RNA molecule as described in any one of claims 1-7; and (II) Lipid nanoparticles; Preferably, the lipid nanoparticles are composed of cationic lipid Dlin-MC3-DMA, distearate phosphatidylcholine (DSPC), cholesterol, and PEGylated lipid DMPE; more preferably, the molar ratio of cationic lipid Dlin-MC3-DMA, distearate phosphatidylcholine (DSPC), cholesterol, and PEGylated lipid DMPE is (40-60):(8-12):(36-40):(1-2), and more preferably 50:10:38.5:1.

5.

16. The vaccine or immunogenic composition according to claim 14 or 15, 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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