Nipah virus mRNA vaccine antigen nv1 and methods of making and using same

Abstract

The application discloses a Nipah virus mRNA vaccine antigen NV1 and a preparation method and application thereof, and belongs to the technical field of mRNA vaccines.The head domain of a Nipah virus G protein is used as a core immunization epitope region, and is directly connected to construct a pseudo-dimer antigen 2Gs, a coding sequence of which is obtained by human codon optimization, and is matched with specific 5'UTR and 3'UTR, mRNA is prepared through in-vitro transcription, and the mRNA is wrapped in a lipid nanoparticle by using a microfluidic technology to obtain the mRNA vaccine NV1.The experimental results show that the vaccine can efficiently induce humoral and cellular immune responses in mice and Syrian hamsters, has good safety, and has better immunogenicity than existing inactivated vaccines, attenuated live vaccines, recombinant subunit vaccines and virus vector vaccines.The vaccine can be used for preventing Nipah virus infection, and has important clinical application and public health values.

Description

Technical Field

[0001] This invention relates to the field of mRNA vaccine technology, specifically to a Nipah virus mRNA vaccine antigen NV1, its preparation method, and its application. Background Technology

[0002] Nipah virus (NiV) is an enveloped, single-stranded, negative-sense RNA virus belonging to the genus Henipavirus in the family Paramyxoviridae. First discovered in 1998-1999 during an outbreak in pigs and humans in Malaysia and Singapore, it is a zoonotic virus primarily affecting fruit bats. It can infect pigs, horses, and other animals, causing severe encephalitis and respiratory illnesses in humans. The mortality rate for humans infected with Nipah virus is as high as 40% to 75%, and it is listed by the World Health Organization (WHO) as one of the priority pathogens for which vaccines are urgently needed. Clinical manifestations of infection are diverse, ranging from asymptomatic infection to acute respiratory infection and fatal encephalitis. Survivors often suffer from severe neurological sequelae such as persistent seizures and personality changes. Despite the significant threat Nipah virus poses to global public health, there are currently no approved human vaccines or specific treatments available globally, and clinical management relies solely on supportive care.

[0003] Nipah virus primarily enters host cells through attachment glycoproteins (G protein) and fusion glycoproteins (F protein) on its envelope. The G protein is the main target for inducing neutralizing antibody production. While traditional full-length antigen designs include all epitopes, they also carry non-neutralizing epitopes or immunosuppressive regions, easily leading to a large number of ineffective antibodies, causing "immune interference," and reducing vaccine efficacy.

[0004] Current Nipah virus vaccine development technologies mainly include: Inactivated vaccines are produced by inactivating Nipah virus through physical or chemical methods. However, Nipah virus is a BSL-4 pathogen, and the cultivation and inactivation of live virus require extremely high biosafety levels in production facilities, resulting in high production costs and risks. The inactivation process can easily damage the natural conformation of G proteins, reduce the ability to induce neutralizing antibodies, and make it difficult to induce an effective cellular immune response.

[0005] Live attenuated vaccines: These are produced by genetic engineering to remove virulence genes or through continuous passage attenuation. However, the attenuated strains can replicate in the body, posing a risk of disease to immunocompromised individuals and carrying the risk of virulence reversion. Due to the high lethality of Nipah virus, the safety verification period is extremely long, and regulatory approval is strict.

[0006] Recombinant subunit protein vaccines: G protein or F protein is expressed using CHO or insect cell expression systems, purified and used with adjuvants, but the preparation cycle is long, the production efficiency is highly dependent on the expression level, and it must be used with adjuvants.

[0007] Viral vector vaccines: These use replication-defective viruses such as adenovirus and VSV as vectors to deliver G or F protein genes. However, pre-existing immunity against common vectors is prevalent in the population, which can neutralize the vector virus, significantly reducing delivery efficiency and immunization effect. Some viral vectors may also induce strong inflammatory responses, posing safety concerns. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a Nipah virus mRNA vaccine antigen NV1, its preparation method, and its application, specifically implemented through the following technical solutions: In a first aspect, the present invention proposes a Nipah virus mRNA vaccine antigen NV1, wherein the antigen is a pseudodimer antigen 2Gs, and the amino acid sequence is shown in SEQ ID NO.1.

[0009] Secondly, the present invention provides a DNA molecule encoding the antigen, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] Thirdly, the present invention proposes an mRNA molecule that encodes the aforementioned dimeric antigen 2Gs and includes a 5'UTR, a coding region, a 3'UTR, and 110 PolyA tails. The 5'UTR sequence is shown in SEQ ID NO.3; The 3'UTR sequence is shown in SEQ ID NO.4.

[0011] Fourthly, the present invention proposes an mRNA-LNP complex comprising the aforementioned mRNA molecule and lipid nanoparticles; The lipid nanoparticles consist of 50% SM102, 1.5% DMG-PEG2000, 10% DSPC, and 38.5% cholesterol by mass percentage.

[0012] Fifthly, the present invention provides a method for preparing the mRNA-LNP complex, comprising the following steps: (1) The head domain of the Nipah virus G protein was selected as the core immune epitope region and tandemly obtained the pseudodimer antigen 2Gs. (2) Human codons were optimized for the pseudodimer antigen 2Gs, and the recombinant plasmid pUC57-T7-5'UTR-2Gs-3'UTR-110PolyA was constructed; (3) The recombinant plasmid pUC57-T7-5'UTR-2Gs-3'UTR-110PolyA was transformed, amplified, extracted, linearized by BsaI restriction enzyme digestion, and purified. (4) Use linearized plasmids as templates for in vitro transcription to prepare modified mRNA; (5) mRNA was encapsulated in lipid nanoparticles using microfluidic technology and purified by ultrafiltration to obtain the mRNA-LNP complex.

[0013] Furthermore, the in vitro transcription system contains N1-Me-Pseudo UTP.

[0014] Furthermore, the buffer phase during microfluidic mixing is: The ethanol flow rate ratio was 15 mL / min: 5 mL / min, and the nitrogen-phosphorus ratio was 1:8.

[0015] Fifthly, the present invention proposes the application of the antigen, the mRNA molecule, and the mRNA-LNP complex in the preparation of a drug for preventing Nipah virus infection.

[0016] Furthermore, the drug is a vaccine.

[0017] Compared with the prior art, the present invention has the following advantages: This invention precisely screens the head domains of G proteins, eliminating non-neutralizing epitopes and immunosuppressive regions to avoid immune interference and significantly increase the proportion of effective antibodies. The dimeric antigen 2Gs efficiently induces high-titer specific IgG and pseudovirus neutralizing antibodies, while simultaneously activating a strong T-cell immune response. Two-dose immunization is superior to the three-dose efficacy of existing recombinant protein vaccines and also surpasses that of chimpanzee adenovirus vector vaccines. Immunization does not cause abnormal biochemical indicators or pathological damage to tissues and organs in mice, with no significant adverse reactions. The prepared mRNA vaccine does not require high-level biosafety facilities, has a short preparation cycle, and is easy to scale up for production, making it suitable for rapid response to sudden outbreaks. It exhibits good neutralizing activity against different Nipah virus strains and possesses broad-spectrum protective potential. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results of B-cell linear epitope prediction in an embodiment of the present invention are shown. Figure 2 The MHC I / II epitope prediction results in an embodiment of the present invention are shown; Figure 3 The results of the docking analysis of G protein with neutralizing antibody (PDB: 8XC4) in an embodiment of the present invention are shown; Figure 4 The design and structure prediction results of the pseudodimer antigen 2Gs in the embodiments of the present invention are shown; Figure 5 The particle size distribution of the vaccine NV1-LNP obtained using microfluidic technology in an embodiment of the present invention is shown. Figure 6 An electron micrograph of the NV1-LNPs vaccine obtained using microfluidic technology in an embodiment of the present invention is shown. Figure 7 The results of Western blotting detection of NV1 expression in vitro are shown in an embodiment of the present invention; Figure 8 The figures show the IgG binding antibody titers induced by NV1 at different time points in Balb / c in embodiments of the present invention; Figure 9 The figure shows the pseudovirus neutralizing antibody titer induced by NV1 on day 28 after the initial immunization in an embodiment of the present invention; Figure 10 This invention illustrates the cellular immunogenicity of NV1 in Balb / c mice, as demonstrated by ELISpot detection of the number of IFN-γ, IL-2, and IL-4 secreting cells. Figure 11 The IgG binding antibody titers induced in Syrian hamsters at different time points after NV1 immunization in embodiments of the present invention are shown. Figure 12 The pseudovirus neutralizing antibody titer induced in Syrian hamsters 28 days after the initial immunization with NV1 in this embodiment of the invention is shown. Figure 13 The following figure shows the IFN-γ secretion level induced in Syrian hamsters 28 days after the initial immunization with NV1 in an embodiment of the present invention. Figure 14 The concentrations of various biochemical indicators of Balb / c were shown 5 days after NV1 immunization in an embodiment of the present invention. Figure 15 The following is an example of the pathological scoring results of multiple tissues and organs 5 days after NV1 immunization in this embodiment of the invention; Figure 16 The results of pathological analysis of multiple tissues and organs 5 days after NV1 immunization in an embodiment of the present invention are shown. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and 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. Example 1

[0021] Identification of Nipah virus G protein immunoepitopes.

[0022] The full-length sequence of the Nipah virus NiV-M strain G protein was obtained from NCBI.

[0023] B-cell linear epitopes were analyzed using the Bepipred Linear Epitope Prediction algorithm from the IEDB database. Epitopes with a score ≥ 0.5 were identified as positive epitopes. Figure 1 ).

[0024] MHC I / II epitopes were predicted using the NetMHCpan 4.1 EL algorithm on the IEDB database, with percentiles <0.02 considered high-confidence epitopes. Figure 2 ).

[0025] Molecular docking prediction of the G protein with the neutralizing antibody (PDB: 8XC4) was performed using AlphaFold3, and the results are as follows: Figure 3 As shown, the vast majority of immunoepitaxes and antigen-antibody interaction regions are located in the head region (170aa-602aa), indicating that the head region of G proteins may have good immunogenicity. Example 2

[0026] Design and vector construction of pseudodimer antigen 2Gs.

[0027] By directly tandemly repeating the head domain of the G protein to obtain more dominant epitopes, a dimeric antigen 2Gs based on the Nipah virus G protein was obtained. Structure prediction was performed using AlphaFold3 (pTM=0.57), and its amino acid sequence is shown in SEQ ID NO.1. The results are as follows: Figure 4 As shown, the pseudodimer antigen 2Gs was successfully constructed and the vector was completed, and the antigen structure was correctly predicted.

[0028] Referring to the NCBI Human Codon Usage Database, the 2Gs codons were optimized according to human codon preferences (GC=50.02%, CAI=0.89) to obtain the DNA sequence (as shown in SEQ ID NO.2).

[0029] The synthesized 5'UTR sequence is shown in SEQ ID NO.3, and the 3'UTR sequence is shown in SEQ ID NO.4.

[0030] SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 were constructed into the pUC57 vector to obtain the recombinant plasmid pUC57-T7-5'UTR-2Gs-3'UTR-110PolyA.

[0031] The sequences involved in this embodiment are shown in Table 1: Table 1 Example 3

[0032] Plasmid linearization and in vitro transcription of mRNA.

[0033] (1) The recombinant plasmid was added to the Top10 bacterial strains and reacted on ice for 30 min, then at 42℃ for 90 s to fully activate it. 700 uL of LB medium was added and cultured at 37℃ with shaking at 220 rpm for 1 h. The cultured bacterial solution was plated and left to stand overnight. Positive single clones were added to 1 mL of LB medium containing ampicillin resistance and cultured at 37℃ with shaking at 220 rpm for 2 h. 400 uL of the shaken bacterial solution was added to 500 mL of LB medium containing ampicillin resistance and cultured at 37℃ with shaking at 220 rpm overnight. The plasmid was extracted from the bacterial solution using a plasmid extraction kit (MN#740414.50) according to the instructions. The final plasmid concentration was determined using NanoDrop, and the OD260 / 280 ratio was kept between 1.8 and 2.0 to ensure plasmid purity.

[0034] (2) Linearization of plasmid was performed using BsaI digestion reaction (total system 500uL; 50μg plasmid; 100U BsaI; 50uL 10*BsaI Buffer; digestion at 37℃ for 16h).

[0035] (3) Using commercial DNA magnetic beads (vazyme#N411-02), the DNA solution after the enzyme digestion reaction was purified according to the instructions. The concentration of the recovered DNA fragments was determined using a NanoDrop instrument, and the OD260 / 280 ratio was analyzed to be between 1.8 and 2.0 to ensure the purity of the DNA fragments. Transcription was performed using a commercial transcription kit (vazyme#DD4203), and purified using RNA magnetic beads (vazyme#N412-02) to obtain pure mRNA. The specific in vitro transcription preparation is as follows: 10 x Co-Reaction Buffer 2ul; N1-Me-Pseudo UTP (100 mM) 1.5ul; ATP Solution (100 mM) 1.5ul; CTP Solution (100 mM) 1.5ul; GTP Solution (100 mM) 1.5ul; CAG Trimer 1ul; T7 RNA Polymerase Mix 2ul; DNA template 1ug; RNase-free ddH2O Up to 20ul. (Please provide the corresponding Chinese translations for the English abbreviations.) The plasmid is completely linearized, with high mRNA purity and good integrity, and can be used for LNP encapsulation. Example 4

[0036] Preparation of mRNA-LNP vaccine NV1.

[0037] Dissolve the mRNA in a 50 mM citrate-sodium citrate buffer solution at pH 4 to achieve a final concentration of 10. 8 ng / μL; Preparation of LNP lipid mixture: SM102, DMG-PEG2000, DSPC, and cholesterol were mixed and dissolved in anhydrous ethanol solution at a mass ratio of 50%, 1.5%, 10%, and 38.5%, respectively. The lipid-ethanol solution and mRNA-citric acid solution were then filtered separately through a 0.22 μm microporous membrane. The mixture was then mixed using a microfluidic instrument at a flow rate of buffer phase:ethanol phase, based on a phosphorus content in mRNA to nitrogen content in SM102 ratio of 1:8. The ratio was 15 mL / min to 5 mL / min. The obtained mRNA-LNP was immediately diluted with 15 mL of the above citrate buffer and ultrafiltered using a 100 KD ultrafiltration tube at a centrifugal force of 3000 g. After ultrafiltration to 1 / 4 volume, 20 mM Tris-HCl buffer (pH=7.5) was added to 15 mL. This process was repeated twice. The particle size and encapsulation efficiency were then measured to ensure that the particle size was around 100 nm and the encapsulation efficiency was above 95%. The morphology of mRNA-LNPs was observed using cryo-electron microscopy.

[0038] Particle size distribution results are as follows Figure 5 As shown, NV1-LNP particles have a diameter of approximately 100 nm and a uniform distribution; cryo-electron microscopy observation results are as follows. Figure 6 As shown, the shape is regular; and the encapsulation rate is ≥95%, which meets the vaccine quality requirements. Example 5

[0039] Validation of NV1 expression in vitro.

[0040] (1) Seed approximately 2 × 10⁶ cells into a 6-well cell culture plate. 5 One HEK-293T cell was cultured for 24 hours, ensuring a density of 70%-80% of the 6-well culture plate.

[0041] (2) The prepared mRNA vaccine NV1 was evenly dropped into the transfection well (5 μg / well); (3) After 24 hours, the total protein in the cells was collected using cell lysis buffer and protease inhibitor. Western blotting was performed using Nipah virus G protein antibody (antibodysystem#PVV07901, 1:4000) to detect the expression of the target antigen.

[0042] The results are as follows Figure 7 As shown, a specific band appeared at the 100Kd position in the NV1 transfection group, while no band was observed in the control group, demonstrating that the 2Gs antigen can be effectively expressed. Example 6

[0043] Immunogenicity evaluation of NV1 in Balb / c mice.

[0044] Balb / c mice were immunized with the NV1 vaccine at a high dose of 10 μg / mouse and a low dose of 2 μg / mouse (6-8 weeks old, intramuscular injection). Balb / c mice that received an equal volume of PBS intramuscularly served as the Placebo control group. Immunization was performed twice, on days 0 and 14. The humoral immunogenicity of the vaccine was assessed by detecting serum antibodies against specific IgG binding to the G protein.

[0045] The results showed that NV1 can induce high levels of specific IgG binding antibodies (such as...) Figure 8 (As shown); it can induce highly efficient pseudovirus neutralizing antibodies and has excellent humoral immunogenicity (e.g. Figure 9 (As shown). Example 7

[0046] NV1 cell immune response detection.

[0047] Splenic lymphocytes were isolated from mice 28 days after the initial immunization.

[0048] The ELiSpot method was used to detect IL-2, INF-γ and IL-4 positive cells in spleen immune cells after antigen stimulation to assess the level of cellular immunity.

[0049] The results are as follows Figure 10 As shown, NV1 can significantly induce the secretion of IFN-γ, IL-2, and IL-4, resulting in a strong cellular immune response. Example 8

[0050] Immunogenicity verification of NV1 in Syrian hamsters.

[0051] Syrian hamsters were grouped and immunized according to the same procedures as mice.

[0052] Serum IgG, pseudovirus neutralizing antibody and IFN-γ levels were detected.

[0053] The results are as follows Figures 11-13 As shown, NV1 can induce high levels of IgG (such as...) in hamsters. Figure 11 As shown); can induce highly efficient pseudovirus neutralizing antibodies (such as... Figure 12 As shown); can induce high levels of IFN-γ (such as... Figure 13 As shown in the figure, this suggests that it has the potential to protect against Nipah virus. Example 9

[0054] Preliminary safety assessment of NV1.

[0055] Blood and organs such as heart, liver, spleen, lungs, and kidneys were collected from mice on day 5 post-immunization for basic physiological and biochemical index testing and pathological damage analysis.

[0056] The results are as follows Figures 14-16 As shown, the results indicate that the NV1 vaccine has a certain degree of safety, does not cause significant changes in physiological and biochemical indicators, and does not cause organ damage due to vaccine immunization.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. 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. Such 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 embodiments of the present invention.

Claims

1. A Nipah virus mRNA vaccine antigen NV1, characterized in that, The antigen is a pseudodimer antigen 2Gs, and its amino acid sequence is shown in SEQ ID NO.

1.

2. A DNA molecule encoding the antigen of claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

3. An mRNA molecule, characterized in that, The mRNA molecule encodes the pseudodimer antigen 2Gs of claim 1 and includes a 5'UTR, a coding region, a 3'UTR, and 110 PolyA tails; The 5'UTR sequence is shown in SEQ ID NO.3; The 3'UTR sequence is shown in SEQ ID NO.

4.

4. An mRNA-LNP complex, characterized in that, Contains the mRNA molecule and lipid nanoparticles as described in claim 3; The lipid nanoparticles consist of 50% SM102, 1.5% DMG-PEG2000, 10% DSPC, and 38.5% cholesterol by mass percentage.

5. A method for preparing the mRNA-LNP complex according to claim 4, characterized in that, Includes the following steps: (1) The head domain of the Nipah virus G protein was selected as the core immune epitope region and tandemly obtained the pseudodimer antigen 2Gs. (2) Human codons were optimized for the pseudodimer antigen 2Gs, and the recombinant plasmid pUC57-T7-5'UTR-2Gs-3'UTR-110PolyA was constructed; (3) The recombinant plasmid pUC57-T7-5'UTR-2Gs-3'UTR-110PolyA was transformed, amplified, extracted, linearized by BsaI restriction enzyme digestion, and purified. (4) Use linearized plasmids as templates for in vitro transcription to prepare modified mRNA; (5) mRNA was encapsulated in lipid nanoparticles using microfluidic technology and purified by ultrafiltration to obtain the mRNA-LNP complex.

6. The preparation method according to claim 5, characterized in that, The in vitro transcription system contains N1-Me-PseudoUTP.

7. The preparation method according to claim 5, characterized in that, The buffer phase for microfluidic mixing is: The ethanol flow rate ratio was 15 mL / min: 5 mL / min, and the nitrogen-phosphorus ratio was 1:

8.

8. The use of the antigen of claim 1, the mRNA molecule of claim 3, and the mRNA-LNP complex of claim 4 in the preparation of a medicament for the prevention of Nipah virus infection.

9. The application according to claim 8, characterized in that, The drug in question is a vaccine.

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